121 results on '"Gabriel Mazzucchelli"'
Search Results
2. HPV infection alters vaginal microbiome through down-regulating host mucosal innate peptides used by Lactobacilli as amino acid sources
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Alizee Lebeau, Diane Bruyere, Patrick Roncarati, Paul Peixoto, Eric Hervouet, Gael Cobraiville, Bernard Taminiau, Murielle Masson, Carmen Gallego, Gabriel Mazzucchelli, Nicolas Smargiasso, Maximilien Fleron, Dominique Baiwir, Elodie Hendrick, Charlotte Pilard, Thomas Lerho, Celia Reynders, Marie Ancion, Roland Greimers, Jean-Claude Twizere, Georges Daube, Geraldine Schlecht-Louf, Françoise Bachelerie, Jean-Damien Combes, Pierrette Melin, Marianne Fillet, Philippe Delvenne, Pascale Hubert, and Michael Herfs
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Science - Abstract
Here, the authors show that HPV infection leads to downregulation of host mucosal innate peptides, which are in turn used by predominant Lactobacillus species as amino acid source, promoting ultimately an imbalance in the vaginal flora.
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- 2022
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3. Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal
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Fernanda Gobbi Amorim, Damien Redureau, Thomas Crasset, Lou Freuville, Dominique Baiwir, Gabriel Mazzucchelli, Stefanie K. Menzies, Nicholas R. Casewell, and Loïc Quinton
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venomics ,proteomic ,mass spectrometry ,snake venom ,toxin ,multi-enzymatic digestion ,Medicine - Abstract
To improve the characterization of snake venom protein profiles, we report the application of a new generation of proteomic methodology to deeply characterize complex protein mixtures. The new approach, combining a synergic multi-enzymatic and a time-limited digestion (MELD), is a versatile and straightforward protocol previously developed by our group. The higher number of overlapping peptides generated during MELD increases the quality of downstream peptide sequencing and of protein identification. In this context, this work aims at applying the MELD strategy to a venomics purpose for the first time, and especially for the characterization of snake venoms. We used four venoms as the test models for this proof of concept: two Elapidae (Dendroaspis polylepis and Naja naja) and two Viperidae (Bitis arietans and Echis ocellatus). Each venom was reduced and alkylated before being submitted to two different protocols: the classical bottom-up proteomics strategy including a digestion step with trypsin only, or MELD, which combines the activities of trypsin, Glu-C and chymotrypsin with a limited digestion approach. The resulting samples were then injected on an M-Class chromatographic system, and hyphenated to a Q-Exactive Mass Spectrometer. Toxins and protein identification were performed by Peaks Studio X+. The results show that MELD considerably improves the number of sequenced (de novo) peptides and identified peptides from protein databases, leading to the unambiguous identification of a greater number of toxins and proteins. For each venom, MELD was successful, not only in terms of the identification of the major toxins (increasing of sequence coverage), but also concerning the less abundant cellular components (identification of new groups of proteins). In light of these results, MELD represents a credible methodology to be applied as the next generation of proteomics approaches dedicated to venomic analysis. It may open new perspectives for the sequencing and inventorying of the venom arsenal and should expand global knowledge about venom composition.
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- 2023
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4. Wobble tRNA modification and hydrophilic amino acid patterns dictate protein fate
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Francesca Rapino, Zhaoli Zhou, Ana Maria Roncero Sanchez, Marc Joiret, Christian Seca, Najla El Hachem, Gianluca Valenti, Sara Latini, Kateryna Shostak, Liesbet Geris, Ping Li, Gang Huang, Gabriel Mazzucchelli, Dominique Baiwir, Christophe J. Desmet, Alain Chariot, Michel Georges, and Pierre Close
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Science - Abstract
Wobble uridine (U34) tRNA modifications are important for the decoding of AA-ending codons. Here the authors show that while the U34-codon content of mRNAs are predictive of changes in ribosome translation elongation, the resulting outcome in protein expression also relies on specific hydrophilic motifs-dependent protein aggregation and clearance.
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- 2021
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5. Lunaemycins, New Cyclic Hexapeptide Antibiotics from the Cave Moonmilk-Dweller Streptomyces lunaelactis MM109T
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Loïc Martinet, Aymeric Naômé, Lucas C. D. Rezende, Déborah Tellatin, Bernard Pignon, Jean-Denis Docquier, Filomena Sannio, Dominique Baiwir, Gabriel Mazzucchelli, Michel Frédérich, and Sébastien Rigali
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antibiotics ,cryptic metabolites ,natural compounds ,Anti-MRSA ,moonmilk ,cave microbiology ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Streptomyces lunaelactis strains have been isolated from moonmilk deposits, which are calcium carbonate speleothems used for centuries in traditional medicine for their antimicrobial properties. Genome mining revealed that these strains are a remarkable example of a Streptomyces species with huge heterogeneity regarding their content in biosynthetic gene clusters (BGCs) for specialized metabolite production. BGC 28a is one of the cryptic BGCs that is only carried by a subgroup of S. lunaelactis strains for which in silico analysis predicted the production of nonribosomal peptide antibiotics containing the non-proteogenic amino acid piperazic acid (Piz). Comparative metabolomics of culture extracts of S. lunaelactis strains either holding or not holding BGC 28a combined with MS/MS-guided peptidogenomics and 1H/13C NMR allowed us to identify the cyclic hexapeptide with the amino acid sequence (D-Phe)-(L-HO-Ile)-(D-Piz)-(L-Piz)-(D-Piz)-(L-Piz), called lunaemycin A, as the main compound synthesized by BGC 28a. Molecular networking further identified 18 additional lunaemycins, with 14 of them having their structure elucidated by HRMS/MS. Antimicrobial assays demonstrated a significant bactericidal activity of lunaemycins against Gram-positive bacteria, including multi-drug resistant clinical isolates. Our work demonstrates how an accurate in silico analysis of a cryptic BGC can highly facilitate the identification, the structural elucidation, and the bioactivity of its associated specialized metabolites.
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- 2023
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6. Glycosylation deficiency of lipopolysaccharide-binding protein and corticosteroid-binding globulin associated with activity and response to treatment for rheumatoid arthritis
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Federica Ciregia, Dominique Baiwir, Gaël Cobraiville, Thibaut Dewael, Gabriel Mazzucchelli, Valérie Badot, Silvana Di Romana, Paschalis Sidiras, Tatiana Sokolova, Patrick Durez, Michel G. Malaise, and Dominique de Seny
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Rheumatoid arthritis ,Post translational modifications ,Glycosylation ,CBG ,LBP ,Biomarkers ,Medicine - Abstract
Abstract Background Serum protein glycosylation is an area of investigation in inflammatory arthritic disorders such as rheumatoid arthritis (RA). Indeed, some studies highlighted abnormalities of protein glycosylation in RA. Considering the numerous types of enzymes, monosaccharides and glycosidic linkages, glycosylation is one of the most complex post translational modifications. By this work, we started with a preliminary screening of glycoproteins in serum from RA patients and controls. Methods In order to isolate glycoproteins from serum, lectin wheat germ agglutinin was used and quantitative differences between patients and controls were investigated by LC–MS/MS. Consequently, we focused our attention on two glycoproteins found in this explorative phase: corticosteroid-binding globulin (CBG) and lipopolysaccharide-binding protein (LBP). The subsequent validation with immunoassays was widened to a larger number of early RA (ERA) patients (n = 90) and well-matched healthy controls (n = 90). Results We observed a significant reduction of CBG and LBP glycosylation in ERA patients compared with healthy controls. Further, after 12 months of treatment, glycosylated CBG and LBP levels increased both to values comparable to those of controls. In addition, these changes were correlated with clinical parameters. Conclusions This study enables to observe that glycosylation changes of CBG and LBP are related to RA disease activity and its response to treatment.
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- 2020
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7. Tumor resistance to ferroptosis driven by Stearoyl-CoA Desaturase-1 (SCD1) in cancer cells and Fatty Acid Biding Protein-4 (FABP4) in tumor microenvironment promote tumor recurrence
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Géraldine Luis, Adrien Godfroid, Shin Nishiumi, Jonathan Cimino, Silvia Blacher, Erik Maquoi, Coline Wery, Alice Collignon, Rémi Longuespée, Laetitia Montero-Ruiz, Isabelle Dassoul, Naima Maloujahmoum, Charles Pottier, Gabriel Mazzucchelli, Edwin Depauw, Akeila Bellahcène, Masaru Yoshida, Agnès Noel, and Nor Eddine Sounni
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Lipid metabolism ,Hypoxia ,Reoxygenation ,Drug-resistance ,Tumor-microenvironment ,ROS-ferroptosis ,Medicine (General) ,R5-920 ,Biology (General) ,QH301-705.5 - Abstract
Problem: Tumor recurrence is a major clinical issue that represents the principal cause of cancer-related deaths, with few targetable common pathways. Mechanisms by which residual tumors persist and progress under a continuous shift between hypoxia-reoxygenation after neoadjuvent-therapy are unknown. In this study, we investigated the role of lipid metabolism and tumor redox balance in tumor recurrence. Methods: Lipidomics, proteomics and mass spectrometry imaging approaches where applied to mouse tumor models of recurrence. Genetic and pharmacological inhibitions of lipid mediators in tumors were used in vivo and in functional assays in vitro. Results: We found that stearoyl-CoA desaturase-1 (SCD1) expressed by cancer cells and fatty acid binding protein-4 (FABP4) produced by tumor endothelial cells (TECs) and adipocytes in the tumor microenvironment (TME) are essential for tumor relapse in response to tyrosine kinase inhibitors (TKI) and chemotherapy. SCD1 and FABP4 were also found upregulated in recurrent human breast cancer samples and correlated with worse prognosis of cancer patients with different types of tumors. Mechanistically, SCD1 leads to fatty acid (FA) desaturation and FABP4 derived from TEM enhances lipid droplet (LD) in cancer cells, which cooperatively protect from oxidative stress-induced ferroptosis. We revealed that lipid mobilization and desaturation elicit tumor intrinsic antioxidant and anti-ferroptotic resources for survival and regrowth in a harsh TME. Inhibition of lipid transport from TME by FABP4 inhibitor reduced tumor regrowth and by genetic — or by pharmacological — targeting SCD1 in vivo, tumor regrowth was abolished completely. Conclusion: This finding unveils that it is worth taking advantage of tumor lipid addiction, as a tumor vulnerability to design novel treatment strategy to prevent cancer recurrence.
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- 2021
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8. Sweat Proteomics in Cystic Fibrosis: Discovering Companion Biomarkers for Precision Medicine and Therapeutic Development
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Bastien Burat, Audrey Reynaerts, Dominique Baiwir, Maximilien Fléron, Sophie Gohy, Gauthier Eppe, Teresinha Leal, and Gabriel Mazzucchelli
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cystic fibrosis ,human eccrine sweat ,shotgun proteomics ,companion biomarkers ,actin cytoskeleton ,Cytology ,QH573-671 - Abstract
In clinical routine, the diagnosis of cystic fibrosis (CF) is still challenging regardless of international consensus on diagnosis guidelines and tests. For decades, the classical Gibson and Cooke test measuring sweat chloride concentration has been a keystone, yet, it may provide normal or equivocal results. As of now, despite the combination of sweat testing, CFTR genotyping, and CFTR functional testing, a small fraction (1–2%) of inconclusive diagnoses are reported and justifies the search for new CF biomarkers. More importantly, in the context of precision medicine, with a view to early diagnosis, better prognosis, appropriate clinical follow-up, and new therapeutic development, discovering companion biomarkers of CF severity and phenotypic rescue are of utmost interest. To date, previous sweat proteomic studies have already documented disease-specific variations of sweat proteins (e.g., in schizophrenia and tuberculosis). In the current study, sweat samples from 28 healthy control subjects and 14 patients with CF were analyzed by nanoUHPLC-Q-Orbitrap-based shotgun proteomics, to look for CF-associated changes in sweat protein composition and abundance. A total of 1057 proteins were identified and quantified at an individual level, by a shotgun label-free approach. Notwithstanding similar proteome composition, enrichment, and functional annotations, control and CF samples featured distinct quantitative proteome profiles significantly correlated with CF, accounting for the respective inter-individual variabilities of control and CF sweat. All in all: (i) 402 sweat proteins were differentially abundant between controls and patients with CF, (ii) 68 proteins varied in abundance between F508del homozygous patients and patients with another genotype, (iii) 71 proteins were differentially abundant according to the pancreatic function, and iv) 54 proteins changed in abundance depending on the lung function. The functional annotation of pathophysiological biomarkers highlighted eccrine gland cell perturbations in: (i) protein biosynthesis and trafficking, (ii) CFTR proteostasis and membrane stability, and (iii) cell-cell adherence, membrane integrity, and cytoskeleton crosstalk. Cytoskeleton-related biomarkers were of utmost interest because of the consistency between variations observed here in CF sweat and variations previously documented in other CF tissues. From a clinical stance, nine candidate biomarkers of CF diagnosis (CUTA, ARG1, EZR, AGA, FLNA, MAN1A1, MIA3, LFNG, SIAE) and seven candidate biomarkers of CF severity (ARG1, GPT, MDH2, EML4 (F508del homozygous), MGAT1 (pancreatic insufficiency), IGJ, TOLLIP (lung function impairment)) were deemed suitable for further verification.
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- 2022
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9. Structure of New Ferroverdins Recruiting Unconventional Ferrous Iron Chelating Agents
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Loïc Martinet, Dominique Baiwir, Gabriel Mazzucchelli, and Sébastien Rigali
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CETP inhibitors ,iron complexes ,Streptomyces ,HDL cholesterol ,metal-nitrosophenolato compounds ,natural products ,Microbiology ,QR1-502 - Abstract
Ferroverdins are ferrous iron (Fe2+)-nitrosophenolato complexes produced by a few Streptomyces species as a response to iron overload. Previously, three ferroverdins were identified: ferroverdin A, in which three molecules of p-vinylphenyl-3-nitroso-4-hydroxybenzoate (p-vinylphenyl-3,4-NHBA) are recruited to bind Fe2+, and Ferroverdin B and Ferroverdin C, in which one molecule of p-vinylphenyl-3,4-NHBA is substituted by hydroxy-p-vinylphenyl-3,4-NHBA, and by carboxy-p-vinylphenyl-3,4-NHBA, respectively. These molecules, especially ferroverdin B, are potent inhibitors of the human cholesteryl ester transfer protein (CETP) and therefore candidate hits for the development of drugs that increase the serum concentration of high-density lipoprotein cholesterol, thereby diminishing the risk of atherosclerotic cardiovascular disease. In this work, we used high-resolution mass spectrometry combined with tandem mass spectrometry to identify 43 novel ferroverdins from the cytosol of two Streptomyces lunaelactis species. For 13 of them (designated ferroverdins C2, C3, D, D2, D3, E, F, G, H, CD, DE, DF, and DG), we could elucidate their structure, and for the other 17 new ferroverdins, ambiguity remains for one of the three ligands. p-formylphenyl-3,4-NHBA, p-benzoic acid-3,4-NHBA, 3,4-NHBA, p-phenylpropionate-3,4-NHBA, and p-phenyacetate-3,4-NHBA were identified as new alternative chelators for Fe2+-binding, and two compounds (C3 and D3) are the first reported ferroverdins that do not recruit p-vinylphenyl-3,4-NHBA. Our work thus uncovered putative novel CETP inhibitors or ferroverdins with novel bioactivities.
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- 2022
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10. In vivo N-Terminomics Highlights Novel Functions of ADAMTS2 and ADAMTS14 in Skin Collagen Matrix Building
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Cédric Leduc, Laura Dupont, Loïc Joannes, Christine Monseur, Dominique Baiwir, Gabriel Mazzucchelli, Christophe Deroanne, Alain Colige, and Mourad Bekhouche
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ADAMTS ,collagen ,Ehlers-Danlos Syndrome (EDS) ,degradomics ,TAILS ,N-Terminomics ,Biology (General) ,QH301-705.5 - Abstract
A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2 and ADAMTS14 were originally known for their ability to cleave the aminopropeptides of fibrillar collagens. Previous work using N-terminomic approach (N-TAILS) in vitro led to the identification of new substrates, including some molecules involved in TGF-β signaling. Here, N-TAILS was used to investigate the substrates of these two enzymes in vivo, by comparing the N-terminomes of the skin of wild type mice, mice deficient in ADAMTS2, in ADAMTS14 and in both ADAMTS2 and ADAMTS14. This study identified 68 potential extracellular and cell surface proteins, with the majority of them being cleaved by both enzymes. These analyses comfort their role in collagen matrix organization and suggest their implication in inflammatory processes. Regarding fibrillar collagen, this study demonstrates that both ADAMTS2 and ADAMTS14 are involved in the processing of the aminopropeptide of alpha1 and alpha2 type V collagen. It also revealed the existence of several cleavage sites in the Col1 domain and in the C-propeptide of type I collagens. In addition to collagens and other extracellular proteins, two major components of the cell cytoskeleton, actin and vimentin, were also identified as potential substrates. The latter data were confirmed in vitro using purified enzymes and could potentially indicate other functions for ADAMTS2 and 14. This original investigation of mouse skin degradomes by N-terminomic highlights the essential role of ADAMTS2 and ADAMTS14 in collagen matrix synthesis and turnover, and gives clues to better understand their functions in skin pathophysiology. Data are available via ProteomeXchange with identifier PXD022179.
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- 2021
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11. Plant defense in response to chewing insects: proteome analysis of Arabidopsis thaliana damaged by Plutella xylostella
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Dieu-Hien Truong, Hoang Chinh Nguyen, Julien Bauwens, Gabriel Mazzucchelli, Georges Lognay, and Frédéric Francis
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Arabidopsis thaliana ,Plutella xylostella ,proteomic expression ,2-DE ,MALDI-TOF MS ,LC-ESI-MS/MS ,Plant culture ,SB1-1110 ,Plant ecology ,QK900-989 - Abstract
The interactions between Arabidopsis thaliana and Plutella xylostella have been considered as a model system to unravel the responses of plants to herbivorous insects. Here, we use a 2-DE proteome approach to detect protein expression changes in the leaves of Arabidopsis plants exposed to P. xylostella larval infestation at 27°C within 8 h. Approximately 450 protein spots were reproducibly detected on gels. Of these, comparing healthy and infested leaves, we identified 18 differentially expressed protein spots. Thirteen proteins were successfully identified by MALDI-TOF/MS and LC-ESI-MS/MS. Functional classification analysis indicated that the differentially identified proteins were associated with amino acid, carbohydrate, energy, lipid metabolism, and photosynthesis. In addition, their relative abundances were assessed according to larval pest feeding on Arabidopsis leaves. These data provide valuable new insights for further works in plant-biotic and environmental stress interaction.
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- 2018
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12. New Proteins Contributing to Immune Cell Infiltration and Pannus Formation of Synovial Membrane from Arthritis Diseases
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Dominique de Seny, Dominique Baiwir, Elettra Bianchi, Gaël Cobraiville, Céline Deroyer, Christophe Poulet, Olivier Malaise, Geneviève Paulissen, Marie-Joëlle Kaiser, Jean-Philippe Hauzeur, Gabriel Mazzucchelli, Philippe Delvenne, and Michel Malaise
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proteomics ,synovial membrane ,inflammation ,LAP3 ,DNAJB11 ,MANF ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
An inflamed synovial membrane plays a major role in joint destruction and is characterized by immune cells infiltration and fibroblast proliferation. This proteomic study considers the inflammatory process at the molecular level by analyzing synovial biopsies presenting a histological inflammatory continuum throughout different arthritis joint diseases. Knee synovial biopsies were obtained from osteoarthritis (OA; n = 9), chronic pyrophosphate arthropathy (CPPA; n = 7) or rheumatoid arthritis (RA; n = 8) patients. The histological inflammatory score was determined using a semi-quantitative scale based on synovial hyperplasia, lymphocytes, plasmocytes, neutrophils and macrophages infiltration. Proteomic analysis was performed by liquid chromatography-mass spectrometry (LC-MS/MS). Differentially expressed proteins were confirmed by immunohistochemistry. Out of the 1871 proteins identified and quantified by LC-MS/MS, 10 proteins (LAP3, MANF, LCP1, CTSZ, PTPRC, DNAJB11, EML4, SCARA5, EIF3K, C1orf123) were differentially expressed in the synovial membrane of at least one of the three disease groups (RA, OA and CPPA). Significant increased expression of the seven first proteins was detected in RA and correlated to the histological inflammatory score. Proteomics is therefore a powerful tool that provides a molecular pattern to the classical histology usually applied for synovitis characterization. Except for LCP1, CTSZ and PTPRC, all proteins have never been described in human synovitis.
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- 2021
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13. Characterization of the Human Eccrine Sweat Proteome—A Focus on the Biological Variability of Individual Sweat Protein Profiles
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Bastien Burat, Audrey Reynaerts, Dominique Baiwir, Maximilien Fléron, Gauthier Eppe, Teresinha Leal, and Gabriel Mazzucchelli
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human eccrine sweat ,shotgun proteomics ,inter-individual variability ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The potential of eccrine sweat as a bio-fluid of interest for diagnosis and personalized therapy has not yet been fully evaluated, due to the lack of in-depth sweat characterization studies. Thanks to recent developments in omics, together with the availability of accredited sweat collection methods, the analysis of human sweat may now be envisioned as a standardized, non-invasive test for individualized monitoring and personalized medicine. Here, we characterized individual sweat samples, collected from 28 healthy adult volunteers under the most standardized sampling methodology, by applying optimized shotgun proteomics. The thorough characterization of the sweat proteome allowed the identification of 983 unique proteins from which 344 were identified across all samples. Annotation-wise, the study of the sweat proteome unveiled the over-representation of newly addressed actin dynamics, oxidative stress and proteasome-related functions, in addition to well-described proteolysis and anti-microbial immunity. The sweat proteome composition correlated with the inter-individual variability of sweat secretion parameters. In addition, both gender-exclusive proteins and gender-specific protein abundances were highlighted, despite the high similarity between human female and male sweat proteomes. In conclusion, standardized sample collection coupled with optimized shotgun proteomics significantly improved the depth of sweat proteome coverage, far beyond previous similar studies. The identified proteins were involved in many diverse biological processes and molecular functions, indicating the potential of this bio-fluid as a valuable biological matrix for further studies. Addressing sweat variability, our results prove the proteomic profiling of sweat to be a promising bio-fluid analysis for individualized, non-invasive monitoring and personalized medicine.
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- 2021
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14. Revisiting Ehrlichia ruminantium Replication Cycle Using Proteomics: The Host and the Bacterium Perspectives
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Isabel Marcelino, Philippe Holzmuller, Ana Coelho, Gabriel Mazzucchelli, Bernard Fernandez, and Nathalie Vachiéry
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Ehrlichia ruminantium ,endothelial cells ,host response ,immunomodulation ,bacterial life cycle ,differential protein expression ,Biology (General) ,QH301-705.5 - Abstract
The Rickettsiales Ehrlichia ruminantium, the causal agent of the fatal tick-borne disease Heartwater, induces severe damage to the vascular endothelium in ruminants. Nevertheless, E. ruminantium-induced pathobiology remains largely unknown. Our work paves the way for understanding this phenomenon by using quantitative proteomic analyses (2D-DIGE-MS/MS, 1DE-nanoLC-MS/MS and biotin-nanoUPLC-MS/MS) of host bovine aorta endothelial cells (BAE) during the in vitro bacterium intracellular replication cycle. We detect 265 bacterial proteins (including virulence factors), at all time-points of the E. ruminantium replication cycle, highlighting a dynamic bacterium–host interaction. We show that E. ruminantium infection modulates the expression of 433 host proteins: 98 being over-expressed, 161 under-expressed, 140 detected only in infected BAE cells and 34 exclusively detected in non-infected cells. Cystoscape integrated data analysis shows that these proteins lead to major changes in host cell immune responses, host cell metabolism and vesicle trafficking, with a clear involvement of inflammation-related proteins in this process. Our findings led to the first model of E. ruminantium infection in host cells in vitro, and we highlight potential biomarkers of E. ruminantium infection in endothelial cells (such as ROCK1, TMEM16K, Albumin and PTPN1), which may be important to further combat Heartwater, namely by developing non-antibiotic-based strategies.
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- 2021
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15. On the Risks of Phylogeny-Based Strain Prioritization for Drug Discovery: Streptomyces lunaelactis as a Case Study
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Loïc Martinet, Aymeric Naômé, Dominique Baiwir, Edwin De Pauw, Gabriel Mazzucchelli, and Sébastien Rigali
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strain prioritization ,metabolomics ,chemical diversity ,natural product ,drug discovery ,genome mining ,Microbiology ,QR1-502 - Abstract
Strain prioritization for drug discovery aims at excluding redundant strains of a collection in order to limit the repetitive identification of the same molecules. In this work, we wanted to estimate what can be unexploited in terms of the amount, diversity, and novelty of compounds if the search is focused on only one single representative strain of a species, taking Streptomyces lunaelactis as a model. For this purpose, we selected 18 S. lunaelactis strains taxonomically clustered with the archetype strain S. lunaelactis MM109T. Genome mining of all S. lunaelactis isolated from the same cave revealed that 54% of the 42 biosynthetic gene clusters (BGCs) are strain specific, and five BGCs are not present in the reference strain MM109T. In addition, even when a BGC is conserved in all strains such as the bag/fev cluster involved in bagremycin and ferroverdin production, the compounds produced highly differ between the strains and previously unreported compounds are not produced by the archetype MM109T. Moreover, metabolomic pattern analysis uncovered important profile heterogeneity, confirming that identical BGC predisposition between two strains does not automatically imply chemical uniformity. In conclusion, trying to avoid strain redundancy based on phylogeny and genome mining information alone can compromise the discovery of new natural products and might prevent the exploitation of the best naturally engineered producers of specific molecules.
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- 2020
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16. Human Liver-Derived Extracellular Matrix for the Culture of Distinct Human Primary Liver Cells
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Niki Alevra Sarika, Valéry L. Payen, Maximilien Fléron, Joachim Ravau, Davide Brusa, Mustapha Najimi, Edwin De Pauw, Gauthier Eppe, Gabriel Mazzucchelli, Etienne M. Sokal, Anne des Rieux, and Adil El Taghdouini
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liver ,primary cells ,extracellular matrix ,decellularization ,proteomics ,Cytology ,QH573-671 - Abstract
The lack of robust methods to preserve, purify and in vitro maintain the phenotype of the human liver’s highly specialized parenchymal and non-parenchymal cell types importantly hampers their exploitation for the development of research and clinical applications. There is in this regard a growing interest in the use of tissue-specific extracellular matrix (ECM) to provide cells with an in vitro environment that more closely resembles that of the native tissue. In the present study, we have developed a method that allows for the isolation and downstream application of the human liver’s main cell types from cryopreserved material. We also isolated and solubilized human liver ECM (HL-ECM), analyzed its peptidomic and proteomic composition by mass spectrometry and evaluated its interest for the culture of distinct primary human liver cells. Our analysis of the HL-ECM revealed proteomic diversity, type 1 collagen abundance and partial loss of integrity following solubilization. Solubilized HL-ECM was evaluated either as a coating or as a medium supplement for the culture of human primary hepatocytes, hepatic stellate cells and liver sinusoidal endothelial cells. Whereas the solubilized HL-ECM was suitable for cell culture, its impact on the phenotype and/or functionality of the human liver cells was limited. Our study provides a first detailed characterization of solubilized HL-ECM and a first report of its influence on the culture of distinct human primary liver cells.
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- 2020
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17. Comparison of secretome from osteoblasts derived from sclerotic versus non-sclerotic subchondral bone in OA: A pilot study.
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Christelle Sanchez, Gabriel Mazzucchelli, Cécile Lambert, Fanny Comblain, Edwin DePauw, and Yves Henrotin
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Medicine ,Science - Abstract
Osteoarthritis (OA) is characterized by cartilage degradation but also by other joint tissues modifications like subchondral bone sclerosis. In this study, we used a proteomic approach to compare secretome of osteoblast isolated from sclerotic (SC) or non sclerotic (NSC) area of OA subchondral bone.Secretome was analyzed using differential quantitative and relative label free analysis on nanoUPLC G2 HDMS system. mRNA of the more differentially secreted proteins were quantified by RT-PCR in cell culture from 5 other patients. Finally, osteomodulin and fibulin-3 sequences were quantified by western blot and immunoassays in serum and culture supernatants.175 proteins were identified in NSC osteoblast secretome. Data are available via ProteomeXchange with identifier PXD008494. Compared to NSC osteoblast secretome, 12 proteins were significantly less secreted (Osteomodulin, IGFBP5, VCAM-1, IGF2, 78 kDa glucose-regulated protein, versican, calumenin, IGFBP2, thrombospondin-4, periostin, reticulocalbin 1 and osteonectin), and 13 proteins were significantly more secreted by SC osteoblasts (CHI3L1, fibulin-3, SERPINE2, IGFBP6, SH3BGRL3, SERPINE1, reticulocalbin3, alpha-2-HS-glycoprotein, TIMP-2, IGFBP3, TIMP-1, SERPINF1, CSF-1). Similar changes in osteomodulin, IGF2, SERPINE1, fibulin-3 and CHI3L1 mRNA levels were observed. ELISAs assays confirm the decrease by half of osteomodulin protein in SC osteoblasts supernatant compared to NSC and in OA patients serum compared to healthy subjects. Fibulin-3 epitopes Fib3-1, Fib3-2 and Fib3-3 were also increased in SC osteoblasts supernatant compared to NSC.We highlighted some proteins differentially secreted by the osteoblasts coming from OA subchondral bone sclerosis. These changes contribute to explain some features observed in OA subchondral bone, like the increase of bone remodeling or abnormalities in bone matrix mineralization. Among identified proteins, osteomodulin was found decreased and fibulin-3 increased in serum of OA patients. These findings suggest that osteomodulin and fibulin-3 fragments could be biomarkers to monitor early changes in subchondral bone metabolism in OA.
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- 2018
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18. Exploring the N-Glycosylation Profile of Glycoprotein B from Human Cytomegalovirus Expressed in CHO and Nicotiana tabacum BY-2 Cells
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Nicolas Smargiasso, Joseph Nader, Stéphane Rioux, Gabriel Mazzucchelli, Marc Boutry, Edwin De Pauw, François Chaumont, and Catherine Navarre
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glycoprotein B ,cytomegalovirus ,plant cell suspension culture ,N-glycosylation ,mass spectrometry ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The ability to control the glycosylation pattern of recombinant viral glycoproteins represents a major prerequisite before their use as vaccines. The aim of this study consisted of expressing the large soluble ectodomain of glycoprotein B (gB) from Human Cytomegalovirus (HMCV) in Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells and of comparing its glycosylation profile with that of gB produced in Chinese hamster ovary (CHO) cells. gB was secreted in the BY-2 culture medium at a concentration of 20 mg/L and directly purified by ammonium sulfate precipitation and size exclusion chromatography. We then measured the relative abundance of N-glycans present on 15 (BY-2) and 17 (CHO) out of the 18 N-sites by multienzymatic proteolysis and mass spectrometry. The glycosylation profile differed at each N-site, some sites being occupied exclusively by oligomannosidic type N-glycans and others by complex N-glycans processed in some cases with additional Lewis A structures (BY-2) or with beta-1,4-galactose and sialic acid (CHO). The profiles were strikingly comparable between BY-2- and CHO-produced gB. These results suggest a similar gB conformation when glycoproteins are expressed in plant cells as site accessibility influences the glycosylation profile at each site. These data thus strengthen the BY-2 suspension cultures as an alternative expression system.
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- 2019
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19. Proteomic analysis of the reproductive organs of the hermaphroditic gastropod Lymnaea stagnalis exposed to different endocrine disrupting chemicals.
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Arnaud Giusti, Pierre Leprince, Gabriel Mazzucchelli, Jean-Pierre Thomé, Laurent Lagadic, Virginie Ducrot, and Célia Joaquim-Justo
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Medicine ,Science - Abstract
Many studies have reported perturbations of mollusc reproduction following exposure to low concentrations (ng/L range) of endocrine disrupting chemicals (EDCs). However, the mechanisms of action of these molecules on molluscs are still poorly understood. Investigation of the modifications of protein expression in organisms exposed to chemicals using proteomic methods can provide a broader and more comprehensive understanding of adverse impacts of pollution on organisms than conventional biochemical biomarkers (e.g., heat-shock proteins, metallothioneins, GST, EROD). In this study we have investigated the impacts of four chemicals, which exhibit different endocrine disrupting properties in vertebrates, on the proteome of the hermaphroditic freshwater pulmonate gastropod Lymnaea stagnalis after 21 days of exposure. Testosterone, tributyltin, chlordecone and cyproterone acetate were chosen as tested compounds as they can induce adverse effects on the reproduction of this snail. The 2D-DIGE method was used to identify proteins whose expression was affected by these compounds. In addition to modifying the expression of proteins involved in the structure and function of the cytoskeleton, chemicals had impacts on the expression of proteins involved in the reproduction of L. stagnalis. Exposure to 19.2 µg/L of chlordecone increased the abundance of ovipostatin, a peptide transmitted during mating through seminal fluid, which reduces oviposition in this species. The expression of yolk ferritin, the vitellogenin equivalent in L. stagnalis, was reduced after exposure to 94.2 ng Sn/L of tributyltin. The identification of yolk ferritin and the modification of its expression in snails exposed to chemicals were refined using western blot analysis. Our results showed that the tested compounds influenced the abundance of yolk ferritin in the reproductive organs. Alteration in proteins involved in reproductive pathways (e.g., ovipostatin and yolk ferritin) could constitute relevant evidence of interaction of EDCs with reproductive pathways that are under the control of the endocrine system of L. stagnalis.
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- 2013
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20. Cation Involvement in Telomestatin Binding to G-Quadruplex DNA
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Frédéric Rosu, Valérie Gabelica, Nicolas Smargiasso, Gabriel Mazzucchelli, Kazuo Shin-Ya, and Edwin De Pauw
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Genetics ,QH426-470 ,Biochemistry ,QD415-436 - Abstract
The binding mode of telomestatin to G-quadruplex DNA has been investigated using electrospray mass spectrometry, by detecting the intact complexes formed in ammonium acetate. The mass measurements show the incorporation of one extra ammonium ion in the telomestatin complexes. Experiments on telomestatin alone also show that the telomestatin alone is able to coordinate cations in a similar way as a crown ether. Finally, density functional theory calculations suggest that in the G-quadruplex-telomestatin complex, potassium or ammonium cations are located between the telomestatin and a G-quartet. This study underlines that monovalent cation coordination capabilities should be integrated in the rational design of G-quadruplex binding ligands.
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- 2010
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21. The structure of DarB in complex with Rel NTD reveals nonribosomal activation of Rel stringent factors
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Andres Ainelo, Julien Caballero-Montes, Ondřej Bulvas, Karin Ernits, Kyo Coppieters ‘t Wallant, Hiraku Takada, Sophie Z. Craig, Gabriel Mazzucchelli, Safia Zedek, Iva Pichová, Gemma C. Atkinson, Ariel Talavera, Chloe Martens, Vasili Hauryliuk, and Abel Garcia-Pino
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Multidisciplinary - Abstract
Rel stringent factors are bifunctional ribosome-associated enzymes that catalyze both synthesis and hydrolysis of the alarmones (p)ppGpp. Besides the allosteric control by starved ribosomes and (p)ppGpp, Rel is regulated by various protein factors depending on specific stress conditions, including the c-di-AMP–binding protein DarB. However, how these effector proteins control Rel remains unknown. We have determined the crystal structure of the DarB 2 :Rel NTD 2 complex, uncovering that DarB directly engages the SYNTH domain of Rel to stimulate (p)ppGpp synthesis. This association with DarB promotes a SYNTH-primed conformation of the N-terminal domain region, markedly increasing the affinity of Rel for ATP while switching off the hydrolase activity of the enzyme. Binding to c-di-AMP rigidifies DarB, imposing an entropic penalty that precludes DarB-mediated control of Rel during normal growth. Our experiments provide the basis for understanding a previously unknown mechanism of allosteric regulation of Rel stringent factors independent of amino acid starvation.
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- 2023
22. Label-Free Higher Order Structure and Dynamic Investigation Method of Proteins in Solution Using an Enzymatic Reactor Coupled to Electrospray High-Resolution Mass Spectrometry Detection
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Elodie Grifnée, Christopher Kune, Cédric Delvaux, Loïc Quinton, Johann Far, Gabriel Mazzucchelli, and Edwin De Pauw
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Protein Denaturation ,Spectrometry, Mass, Electrospray Ionization ,Protein Conformation ,Caseins ,Cytochromes c ,Proteins ,Equipment Design ,Lactoglobulins ,Sensitivity and Specificity ,Bioreactors ,Structural Biology ,Animals ,Cattle ,Trypsin ,Horses ,Spectroscopy ,Peptide Hydrolases - Abstract
For decades, structural analysis of proteins have received considerable attention, from their sequencing to the determination of their 3D structures either in the free state (e.g., no host-guest system, apoproteins) or (non)covalently bound complexes. The elucidation of the 3D structures and the mapping of intra- and intermolecular interactions are valuable sources of information to understand the physicochemical properties of such systems. X-ray crystallography and nuclear magnetic resonance are methods of choice for obtaining structures at the atomic level. Nonetheless, they still present drawbacks which limit their use to highly purified systems in a relatively high amount. On the contrary, mass spectrometry (MS) has become a powerful tool thanks to its selectivity, sensitivity, and the development of structural methods both at the global shape and the residue level. The combination of several MS-based methods is mandatory to fully assign a putative structure in combination with computational chemistry and bioinformatics. In that context, we propose a strategy which complements the existing methods of structural studies (e.g., circular dichroism, hydrogen/deuterium exchange and cross-links experiments, nuclear magnetic resonance). The workflow is based on the collection of structural information on proteins from the apparition rates and the time of appearance of released peptides generated by a protease in controlled experimental conditions with online detection by electrospray high-resolution mass spectrometry. Nondenaturing, partially or fully denatured proteins were digested by the enzymatic reactor, i.e., β-lactoglobulin, cytochrome
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- 2021
23. Lunaemycins, new cyclic hexapeptide antibiotics from the cave moonmilk-dwellerStreptomyces lunaelactisMM109T
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Loïc Martinet, Aymeric Naômé, Lucas C. D. Rezende, Déborah Tellatin, Bernard Pignon, Jean-Denis Docquier, Filomena Sannio, Dominique Baiwir, Gabriel Mazzucchelli, Michel Frédérich, and Sébastien Rigali
- Abstract
Streptomyces lunaelactisstrains have been isolated from moonmilk deposits which are calcium carbonate speleothems used for centuries in traditional medicine for their antimicrobial properties. Genome mining revealed that these strains are a remarkable example of aStreptomycesspecies with huge heterogeneity regarding their content in biosynthetic gene clusters (BGCs) for specialized metabolite production. BGC 28a is one of the cryptic BGCs that is only carried by a subgroup ofS. lunaelactisstrains for whichin silicoanalysis predicted the production of nonribo-somal peptide antibiotics containing the non-proteogenic amino acid piperazic acid (Piz). Comparative metabolomics of culture extracts ofS. lunaelactisstrains either or not holding BGC 28a combined with MS/MS-guided peptidogenomics and1H/13C NMR allowed to identify the cyclic hexapeptide with the amino acid sequence (D-Phe)-(L-HO-Ile)-(D-Piz)-(L-Piz)-(D-Piz)-(L-Piz), called lunaemycin A, as the main compound synthesized by BGC 28a. Molecular networking further identified 18 additional lunaemycins, 14 of them having their structure elucidated by HRMS/MS. Antimicrobial assays demonstrated a huge bactericidal activity of lunaemycins against Gram-positive bacteria including multi-drug resistant clinical isolates. Our work demonstrates how accuratein silicoanalysis of a cryptic BGC can highly facilitate the identification, the structural elucidation, and the bioactivity of its associated specialized metabolites.
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- 2022
24. Up-Regulated Salivary Proteins of Brown Marmorated Stink Bug Halyomorpha halys on Plant Growth-Promoting Rhizobacteria-Treated Plants
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Gabriel Mazzucchelli, Lisa Iannello, Laurent Serteyn, Frédéric Francis, Dominique Baiwir, Olivier Lourme, and Marc Ongena
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media_common.quotation_subject ,Bacillus ,Insect ,Biology ,Rhizobacteria ,Biochemistry ,Salivary Glands ,Microbiology ,Heteroptera ,Stress, Physiological ,Tandem Mass Spectrometry ,Plant defense against herbivory ,medicine ,Animals ,Salivary Proteins and Peptides ,Brown marmorated stink bug ,Chromatography, High Pressure Liquid ,Ecology, Evolution, Behavior and Systematics ,Allelopathy ,media_common ,Salivary gland ,fungi ,food and beverages ,General Medicine ,biology.organism_classification ,Up-Regulation ,Vicia faba ,medicine.anatomical_structure ,Larva ,PEST analysis - Abstract
Plant Growth-Promoting Rhizobacteria (PGPR) induce systemic resistance (SR) in plants, decreasing the development of phytopathogens. The FZB42 strain of Bacillus velezensis is known to induce an SR against pathogens in various plant species. Previous studies suggested that it could also influence the interactions between plants and associated pests. However, insects have developed several strategies to counteract plant defenses, including salivary proteins that allow the insect escaping detection, manipulating defensive pathways to its advantage, deactivating early signaling processes, or detoxifying secondary metabolites. Because Brown Marmorated Stink Bug (BMSB) Halyomorpha halys is highly invasive and polyphagous, we hypothesized that it could detect the PGPR-induced systemic defenses in the plant, and efficiently adapt its salivary compounds to counteract them. Therefore, we inoculated a beneficial rhizobacterium on Vicia faba roots and soil, previous to plant infestation with BMSB. Salivary gland proteome of BMSB was analyzed by LC-MS/MS and a label-free quantitative proteomic method. Among the differentially expressed proteins, most were up-regulated in salivary glands of insects exposed to PGPR-treated plants for 24 h. We could confirm that BMSB was confronted with a stress during feeding on PGPR-treated plants. The to-be-confirmed defensive state of the plant would have been rapidly detected by the invasive H. halys pest, which consequently modified its salivary proteins. Among the up-regulated proteins, many could be associated with a role in plant defense counteraction, and more especially in allelochemicals detoxification or sequestration.
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- 2021
25. Potential Role of Epithelial Endoplasmic Reticulum Stress and Anterior Gradient Protein 2 Homologue in Crohn’s Disease Fibrosis
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Florence Quesada Calvo, Florian Rieder, Dominique Baiwir, Marie-Alice Meuwis, Charlotte Massot, Noëlla Bletard, Edwin De Pauw, Philippe Delvenne, Shurong Hu, Nicolas Pierre, Sophie Vieujean, Emeline Bequet, Edouard Louis, Carla Coimbra Marques, Gabriel Mazzucchelli, and Catherine Salee
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Proteomics ,0301 basic medicine ,Colon ,AGR2 ,Pilot Projects ,Cell Line ,03 medical and health sciences ,HT29 Cells ,Mucoproteins ,0302 clinical medicine ,Crohn Disease ,Ileum ,Humans ,Medicine ,Intestinal Mucosa ,Fibroblast ,Anterior Gradient Protein 2 Homolog ,Oncogene Proteins ,business.industry ,Endoplasmic reticulum ,Gastroenterology ,Original Articles ,General Medicine ,Endoplasmic Reticulum Stress ,Fibrosis ,Molecular biology ,Intestinal epithelium ,Epithelium ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Unfolded protein response ,business - Abstract
Background and Aims Intestinal fibrosis is a common complication of Crohn’s disease [CD]. It is characterised by an accumulation of fibroblasts differentiating into myofibroblasts secreting excessive extracellular matrix. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. Methods We performed a pilot proteomic study comparing the proteome of surface epithelium, isolated by laser-capture microdissection, in normal and fibrotic zones of resected ileal CD strictures [13 zones collected in five patients]. Proteins of interests were validated by immunohistochemistry [IHC] in ileal and colonic samples of stricturing CD [n = 44], pure inflammatory CD [n = 29], and control [n = 40] subjects. The pro-fibrotic role of one selected epithelial protein was investigated through in-vitro experiments using HT-29 epithelial cells and a CCD-18Co fibroblast to myofibroblast differentiation model. Results Proteomic study revealed an endoplasmic reticulum [ER] stress proteins increase in the epithelium of CD ileal fibrotic strictures, including anterior gradient protein 2 homologue [AGR2] and binding-immunoglobulin protein [BiP]. This was confirmed by IHC. In HT-29 cells, tunicamycin-induced ER stress triggered AGR2 intracellular expression and its secretion. Supernatant of these HT-29 cells, pre-conditioned by tunicamycin, led to a myofibroblastic differentiation when applied on CCD-18Co fibroblasts. By using recombinant protein and blocking agent for AGR2, we demonstrated that the secretion of this protein by epithelial cells can play a role in the myofibroblastic differentiation. Conclusions The development of CD fibrotic strictures could involve epithelial ER stress and particularly the secretion of AGR2.
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- 2021
26. Oligodendrocyte precursors guide interneuron migration by unidirectional contact repulsion
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Fanny Lepiemme, Julie Stoufflet, Míriam Javier-Torrent, Gabriel Mazzucchelli, Carla G. Silva, and Laurent Nguyen
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Cerebral Cortex ,Oligodendrocyte Precursor Cells ,Mice ,Multidisciplinary ,Models, Genetic ,Cell Movement ,Interneurons ,Neurogenesis ,Animals ,Chemokine CXCL12 - Abstract
In the forebrain, ventrally derived oligodendrocyte precursor cells (vOPCs) travel tangentially toward the cortex together with cortical interneurons. Here, we tested in the mouse whether these populations interact during embryogenesis while migrating. By coupling histological analysis of genetic models with live imaging, we show that although they are both attracted by the chemokine Cxcl12, vOPCs and cortical interneurons occupy mutually exclusive forebrain territories enriched in this chemokine. Moreover, first-wave vOPC depletion selectively disrupts the migration and distribution of cortical interneurons. At the cellular level, we found that by promoting unidirectional contact repulsion, first-wave vOPCs steered the migration of cortical interneurons away from the blood vessels to which they were both attracted, thereby allowing interneurons to reach their proper cortical territories.
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- 2022
27. Discovery of biomarker candidates associated with the risk of short-term and mid/long-term relapse after infliximab withdrawal in Crohn’s patients: a proteomics-based study
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Dominique Baiwir, Edwin De Pauw, Nicolas Pierre, Marie-Alice Meuwis, D. Laharie, Yoram Bouhnik, Edouard Louis, Vân Anh Huynh-Thu, Gabriel Mazzucchelli, Nicolas Smargiasso, and Jean-Frederic Colombel
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Oncology ,medicine.medical_specialty ,Crohn's disease ,business.industry ,Proportional hazards model ,medicine.drug_class ,Gastroenterology ,Disease ,medicine.disease ,Antimetabolite ,Inflammatory bowel disease ,Infliximab ,Discontinuation ,Internal medicine ,medicine ,Biomarker (medicine) ,business ,medicine.drug - Abstract
ObjectiveA subset of Crohn’s disease (CD) patients experiences mid/long-term remission after infliximab withdrawal. Biomarkers are needed to identify those patients.DesignNew biomarkers of relapse were searched in the baseline serum of CD patients stopping infliximab when they were under combined therapy (antimetabolite and infliximab) and stable clinical remission (diSconTinuation in CrOhn’s disease patients in stable Remission on combined therapy with Immunosuppressors cohort, n=102). From shotgun proteomics experiment (discovery step), biomarker candidates were identified and further targeted by selected reaction monitoring (verification step). The dataset was stratified to search for markers of short-term (6 months). The risk of relapse and the predicting capacity associated with biomarker candidates were evaluated using univariate Cox model and log-rank statistic, respectively. To test their complementary predicting capacity, biomarker candidates were systematically combined in pairs.ResultsDistinct biomarker candidates were associated with the risk (HR) of short-term (15 proteins, 2.9ConclusionWe identified for the first time circulating biomarker candidates associated with the risk of mid/long-term relapse in CD patients stopping infliximab. We also highlight a sequence of pathophysiological processes leading to relapse, this could help to better understand the disease progression. Our findings may pave the way for a better non-invasive evaluation of the risk of relapse when contemplating antitumour necrosis factor α withdrawal in CD patients.
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- 2020
28. Solute Carrier Family 12 Member 2 as a Proteomic and Histological Biomarker of Dysplasia and Neoplasia in Ulcerative Colitis
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Sena Bluemel, A Vijverman, Marie-Alice Meuwis, Cécile Oury, Laurence Servais, Florence Quesada Calvo, Dominique Baiwir, Michael Scharl, Christine Sempoux, Odile Wéra, Edouard Louis, Noëlla Bletard, Gabriel Mazzucchelli, Carla Coimbra Marques, Laurence de Leval, Roberto Manzini, Sophie Vieujean, Angela-Maria Merli, Gerhard Rogler, Edwin De Pauw, Charlotte Massot, Philippe Delvenne, Arnaud Colard, and University of Zurich
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0303 health sciences ,Pathology ,medicine.medical_specialty ,business.industry ,Colorectal cancer ,Gastroenterology ,Cancer ,610 Medicine & health ,General Medicine ,medicine.disease ,Ulcerative colitis ,3. Good health ,Staining ,03 medical and health sciences ,10219 Clinic for Gastroenterology and Hepatology ,0302 clinical medicine ,Dysplasia ,030220 oncology & carcinogenesis ,medicine ,Biomarker (medicine) ,Immunohistochemistry ,Clinical significance ,business ,030304 developmental biology - Abstract
Background and Aims Ulcerative colitis [UC] patients have a greater risk of developing colorectal cancer through inflammation-dysplasia-carcinoma sequence of transformation. The histopathological diagnosis of dysplasia is therefore of critical clinical relevance, but dysplasia may be difficult to distinguish from inflammatory changes. Methods A proteomic pilot study on five UC colorectal dysplastic patients highlighted proteins differentially distributed between paired dysplastic, inflammatory, and normal tissues. The best candidate marker was selected and immunohistochemistry confirmation was performed on azoxymethane/dextran sulphate sodium [AOM/DSS] mouse model lesions, 37 UC-dysplasias, 14 UC-cancers, 23 cases of long-standing UC, 35 sporadic conventional adenomas, 57 sporadic serrated lesions, and 82 sporadic colorectal cancers. Results Differential proteomics found 11 proteins significantly more abundant in dysplasia compared with inflammation, including Solute carrier family 12 member 2 [SLC12A2] which was confidently identified with eight specific peptides and was below the limit of quantitation in both inflammatory and normal colon. SLC12A2 immunohistochemical analysis confirmed the discrimination of preneoplastic and neoplastic lesions from inflammatory lesions in mice, in UC, and in sporadic contexts. A specific SLC12A2 staining pattern termed ‘loss of gradient’ reached 89% sensitivity, 95% specificity, and 92% accuracy for UC-dysplasia diagnosis together with an inter-observer agreement of 95.24% [multirater κ free of 0.90; 95% CI: 0.78 - 1.00]. Such discrimination could not be obtained by Ki67 staining. This specific pattern was also associated with sporadic colorectal adenomas and cancers. Conclusions We found a specific SLC12A2 immunohistochemical staining pattern in precancerous and cancerous colonic UC lesions which could be helpful for diagnosing dysplasia and cancer in UC and non-UC patients.
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- 2020
29. Proteins involved in the endoplasmic reticulum stress are modulated in synovitis of osteoarthritis, chronic pyrophosphate arthropathy and rheumatoid arthritis, and correlate with the histological inflammatory score
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Gaël Cobraiville, Philippe Delvenne, Dominique de Seny, Elettra Bianchi, Marie-Joëlle Kaiser, Michel Malaise, Gabriel Mazzucchelli, Jean-Philippe Hauzeur, Dominique Baiwir, Charlotte Collin, and Mégane Deliège
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Adult ,Male ,0301 basic medicine ,Pathology ,medicine.medical_specialty ,Proteome ,lcsh:Medicine ,Chondrocalcinosis ,Osteoarthritis ,Article ,S100A9 ,S100A8 ,Arthritis, Rheumatoid ,03 medical and health sciences ,Rheumatic diseases ,0302 clinical medicine ,Rheumatology ,Synovitis ,Arthropathy ,medicine ,Humans ,lcsh:Science ,Endoplasmic Reticulum Chaperone BiP ,Aged ,Retrospective Studies ,Aged, 80 and over ,Inflammation ,Multidisciplinary ,business.industry ,lcsh:R ,Proteins ,Middle Aged ,Endoplasmic Reticulum Stress ,medicine.disease ,HSPA1A ,Diphosphates ,030104 developmental biology ,medicine.anatomical_structure ,Rheumatoid arthritis ,Female ,lcsh:Q ,Inflammation Mediators ,Synovial membrane ,business ,Biomarkers ,030217 neurology & neurosurgery - Abstract
It is now well recognized that osteoarthritis (OA) synovial membrane presents inflammatory components. The aim of this work is to provide evidence that similar inflammatory mechanisms exist in synovial membrane (n = 24) obtained from three pathologies presenting altogether an inflammatory gradient: OA, chronic pyrophosphate arthropathy (CPPA) and rheumatoid arthritis (RA). Synovial biopsies were first characterized by a histological score based on synovial hyperplasia and infiltration of lymphocytes, plasma cells, polymorphonuclear and macrophages. All biopsies were also analyzed by 2D-nano-UPLC-ESI-Q-Orbitrap for protein identification and quantification. Protein levels were correlated with the histological score. Histological score was in the range of 3 to 8 for OA, 5 to 13 for CPPA and 12 to 17 for RA. Of the 4,336 proteins identified by mass spectrometry, 51 proteins were selected for their strong correlation (p DNAJB11, CALR, ERP29, GANAB, HSP90B1, HSPA1A, HSPA5, HYOU1, LMAN1, PDIA4, and TXNDC5) were involved in the endoplasmic reticulum (ER) stress. Protein levels of S100A8 and S100A9 were significantly higher in RA compared to OA (for both) or to CPPA (for S100A8 only) and also significantly correlated with the histological score. Eighteen complement component proteins were identified, but only C1QB and C1QBP were weakly correlated with the histological score. This study highlights the inflammatory gradient existing between OA, CPPA and RA synovitis either at the protein level or at the histological level. Inflamed synovitis was characterized by the overexpression of ER stress proteins.
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- 2020
30. Glycosylation deficiency of lipopolysaccharide-binding protein and corticosteroid-binding globulin associated with activity and response to treatment for rheumatoid arthritis
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Valérie Badot, Federica Ciregia, Dominique Baiwir, Gaël Cobraiville, Gabriel Mazzucchelli, Thibaut Dewael, T. Sokolova, Michel Malaise, Paschalis Sidiras, Patrick Durez, Silvana Di Romana, and Dominique de Seny
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0301 basic medicine ,medicine.medical_specialty ,Glycosylation ,Globulin ,lcsh:Medicine ,General Biochemistry, Genetics and Molecular Biology ,Arthritis, Rheumatoid ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Transcortin ,Tandem Mass Spectrometry ,Internal medicine ,LBP ,medicine ,Humans ,Rheumatoid arthritis ,030203 arthritis & rheumatology ,chemistry.chemical_classification ,Membrane Glycoproteins ,biology ,business.industry ,Research ,C-reactive protein ,lcsh:R ,Lectin ,General Medicine ,Sciences bio-médicales et agricoles ,medicine.disease ,Post translational modifications ,030104 developmental biology ,Endocrinology ,chemistry ,biology.protein ,CBG ,Carrier Proteins ,business ,Glycoprotein ,Biologie ,Lipopolysaccharide binding protein ,Biomarkers ,Acute-Phase Proteins ,Chromatography, Liquid - Abstract
Background: Serum protein glycosylation is an area of investigation in inflammatory arthritic disorders such as rheumatoid arthritis (RA). Indeed, some studies highlighted abnormalities of protein glycosylation in RA. Considering the numerous types of enzymes, monosaccharides and glycosidic linkages, glycosylation is one of the most complex post translational modifications. By this work, we started with a preliminary screening of glycoproteins in serum from RA patients and controls. Methods: In order to isolate glycoproteins from serum, lectin wheat germ agglutinin was used and quantitative differences between patients and controls were investigated by LC-MS/MS. Consequently, we focused our attention on two glycoproteins found in this explorative phase: corticosteroid-binding globulin (CBG) and lipopolysaccharide-binding protein (LBP). The subsequent validation with immunoassays was widened to a larger number of early RA (ERA) patients (n = 90) and well-matched healthy controls (n = 90). Results: We observed a significant reduction of CBG and LBP glycosylation in ERA patients compared with healthy controls. Further, after 12 months of treatment, glycosylated CBG and LBP levels increased both to values comparable to those of controls. In addition, these changes were correlated with clinical parameters. Conclusions: This study enables to observe that glycosylation changes of CBG and LBP are related to RA disease activity and its response to treatment., SCOPUS: ar.j, info:eu-repo/semantics/published
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- 2020
31. Periostin in lymph node pre-metastatic niches governs lymphatic endothelial cell functions and metastatic colonization
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Lionel Gillot, Alizée Lebeau, Louis Baudin, Charles Pottier, Thomas Louis, Tania Durré, Rémi Longuespée, Gabriel Mazzucchelli, Christophe Nizet, Silvia Blacher, Frédéric Kridelka, and Agnès Noël
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Pharmacology ,Proteomics ,Vascular Endothelial Growth Factor C ,Endothelial Cells ,Uterine Cervical Neoplasms ,Cell Biology ,Cellular and Molecular Neuroscience ,Mice ,Lymphatic Metastasis ,Molecular Medicine ,Animals ,Humans ,Female ,Lymph Nodes ,Molecular Biology ,Cell Adhesion Molecules - Abstract
Although lymph node (LN) metastasis is an important prognostic parameter in cervical cancer, the tissue remodeling at a pre-metastatic state is poorly documented in LNs. We here identified periostin (POSTN) as a component of non-metastatic LNs by applying proteomic analyses and computerized image quantifications on LNs of patients with cervical cancer. We provide evidence for remarkable modifications of POSTN and lymphatic vessel distributions and densities in non-metastatic sentinel and metastatic human LNs, when compared to distant non-metastatic LNs. POSTN deposition at a pre-metastatic stage was demonstrated in a pre-clinical murine model (the ear sponge assay). Its expression by fibroblastic LN cells was assessed by in situ hybridization and in vitro cultures. In vitro, POSTN promoted lymphatic endothelial cell functions and tumor cell proliferation. Accordingly, the in vivo injection of recombinant POSTN together with VEGF-C boosted the lymphangiogenic response, while the metastatic potential of tumor cells was drastically reduced using a POSTN blocking antibody. This translational study also supports the existence of an unprecedented dialog “in cascade”, between the primary tumor and the first pelvic nodal relay in early cervical cancer, and subsequently from pelvic LN to para-aortic LNs in locally advanced cervical cancers. Collectively, this work highlights the association of POSTN deposition with lymphangiogenesis in LNs, and provides evidence for a key contribution of POSTN in promoting VEGF-C driven lymphangiogenesis and the seeding of metastatic cells.
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- 2021
32. Precise co-registration of mass spectrometry imaging, histology, and laser microdissection-based omics
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Dominique Baiwir, Gabriel Mazzucchelli, Ron M. A. Heeren, Benjamin Balluff, Edwin De Pauw, Frédéric Dewez, Marta Martin-Lorenzo, Michael Herfs, Imaging Mass Spectrometry (IMS), and RS: M4I - Imaging Mass Spectrometry (IMS)
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Proteomics ,Co registration ,Microproteomics ,Breast Neoplasms ,02 engineering and technology ,Computational biology ,01 natural sciences ,Biochemistry ,Mass Spectrometry ,Mass spectrometry imaging ,Total error ,Analytical Chemistry ,Humans ,Segmentation ,Multiplex ,neoplasms ,Laser capture microdissection ,Co-registration ,Chemistry ,Communication ,Lasers ,010401 analytical chemistry ,Histology ,Biological tissue ,021001 nanoscience & nanotechnology ,digestive system diseases ,Neoplasm Proteins ,0104 chemical sciences ,Intratumor heterogeneity ,Female ,Laser microdissection ,0210 nano-technology - Abstract
Mass spectrometry imaging (MSI) is an analytical technique for the unlabeled and multiplex imaging of molecules in biological tissue sections. It therefore enables the spatial and molecular annotations of tissues complementary to histology. It has already been shown that MSI can guide subsequent material isolation technologies such as laser microdissection (LMD) to enable a more in-depth molecular characterization of MSI-highlighted tissue regions. However, with MSI now reaching spatial resolutions at the single-cell scale, there is a need for a precise co-registration between MSI and the LMD. As proof-of-principle, MSI of lipids was performed on a breast cancer tissue followed by a segmentation of the data to detect molecularly distinct segments within its tumor areas. After image processing of the segmentation results, the coordinates of the MSI-detected segments were passed to the LMD system by three co-registration steps. The errors of each co-registration step were quantified and the total error was found to be less than 13 μm. With this link established, MSI data can now accurately guide LMD to excise MSI-defined regions of interest for subsequent extract-based analyses. In our example, the excised tissue material was then subjected to ultrasensitive microproteomics in order to determine predominant molecular mechanisms in each of the MSI-highlighted intratumor segments. This work shows how the strengths of MSI, histology, and extract-based omics can be combined to enable a more comprehensive molecular characterization of in situ biological processes. Electronic supplementary material The online version of this article (10.1007/s00216-019-01983-z) contains supplementary material, which is available to authorized users.
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- 2019
33. Multi-Enzymatic Limited Digestion: The Next-Generation Sequencing for Proteomics?
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Raphaël La Rocca, Nicolas Smargiasso, Elodie Grifnée, Dominique Baiwir, Rémi Longuespée, Denis Morsa, Edwin De Pauw, Tyler A. Zimmerman, Marie-Alice Meuwis, Emeline Hanozin, and Gabriel Mazzucchelli
- Subjects
Proteomics ,0301 basic medicine ,Flexibility (engineering) ,030102 biochemistry & molecular biology ,Computer science ,High-Throughput Nucleotide Sequencing ,General Chemistry ,Computational biology ,Biochemistry ,DNA sequencing ,03 medical and health sciences ,030104 developmental biology ,Sequence Analysis, Protein ,Tandem Mass Spectrometry ,Redundancy (engineering) ,Humans ,De novo sequencing ,Amino Acid Sequence ,Peptides ,Protein Processing, Post-Translational ,Algorithms - Abstract
Over the past 40 years, proteomics, generically defined as the field dedicated to the identification and analysis of proteins, has tremendously gained in popularity and potency through advancements in genome sequencing, separative techniques, mass spectrometry, and bioinformatics algorithms. As a consequence, its scope of application has gradually enlarged and diversified to meet specialized topical biomedical subjects. Although the tryptic bottom-up approach is widely regarded as the gold standard for rapid screening of complex samples, its application for precise and confident mapping of protein modifications is often hindered due to partial sequence coverage, poor redundancy in indicative peptides, and lack of method flexibility. We here show how the synergic and time-limited action of a properly diluted mix of multiple enzymes can be exploited in a versatile yet straightforward protocol to alleviate present-day drawbacks. Merging bottom-up and middle-down ideologies, our results highlight broad assemblies of overlapping peptides that enable refined and reliable characterizations of proteins, including variant identification, and their carried modifications, including post-translational modifications, truncations, and cleavages. Beyond this boost in performance, our methodology also offers efficient de novo sequencing capabilities, in view of which we here present a dedicated custom assembly algorithm.
- Published
- 2019
34. Limited cross reactivity among arginine kinase allergens from mealworm and cricket edible insects
- Author
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Taofic Alabi, Eric Haubruge, Francis Corazza, Rudy Caparros, Frédéric Francis, Gabriel Mazzucchelli, Virginie Doyen, F. Debaugnies, and Christophe Blecker
- Subjects
Adult ,Electrophoresis ,Male ,Mealworm ,Entomophagy ,Entomology ,media_common.quotation_subject ,Insect ,Cross Reactions ,medicine.disease_cause ,01 natural sciences ,Cross-reactivity ,Analytical Chemistry ,Gryllidae ,0404 agricultural biotechnology ,Cricket ,medicine ,Animals ,Humans ,Cooking ,Food science ,Tenebrio ,media_common ,biology ,010401 analytical chemistry ,Arginine Kinase ,04 agricultural and veterinary sciences ,General Medicine ,Allergens ,Immunoglobulin E ,Middle Aged ,Arginine kinase ,biology.organism_classification ,040401 food science ,0104 chemical sciences ,Acheta ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,biology.protein ,Insect Proteins ,Female ,Food Hypersensitivity ,Food Science - Abstract
Insects are seen as a solution to the increasing demand for protein sources for food. However, entomophagy has unfortunately been linked to allergic reactions in Europe with people with professional contacts. As mealworms (Tenebrio molitor) and crickets (Acheta domesticus) have recently become commercially available (both whole or in food formulation) in several European countries, this research assessed the cross allergenicity of arginine kinase (AK). Based on the collection of sera from a entomology laboratory staff, oven cooked insects but also purified AK fractions were tested. Immunoblotting against the protein extracts revealed different Immunoglobulin E reactivity of sera according to the insect target species: two bands (40 and 14 kDa) for crickets and a pattern including light responses at 17, 25 and 37 kDa for mealworms. Focusing on AK, low specific allergenicity was here illustrated and discussed in relation to the development of a safe edible insect consumption by humans.
- Published
- 2019
35. Revisiting
- Author
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Isabel, Marcelino, Philippe, Holzmuller, Ana, Coelho, Gabriel, Mazzucchelli, Bernard, Fernandez, and Nathalie, Vachiéry
- Subjects
infection biomarkers ,animal structures ,virulence factors ,host response ,Ehrlichia ruminantium ,immunomodulation ,Article ,endothelial cells ,bacterial life cycle ,differential protein expression - Abstract
The Rickettsiales Ehrlichia ruminantium, the causal agent of the fatal tick-borne disease Heartwater, induces severe damage to the vascular endothelium in ruminants. Nevertheless, E. ruminantium-induced pathobiology remains largely unknown. Our work paves the way for understanding this phenomenon by using quantitative proteomic analyses (2D-DIGE-MS/MS, 1DE-nanoLC-MS/MS and biotin-nanoUPLC-MS/MS) of host bovine aorta endothelial cells (BAE) during the in vitro bacterium intracellular replication cycle. We detect 265 bacterial proteins (including virulence factors), at all time-points of the E. ruminantium replication cycle, highlighting a dynamic bacterium–host interaction. We show that E. ruminantium infection modulates the expression of 433 host proteins: 98 being over-expressed, 161 under-expressed, 140 detected only in infected BAE cells and 34 exclusively detected in non-infected cells. Cystoscape integrated data analysis shows that these proteins lead to major changes in host cell immune responses, host cell metabolism and vesicle trafficking, with a clear involvement of inflammation-related proteins in this process. Our findings led to the first model of E. ruminantium infection in host cells in vitro, and we highlight potential biomarkers of E. ruminantium infection in endothelial cells (such as ROCK1, TMEM16K, Albumin and PTPN1), which may be important to further combat Heartwater, namely by developing non-antibiotic-based strategies.
- Published
- 2021
36. Are physicochemical properties shaping the allergenic potency of plant allergens?
- Author
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Laura Martín-Pedraza, Colette Larré, Thomas Holzhauser, Pedro M. Rodrigues, Daniel Lozano-Ojalvo, Karin Hoffmann-Sommergruber, Sara Benedé, Isabel Mafra, Gabriel Mazzucchelli, Tanja Cirkovic-Velickovic, Annette Kuehn, Cristian Piras, Joana Costa, Denise Schrama, Eva Gelencser, Kitty C.M. Verhoeckx, Cristina Bueno-Díaz, Roberta Lupi, Linda Monaci, Araceli Díaz-Perales, Julia Klueber, Caterina Villa, Simona L. Bavaro, Elena Molina, Paola Roncada, European Commission, Fundação para a Ciência e a Tecnologia (Portugal), Ministry of Education, Science and Technological Development (Serbia), Ministério da Ciência, Tecnologia e Ensino Superior (Portugal), Fonds National de la Recherche Luxembourg, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Universidade do Porto, Istituto di Bioscienze e BioRisorse [Palermo] (IBBR), Consiglio Nazionale delle Ricerche (CNR), Teagasc Food Research Centre, Food Chemistry and Technology department, Moorepark, Fermoy, Country Cork, Unité de recherche sur les Biopolymères, Interactions Assemblages (BIA), European Cooperation in Science and Technology (COST) Office, European Cooperation in Science and Technology (COST) : FA1402, Portuguese Foundation for Science and Technology, European Commission : UIDB 50006/2020, projects AlleRiskAssess, PTDC/BAA-AGR/31720/2017, NORTE-01-0145-FEDER-00001, FCT - POPH-QREN, PD/BD/114576/2016, Ministry of Education, Science and Technological Development of the Republic of Serbia : OI172024, Portuguese Foundation for Science and Technology European Commission : UIDB/04326/2020, and 16-02-01-FMP0014.Luxembourg National Research FundPRIDE/11012546/NEXTIMMUNEPersonalised Medicine Consortium (PMC), Luxembourg PMC/2017/02
- Subjects
Allergy ,LIPID TRANSFER PROTEIN ,PRU P 3 ,Food processing ,Protein family ,[SDV]Life Sciences [q-bio] ,Plant allergens ,Clinical manifestation ,Computational biology ,Protein aggregation ,Biology ,PEANUT ALLERGENS ,03 medical and health sciences ,Mice ,0302 clinical medicine ,medicine ,Medicine and Health Sciences ,Immunology and Allergy ,Potency ,Animals ,Humans ,ARA H 1 ,SEED STORAGE PROTEINS ,Matrix effect ,ComputingMilieux_MISCELLANEOUS ,Plant Proteins ,2. Zero hunger ,030203 arthritis & rheumatology ,ALPHA-AMYLASE INHIBITOR ,General Medicine ,IN-VITRO DIGESTION ,Allergens ,COMPONENT-RESOLVED DIAGNOSIS ,medicine.disease ,3. Good health ,EXERCISE-INDUCED ANAPHYLAXIS ,Animal allergens ,WHEAT GLUTEN PROTEINS ,Protein families ,Plant protein ,Allergenicity ,Pollen ,Food Hypersensitivity ,030215 immunology - Abstract
This review searched for published evidence that could explain how different physicochemical properties impact on the allergenicity of food proteins and if their effects would follow specific patterns among distinct protein families. Owing to the amount and complexity of the collected information, this literature overview was divided in two articles, the current one dedicated to protein families of plant allergens and a second one focused on animal allergens. Our extensive analysis of the available literature revealed that physicochemical characteristics had consistent effects on protein allergenicity for allergens belonging to the same protein family. For example, protein aggregation contributes to increased allergenicity of 2S albumins, while for legumins and cereal prolamins, the same phenomenon leads to a reduction. Molecular stability, related to structural resistance to heat and proteolysis, was identified as the most common feature promoting plant protein allergenicity, although it fails to explain the potency of some unstable allergens (e.g. pollen-related food allergens). Furthermore, data on physicochemical characteristics translating into clinical effects are limited, mainly because most studies are focused on in vitro IgE binding. Clinical data assessing how these parameters affect the development and clinical manifestation of allergies is minimal, with only few reports evaluating the sensitising capacity of modified proteins (addressing different physicochemical properties) in murine allergy models. In vivo testing of modified pure proteins by SPT or DBPCFC is scarce. At this stage, a systematic approach to link the physicochemical properties with clinical plant allergenicity in real-life scenarios is still missing., The authors highly appreciate the support from the European Cooperation in Science and Technology (COST) Office. This article is based upon work from COST Action FA1402, supported by COST (www.cost.eu). This work was also supported by Fundação para a Ciência e Tecnologia under the Partnership Agreement UIDB 50006/2020 and by the projects AlleRiskAssess - PTDC/BAA-AGR/31720/2017 and NORTE-01-0145-FEDER-00001. C.V. is grateful to FCT grants (PD/BD/114576/2016) financed by POPH-QREN (subsidised by FSE and MCTES). T.C.V. is grateful to the Ministry of Education, Science and Technological Development of the Republic of Serbia through grant number OI172024. P.M.R. and D.S. are grateful to FCT through project UIDB/04326/2020 and Mar2020 16–02-01-FMP-0014 – ‘ALLYFISH’. J.K. and A.K. acknowledge the PRIDE program grant (PRIDE/11012546/NEXTIMMUNE) by the Fonds National de la Recherche (FNR), Luxembourg and a translational grant (APSIS, PMC/2017/02) by the Personalised Medicine Consortium (PMC), Luxembourg.
- Published
- 2021
37. P013 Potential Role of Epithelial Protein Disulphide Isomerases in Crohn’s Disease Fibrosis
- Author
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Nicolas Pierre, L Edouard, Marie-Alice Meuwis, Shurong Hu, Dominique Baiwir, E. De Pauw, Philippe Delvenne, C Coimbra Marques, Sophie Vieujean, Noëlla Bletard, F Quesada Calvo, Charlotte Massot, Emeline Bequet, Gabriel Mazzucchelli, and Catherine Salee
- Subjects
Crohn's disease ,business.industry ,Gastroenterology ,General Medicine ,Isomerase ,medicine.disease ,Intestinal epithelium ,Extracellular matrix ,Paracrine signalling ,Fibrosis ,Heat shock protein ,medicine ,Cancer research ,Protein disulfide-isomerase ,business - Abstract
Background Intestinal fibrosis is a common complication of Crohn’s disease (CD) characterized by an accumulation of fibroblasts differentiating into activated myofibroblasts secreting excessive extracellular matrix. In in-vitro experiments, this myofibroblastic differentiation is elicited by a whole series of factors among which transforming growth factor-β1 (TGF-β1) seems to play a key role. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. Methods We performed a pilot proteomic study comparing the proteome of surface epithelium isolated by laser-capture microdissection in normal and fibrotic zones of resected ileal CD strictures (13 zones collected in 5 patients). The pro-fibrotic role of selected epithelial proteins was investigated through in-vitro experiments using HT-29 epithelial cells and a CCD-18Co fibroblast to myofibroblast differentiation model. Results Proteomic study revealed an endoplasmic reticulum (ER) stress proteins increase in the epithelium of CD ileal fibrotic strictures, including Anterior gradient protein 2 homolog (AGR2), Protein disulphide isomerase A6 (PDIA6) and Endoplasmic reticulum resident protein 44 (ERP44) which are 3 protein disulphide isomerases. In HT-29 cells, tunicamycin-induced ER stress triggered AGR2, PDIA6, ERP44 as well as TGF-β1 intracellular expression and their secretion. Supernatant of these HT-29 cells, pre-conditioned by tunicamycin (Tm), led to a myofibroblastic differentiation when applied on CCD-18Co fibroblasts. The application of blocking agents for AGR2, PDIA6, ERP44 or TGF-β1 in the supernatant of these Tm-pre-conditioned HT-29 cells, attenuated the myofibroblastic differentiation induced by this supernatant, suggesting a pro-fibrotic role of these secreted epithelial proteins. Conclusion The development of CD fibrotic strictures may involve ER stress in epithelial cells, releasing a whole set of proteins into their environment, including AGR2, PDIA6, ERP44 as well as TGF-β1, which could exercise a pro-fibrotic role through a paracrine action.
- Published
- 2021
38. Human Liver-Derived Extracellular Matrix for the Culture of Distinct Human Primary Liver Cells
- Author
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Adil El Taghdouini, Edwin De Pauw, Valéry Payen, Etienne Sokal, Gabriel Mazzucchelli, Anne des Rieux, Gauthier Eppe, Davide Brusa, Joachim Ravau, Niki Alevra Sarika, Mustapha Najimi, Maximilien Fleron, UCL - SSS/LDRI - Louvain Drug Research Institute, UCL - SSS/IREC/PEDI - Pôle de Pédiatrie, and UCL - (SLuc) Service de gastro-entérologie et hépatologie pédiatrique
- Subjects
Proteomics ,Cell type ,extracellular matrix ,Cell Culture Techniques ,primary cells ,liver ,Article ,Extracellular matrix ,proteomics ,Humans ,lcsh:QH301-705.5 ,Cells, Cultured ,Cryopreservation ,Decellularization ,Chemistry ,General Medicine ,Phenotype ,In vitro ,Cell biology ,lcsh:Biology (General) ,Solubility ,Cell culture ,Hepatic stellate cell ,decellularization ,Peptides - Abstract
The lack of robust methods to preserve, purify and in vitro maintain the phenotype of the human liver&rsquo, s highly specialized parenchymal and non-parenchymal cell types importantly hampers their exploitation for the development of research and clinical applications. There is in this regard a growing interest in the use of tissue-specific extracellular matrix (ECM) to provide cells with an in vitro environment that more closely resembles that of the native tissue. In the present study, we have developed a method that allows for the isolation and downstream application of the human liver&rsquo, s main cell types from cryopreserved material. We also isolated and solubilized human liver ECM (HL-ECM), analyzed its peptidomic and proteomic composition by mass spectrometry and evaluated its interest for the culture of distinct primary human liver cells. Our analysis of the HL-ECM revealed proteomic diversity, type 1 collagen abundance and partial loss of integrity following solubilization. Solubilized HL-ECM was evaluated either as a coating or as a medium supplement for the culture of human primary hepatocytes, hepatic stellate cells and liver sinusoidal endothelial cells. Whereas the solubilized HL-ECM was suitable for cell culture, its impact on the phenotype and/or functionality of the human liver cells was limited. Our study provides a first detailed characterization of solubilized HL-ECM and a first report of its influence on the culture of distinct human primary liver cells.
- Published
- 2020
39. Proteins involved in the endoplasmic reticulum stress are key players in synovitis of osteoarthritis, chronic pyrophosphate arthropathy and rheumatoid arthritis, and correlate with the histological score
- Author
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Dominique de Seny, Elettra Bianchi, Dominique Baiwir, Gaël Cobraiville, Charlotte Collin, Mégane Deliège, Marie-Joëlle Kaiser, Gabriel Mazzucchelli, Jean-Philippe Hauzeur, Philippe Delvenne, and Michel Malaise
- Abstract
Background: It is now well recognized that osteoarthritis (OA) synovial membrane presents inflammatory components. The aim of this work is to provide evidence that similar inflammatory mechanisms exist among the three pathologies presenting an inflammatory gradient: OA, chronic pyrophosphate arthropathy (CPPA) and rheumatoid arthritis (RA).Methods: Synovial biopsies of OA (n=9), CPPA (n=7) and RA (n=8) patients were first characterized by a histological score based on synovial hyperplasia and infiltration of lymphocytes, plasma cells, polymorphonuclear and macrophages. All biopsies were also analyzed by 2D-nano-UPLC-ESI-Q-Orbitrap for protein identification and quantification. Protein levels were correlated with the histological score.Results: Histological score was in the range of 3 to 8 for OA, 5 to 13 for CPPA and 12 to 17 for RA. Of the 4336 proteins identified by mass spectrometry, 51 proteins were selected for their strong correlation (p
- Published
- 2020
40. Accelerated pre-senile systemic amyloidosis in PACAP knockout mice - a protective role of PACAP in age-related degenerative processes
- Author
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Balazs D. Fulop, Attila Bardosi, Tamás Juhász, Dora Reglodi, Mark Kriegsmann, Jason Sparks, Sebastian Bardosi, Zsuzsanna Nagy, Rémi Longuespée, Krisztina Kovacs, Joerg Kriegsmann, Attila Miseta, Adel Jungling, Andrea Tamas, Hitoshi Hashimoto, Gabriel Mazzucchelli, and Rita Casadonte
- Subjects
0301 basic medicine ,endocrine system ,Kidney ,medicine.medical_specialty ,Amyloid ,business.industry ,Amyloidosis ,Neuropeptide ,Spleen ,medicine.disease ,Pathophysiology ,Pathology and Forensic Medicine ,03 medical and health sciences ,030104 developmental biology ,medicine.anatomical_structure ,Endocrinology ,Internal medicine ,Knockout mouse ,medicine ,Immunohistochemistry ,business ,hormones, hormone substitutes, and hormone antagonists - Abstract
Dysregulation of neuropeptides may play an important role in aging-induced impairments. Among them, pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent cytoprotective peptide that provides an endogenous control against a variety of tissue-damaging stimuli. We hypothesized that the progressive decline of PACAP throughout life and the well-known general cytoprotective effects of PACAP lead to age-related pathophysiological changes in PACAP deficiency, supported by the increased vulnerability to various stressors of animals partially or totally lacking PACAP. Using young and aging CD1 PACAP knockout (KO) and wild type (WT) mice, we demonstrated pre-senile amyloidosis in young PACAP KO animals and showed that senile amyloidosis appeared accelerated, more generalized, more severe, and affected more individuals. Histopathology showed age-related systemic amyloidosis with mainly kidney, spleen, liver, skin, thyroid, intestinal, tracheal, and esophageal involvement. Mass spectrometry-based proteomic analysis, reconfirmed with immunohistochemistry, revealed that apolipoprotein-AIV was the main amyloid protein in the deposits together with several accompanying proteins. Although the local amyloidogenic protein expression was disturbed in KO animals, no difference was found in laboratory lipid parameters, suggesting a complex pathway leading to increased age-related degeneration with amyloid deposits in the absence of PACAP. In spite of no marked inflammatory histological changes or blood test parameters, we detected a disturbed cytokine profile that possibly creates a pro-inflammatory milieu favoring amyloid deposition. In summary, here we describe accelerated systemic senile amyloidosis in PACAP gene-deficient mice, which might indicate an early aging phenomenon in this mouse strain. Thus, PACAP KO mice could serve as a model of accelerated aging with human relevance. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
- Published
- 2018
41. Plant defense in response to chewing insects: proteome analysis of Arabidopsis thaliana damaged by Plutella xylostella
- Author
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Georges Lognay, Frédéric Francis, Julien Bauwens, Dieu-Hien Truong, Gabriel Mazzucchelli, and Hoang Chinh Nguyen
- Subjects
0106 biological sciences ,0301 basic medicine ,Arabidopsis thaliana ,Plant Science ,lcsh:Plant culture ,Photosynthesis ,01 natural sciences ,proteomic expression ,03 medical and health sciences ,Arabidopsis ,Botany ,Plant defense against herbivory ,MALDI-TOF MS ,lcsh:SB1-1110 ,Plutella xylostella ,Ecology, Evolution, Behavior and Systematics ,Larva ,biology ,fungi ,Plutella ,food and beverages ,lcsh:QK900-989 ,LC-ESI-MS/MS ,biology.organism_classification ,2-DE ,030104 developmental biology ,Proteome ,lcsh:Plant ecology ,PEST analysis ,010606 plant biology & botany - Abstract
The interactions between Arabidopsis thaliana and Plutella xylostella have been considered as a model system to unravel the responses of plants to herbivorous insects. Here, we use a 2-DE proteome approach to detect protein expression changes in the leaves of Arabidopsis plants exposed to P. xylostella larval infestation at 27°C within 8 h. Approximately 450 protein spots were reproducibly detected on gels. Of these, comparing healthy and infested leaves, we identified 18 differentially expressed protein spots. Thirteen proteins were successfully identified by MALDI-TOF/MS and LC-ESI-MS/MS. Functional classification analysis indicated that the differentially identified proteins were associated with amino acid, carbohydrate, energy, lipid metabolism, and photosynthesis. In addition, their relative abundances were assessed according to larval pest feeding on Arabidopsis leaves. These data provide valuable new insights for further works in plant-biotic and environmental stress interaction.
- Published
- 2018
42. Acid Hydrolysis of Gluten Enhances the Skin Sensitizing Potential and Drives Diversification of IgE Reactivity to Unmodified Gluten Proteins
- Author
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Katrine Lindholm Bøgh, Paola Roncada, Sandra Denery-Papini, Jeppe Madura Larsen, Cristian Piras, Anne-Sofie Ravn Ballegaard, Daniel Andersen, Grégory Bouchaud, Susanne Brix, Clélia Villemin, Laure Castan, Gabriel Mazzucchelli, Charlotte Bernhard Madsen, Danmarks Tekniske Universitet = Technical University of Denmark (DTU), Unité de recherche sur les Biopolymères, Interactions Assemblages (BIA), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Department of Chemistry, University of Reading (UOR), Department of Biomedical Sciences [Copenhagen], Faculty of Health and Medical Sciences, University of Copenhagen = Københavns Universitet (UCPH)-University of Copenhagen = Københavns Universitet (UCPH), Università degli Studi 'Magna Graecia' di Catanzaro = University of Catanzaro (UMG), Center for Biological Sequence Analysis - Department of Systems Biology, Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA-Research), Université de Liège, National Food Institute Lyngby, Lundbeckfonden : R181-2014-2495 (Danish Environmental Protection Agency)., and European Project
- Subjects
Brown norway rats ,Glutens ,Wheat Hypersensitivity ,digestive system ,Hydrolysate ,Hydrolysis ,[SDV.IDA]Life Sciences [q-bio]/Food engineering ,medicine ,Animals ,skin sensitization ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Food science ,Deamidation ,Sensitization ,chemistry.chemical_classification ,nutritional and metabolic diseases ,wheat allergy ,Allergens ,Immunoglobulin E ,Skin sensitisation ,medicine.disease ,Gluten ,Wheat allergy ,digestive system diseases ,Rats ,deamidation ,Enzyme ,medicine.anatomical_structure ,chemistry ,Acid hydrolysis ,Brown Norway rats ,[SDV.AEN]Life Sciences [q-bio]/Food and Nutrition ,Food Science ,Biotechnology - Abstract
ScopePersonal care products containing hydrolysed gluten have been linked to spontaneous sensitisation through the skin, however the impact of the hydrolysate characteristics on the sensitising capacity is generally unknown. Methods and ResultsThe physicochemical properties of five different wheat-derived gluten products (1 unmodified, 1 enzyme hydrolysed, and 3 acid hydrolysed) were investigated, and the skin sensitising capacity was determined in allergy-prone Brown Norway rats. Acid hydrolysed gluten products exhibited the strongest intrinsic sensitising capacity via the skin. All hydrolysed gluten products induced cross-reactivity to unmodified gluten in the absence of oral tolerance to wheat, but were unable to break tolerance in animals on a wheat-containing diet. Still, the degree of deamidation in acid hydrolysed products was associated with product-specific sensitisation in wheat tolerant rats. Sensitisation to acid hydrolysed gluten products was associated with a more diverse IgE reactivity profile to unmodified gluten proteins compared to sensitisation induced by unmodified gluten or enzyme hydrolysed gluten. ConclusionAcid hydrolysis enhances the skin sensitising capacity of gluten and drives IgE reactivity to more gluten proteins. This property of acid hydrolysed gluten may be related to the degree of product deamidation, and could be a strong trigger of wheat allergy in susceptible individuals.
- Published
- 2021
43. Liquid chromatography setup-dependent artefactual methionine oxidation of peptides: The importance of an adapted quality control process
- Author
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France Baumans, Emeline Hanozin, Dominique Baiwir, Corentin Decroo, Ruddy Wattiez, Edwin De Pauw, Gauthier Eppe, and Gabriel Mazzucchelli
- Subjects
Quality Control ,chemistry.chemical_classification ,Chromatography, Reverse-Phase ,Methionine ,Chromatography ,Chemistry ,Organic Chemistry ,Peptide ,General Medicine ,Mass spectrometry ,Biochemistry ,Analytical Chemistry ,Chromatographic separation ,chemistry.chemical_compound ,Stationary phase ,Scientific method ,Phase (matter) ,Peptides ,Oxidation-Reduction - Abstract
In both biologics quality control experiments and protein post-translational modification studies, the analytical system used is not supposed to bring any artefactual modifications which could impair the results. In this work, we investigated oxidation of methionine-containing peptides during reversed-phase (RP) chromatographic separation. We first used a synthetic methionine-containing peptide to evaluate this artefactual phenomenon and then considered more complex samples (i.e., plasma and HeLa protein digests). The methionine oxidation levels of the peptides were systematically assessed and compared for the long-term use of the analytical column, the sample trapping time, the gradient length, the sample load and the nature of the stationary phase (HSS T3 from Waters, YMC Triart C18 from YMC Europe GmbH and BEH130 C18 from Waters). In addition to the oxidation of methionine in solution, we observed on the HSS T3 and the BEH130 stationary phases an additional broad peak corresponding to an on-column oxidized species. Considering the HSS T3 phase, our results highlight that the on-column oxidation level significantly increases with the age of the analytical column and the gradient length and reaches 56 % when a 1-year-old column set is used with a 180 min-long LC method. These levels go to 0 % and 18 % for the YMC Triart C18 and the BEH130 C18 phases respectively. Interestingly, the on-column oxidation proportion decreases as the injected sample load increases suggesting the presence of a discrete number of oxidation sites within the stationary phase of the analytical column. Those findings observed in different laboratories using distinct set of columns, albeit to varying degrees, strengthen the need for a standard of methionine-containing peptide that could be used as a quality control to appraise the status of the liquid chromatographic columns.
- Published
- 2021
44. Proteomic signatures reveal a dualistic and clinically relevant classification of anal canal carcinoma
- Author
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Michael Herfs, Fabiola Obregon, Patrick Roncarati, Edwin De Pauw, Alizée Lebeau, Christopher P. Crum, Rémi Longuespée, Andrew Dunn, Philippe Delvenne, Meggy Suarez-Carmona, Pascale Hubert, Diane Bruyère, Dominique Baiwir, Charles M. Quick, Nicolas Smargiasso, Eric J Yang, Keith Lai, and Gabriel Mazzucchelli
- Subjects
0301 basic medicine ,education.field_of_study ,Pathology ,medicine.medical_specialty ,Anal Carcinoma ,General surgery ,Population ,HPV infection ,Biology ,Anal canal ,medicine.disease ,Pathology and Forensic Medicine ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,medicine ,Anal cancer ,education ,Cervix ,Pathological ,Anal Transitional Zone - Abstract
Aetiologically linked to HPV infection, malignancies of the anal canal have substantially increased in incidence over the last 20 years. Although most anal squamous cell carcinomas (SCCs) respond well to chemoradiotherapy, about 30% of patients experience a poor outcome, for undetermined reasons. Despite cumulative efforts for discovering independent predictors of overall survival, both nodal status and tumour size are still the only reliable factors predicting patient outcome. Recent efforts have revealed that the biology of HPV-related lesions in the cervix is strongly linked to the originally infected cell population. To address the hypothesis that topography also influences both gene expression profile and behaviour of anal (pre)neoplastic lesions, we correlated both proteomic signatures and clinicopathological features of tumours arising from two distinct portions of the anal canal: the lower part (squamous zone) and the more proximal anal transitional zone. Although microdissected cancer cells appeared indistinguishable by morphology (squamous phenotype), unsupervised clustering analysis of the whole proteome significantly highlighted the heterogeneity that exists within anal canal tumours. More importantly, two region-specific subtypes of SCC were revealed. The expression profile (sensitivity/specificity) of several selected biomarkers (keratin filaments) further confirmed the subclassification of anal (pre)cancers based on their cellular origin. Less commonly detected compared to their counterparts located in the squamous mucosa, SCCs originating in the transitional zone more frequently displayed a poor or basaloid differentiation, and were significantly correlated with reduced disease-free and overall survivals. Taken together, we present direct evidence that anal canal SCC comprises two distinct entities with different cells of origin, proteomic signatures, and survival rates. This study forms the basis for a dualistic classification of anal carcinoma, with implications for management, outcome expectations, and possibly therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
- Published
- 2017
45. Proteomic investigation of human cystic echinococcosis in the liver
- Author
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Rémi Longuespée, Jörg Kriegsmann, Rita Casadonte, Petra Wandernoth, Katharina Lisenko, Mark Kriegsmann, Michael Becker, and Gabriel Mazzucchelli
- Subjects
Proteomics ,0301 basic medicine ,Echinococcosis, Hepatic ,Pathology ,medicine.medical_specialty ,Proteome ,Laser Capture Microdissection ,Mass spectrometry imaging ,03 medical and health sciences ,0302 clinical medicine ,Tandem Mass Spectrometry ,parasitic diseases ,medicine ,Animals ,Humans ,Echinococcus granulosus ,Molecular Biology ,Laser capture microdissection ,biology ,Cystic echinococcosis ,Parasitic cyst ,biology.organism_classification ,medicine.disease ,030104 developmental biology ,Infectious disease (medical specialty) ,030220 oncology & carcinogenesis ,Parasitic disease ,Immunology ,Parasitology ,Chromatography, Liquid - Abstract
Cystic echinococcosis (CE) is a pandemic infectious disease caused by the tapeworm Echinococcus granulosus that forms cysts in different organs such as lungs and liver. Imaging examination and serological tests have some drawbacks such as low sensitivity. In this study, we used an up-to-date workflow of laser microdissection-based microproteomics and matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry in order to depict the proteomic pattern of CE in the liver. This investigation revealed specific markers of a parasitic cyst in liver. This proteomic pattern could facilitate diagnosis of CE in the future.
- Published
- 2017
46. P032 Increased endoplasmic reticulum stress-specific chaperones characterise CD fibrosis epithelium tissues and participate to in vitro induction of intestinal fibrobast differentiation
- Author
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Nicolas Pierre, Edouard Louis, Charlotte Massot, Gabriel Mazzucchelli, Noëlla Bletard, Florence Quesada Calvo, Sophie Vieujean, Catherine Salee, Dominique Baiwir, Marie-Alice Meuwis, Emeline Bequet, Shurong Hu, Philippe Delvenne, and Edwin De Pauw
- Subjects
business.industry ,Endoplasmic reticulum ,Gastroenterology ,General Medicine ,medicine.disease ,Intestinal epithelium ,Epithelium ,Cell biology ,Extracellular matrix ,Paracrine signalling ,medicine.anatomical_structure ,Fibrosis ,Heat shock protein ,medicine ,Unfolded protein response ,business - Abstract
Background Intestinal fibrosis is a complication of Crohn’s disease (CD) characterised by myofibroblasts and extracellular matrix accumulation within the submucosa and smooth muscles, leading to bowel strictures. No medical treatment exists to treat or reverse intestinal fibrosis leading often to surgical resection. The potential role of intestinal epithelium in the fibrotic process remains poorly defined. Methods We performed a pilot study on ileal fibrostricturing CD surgical samples (n = 5), comparing the proteome of surface epithelium isolated by laser capture microdissection in normal and fibrotic zones. Confirmation of the specific protein increases was obtained by immunohistochemistry in colonic and ileal samples of CD (n = 44) compared with healthy subjects (n = 40), as well as in intestinal epithelial cell line under induced endoplasmic reticulum (ER) stress. A model of fibroblast to myofibroblast differentiation induction was also challenged using preconditioned media of intestinal epithelial cells after a pulsed ER stress. Results Label-free proteomics revealed high ER stress in the epithelium surrounding fibrotic bowel wall, involving anterior gradient protein 2 homolog (AGR2) and 78 kDA glucose-regulated protein (BiP). Confirmation of both proteins increase was obtained by immunohistochemistry. ER stress induction in intestinal epithelial cells was associated with an intracellular increase of AGR2, BiP and ER stress markers as sXPB1 and CHOP. AGR2 was also detected in the culture medium of these epithelial cells and myofibroblast differentiation was obtained using this culture medium. Conclusion The increase of ER stress proteins observed in fibrostenosing tissues together with these preliminary evidences of fibroblast to myofibrobast differentiation obtained by paracrine action of intestinal epithelial cell preconditioned to ER stress induction suggest a role of epithelial ER stress in Crohn’s disease intestinal fibrosis.
- Published
- 2020
47. Exploring the N-Glycosylation Profile of Glycoprotein B from Human Cytomegalovirus Expressed in CHO and Nicotiana tabacum BY-2 Cells
- Author
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Edwin De Pauw, Catherine Navarre, François Chaumont, Gabriel Mazzucchelli, Nicolas Smargiasso, Marc Boutry, Joseph Nader, Stéphane Rioux, and UCL - SST/LIBST - Louvain Institute of Biomolecular Science and Technology
- Subjects
0106 biological sciences ,0301 basic medicine ,Glycosylation ,Nicotiana tabacum ,Gene Expression ,N-glycosylation ,01 natural sciences ,lcsh:Chemistry ,chemistry.chemical_compound ,N-linked glycosylation ,Viral Envelope Proteins ,Chemical Precipitation ,glycoprotein B ,lcsh:QH301-705.5 ,plant cell suspension culture ,Spectroscopy ,mass spectrometry ,chemistry.chemical_classification ,biology ,Chinese hamster ovary cell ,General Medicine ,Recombinant Proteins ,Computer Science Applications ,Ectodomain ,Carbohydrate Sequence ,Ammonium Sulfate ,Chromatography, Gel ,CHO Cells ,Article ,Catalysis ,Inorganic Chemistry ,03 medical and health sciences ,Cricetulus ,Polysaccharides ,Plant Cells ,Tobacco ,Animals ,Humans ,Physical and Theoretical Chemistry ,Molecular Biology ,cytomegalovirus ,Ammonium sulfate precipitation ,Organic Chemistry ,Galactose ,biology.organism_classification ,Molecular biology ,N-Acetylneuraminic Acid ,Peptide Fragments ,Sialic acid ,030104 developmental biology ,lcsh:Biology (General) ,lcsh:QD1-999 ,chemistry ,Proteolysis ,Glycoprotein ,010606 plant biology & botany - Abstract
The ability to control the glycosylation pattern of recombinant viral glycoproteins represents a major prerequisite before their use as vaccines. The aim of this study consisted of expressing the large soluble ectodomain of glycoprotein B (gB) from Human Cytomegalovirus (HMCV) in Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells and of comparing its glycosylation profile with that of gB produced in Chinese hamster ovary (CHO) cells. gB was secreted in the BY-2 culture medium at a concentration of 20 mg/L and directly purified by ammonium sulfate precipitation and size exclusion chromatography. We then measured the relative abundance of N-glycans present on 15 (BY-2) and 17 (CHO) out of the 18 N-sites by multienzymatic proteolysis and mass spectrometry. The glycosylation profile differed at each N-site, some sites being occupied exclusively by oligomannosidic type N-glycans and others by complex N-glycans processed in some cases with additional Lewis A structures (BY-2) or with beta-1,4-galactose and sialic acid (CHO). The profiles were strikingly comparable between BY-2- and CHO-produced gB. These results suggest a similar gB conformation when glycoproteins are expressed in plant cells as site accessibility influences the glycosylation profile at each site. These data thus strengthen the BY-2 suspension cultures as an alternative expression system.
- Published
- 2019
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48. Resolution of the proteome, transcript and ionome dynamics upon Zn re-supply in Zn-deficient Arabidopsis
- Author
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Borjana Arsova, Sahand Amini, Maxime Scheepers, Dominique Baiwir, Gabriel Mazzucchelli, Monique Carnol, Bernard Bosman, Patrick Motte, Edwin de Pauw, Michelle Watt, and Marc Hanikenne
- Abstract
SummaryRegulation of plant Zn acquisition is poorly understood, while Zn deficiency affects over 2 billion people worldwide. We therefore dissected the dynamic response to changes in Zn supply in Arabidopsis.Hydroponically-grown Zn starved plants were re-supplied with Zn. Subsequent time-resolved sampling strategy allowed concomitant quantification of the dynamics of Zn uptake, microsomal and soluble proteins, and specific transcripts, in space (roots and shoots) and time.Zn accumulates in roots within 10min, but 8h are needed before shoot Zn increases. By 8h, root Zn concentration was ~60% of non-starved plants. Overexpressed root Zn transporters further peaked in 10-30min post re-supply, before reaching a minimum in 120min and 200 ppm Zn. Zn-responding signaling/regulatory molecules include receptor and MAP kinases, calcium signaling proteins, phosphoinositides, G-proteins, COP9 signalosome members, as well as multiple transcription factors.Zn acquisition is a highly controlled dynamic process. Our study identifies novel players in Zn homeostasis and points to cross-talk with other nutrients. It paves the way for directed investigation of so far omitted candidates which dynamically respond to sudden changes in Zn supply but are expressed at similar levels at steady-state Zn deficiency and sufficiency.
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- 2019
49. A laser microdissection-based workflow for FFPE tissue microproteomics: Important considerations for small sample processing
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Edwin De Pauw, Philippe Delvenne, Rémi Longuespée, Gabriel Mazzucchelli, Mark Kriegsmann, Michael Herfs, Dominique Baiwir, Jörg Kriegsmann, Charles Pottier, Nicolas Smargiasso, and Deborah Alberts
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Proteomics ,0301 basic medicine ,Tissue Fixation ,Formalin fixed paraffin embedded ,Computational biology ,Bioinformatics ,Citric Acid ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Breast cancer ,Tandem Mass Spectrometry ,Formaldehyde ,medicine ,Humans ,Antigens ,Biomarker discovery ,Molecular Biology ,Laser capture microdissection ,Paraffin Embedding ,Molecular pathology ,Lasers ,Proteins ,Small sample ,medicine.disease ,030104 developmental biology ,Antigen retrieval ,chemistry ,Microdissection ,Chromatography, Liquid - Abstract
Proteomic methods are today widely applied to formalin-fixed paraffin-embedded (FFPE) tissue samples for several applications in research, especially in molecular pathology. To date, there is an unmet need for the analysis of small tissue samples, such as for early cancerous lesions. Indeed, no method has yet been proposed for the reproducible processing of small FFPE tissue samples to allow biomarker discovery. In this work, we tested several procedures to process laser microdissected tissue pieces bearing less than 3000 cells. Combined with appropriate settings for liquid chromatography mass spectrometry–mass spectrometry (LC–MS/MS) analysis, a citric acid antigen retrieval (CAAR)-based procedure was established, allowing to identify more than 1400 proteins from a single microdissected breast cancer tissue biopsy. This work demonstrates important considerations concerning the handling and processing of laser microdissected tissue samples of extremely limited size, in the process opening new perspectives in molecular pathology. A proof of the proposed method for biomarker discovery, with respect to these specific handling considerations, is illustrated using the differential proteomic analysis of invasive breast carcinoma of no special type and invasive lobular triple-negative breast cancer tissues. This work will be of utmost importance for early biomarker discovery or in support of matrix-assisted laser desorption/ionization (MALDI) imaging for microproteomics from small regions of interest.
- Published
- 2016
50. P202 Sweat proteomics for cystic fibrosis diagnosis and personalised therapy
- Author
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B. Burat, Teresinha Leal, Dominique Baiwir, Gauthier Eppe, A. Reynaerts, Gabriel Mazzucchelli, and Maximilien Fleron
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Pulmonary and Respiratory Medicine ,SWEAT ,Pathology ,medicine.medical_specialty ,business.industry ,Pediatrics, Perinatology and Child Health ,Medicine ,business ,medicine.disease ,Proteomics ,Cystic fibrosis - Published
- 2020
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