References of "Mazzucchelli, Gabriel"
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See detailGender differences in responses in Gammarus pulex exposed to BDE-47: a gel-free proteomic approach
Gismondi, Eric ULg; Mazzucchelli, Gabriel ULg; De Pauw, Edwin ULg et al

in Ecotoxicology & Environmental Safety (2015), 122

Very few ecotoxicological studies have considered differences in toxic effects on male and female organisms. Here, we investigated protein expression differences in caeca of Gammarus pulex males and ... [more ▼]

Very few ecotoxicological studies have considered differences in toxic effects on male and female organisms. Here, we investigated protein expression differences in caeca of Gammarus pulex males and females under control conditions (unexposed) and after 96 h exposure to BDE-47. Using gel-free proteomic analysis, we have identified 45 proteins, of which 25 were significantly differently expressed according to sex and/or BDE-47 exposure. These proteins were involved in several biological processes such as energy metabolism, chaperone proteins, or transcription/translation. In unexposed amphipods, 11 proteins were significantly over-expressed in females, and 6 proteins were over-expressed in males. Under BDE-47 stress, 7 proteins were differently impacted according to sex. For example, catalase was over-expressed in exposed females and under-expressed in exposed males, as compared to respective controls. Conversely, proteins involved in energy metabolism were up-regulated in males and down-regulated in females. Our proteomic study showed differences in responses of males and females to BDE-47 exposure, emphasizing that sex is a confounding factor in ecotoxicological assessment. However, due to the limited information existing in databases on Gammarids, it was difficult to define a BDE-47 mechanism of action. The gel-free proteomic seems to be a promising method to develop in future ecotoxicological studies and thus, to improve our understanding of the mechanism of action of xenobiotics. [less ▲]

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See detailDe novo sequencing using MELD proteolysis coupled to a "sequence assembly" algorithm
Mazzucchelli, Gabriel ULg; Zimmerman, Tyler; Smargiasso, Nicolas ULg et al

in 63rd ASMS Conference Proceedings, May 30 - June 4 2015, St. Louis, MO (2015, June)

Introduction Protein de novo sequencing requires a method that combines extensive MSMS fragmentation and an appropriate data processing. This can be applied either on intact protein or on its proteolytic ... [more ▼]

Introduction Protein de novo sequencing requires a method that combines extensive MSMS fragmentation and an appropriate data processing. This can be applied either on intact protein or on its proteolytic peptides. Peptide analysis has the advantage to be compatible with well-known workflows. Nevertheless the connectivity between peptides is lost. Taking these considerations into account, a specific digestion method and a sequence assembly software were developed. The MELD method relies on a combination of MultiEnzymatic AND Limited proteolytic Digestions. The MELD generates in a single experiment numerous different peptides with miss-cleavages that overlap when aligned on the matching protein sequence. The two major benefices are an increased probability to obtain the entire protein sequence and a redundancy in the protein sequence matches. Methods The MELD consists in two parallel 2h digestions both using an optimized protease mixture. The mixtures are composed of the same proteases but in different relative quantities. Analyses were performed by UPLC-Orbitrap (IClass, Waters, QExactive, Thermo). Data were processed with PEAKS (BSI) to generate de novo sequence candidates. After data importation into our software, a seed sequence is set or can be found automatically. The software extends the seed sequence in both directions with moving windows of three amino acids, plus a fourth one to be added. The added amino acid is validated in several ways, including by the total frequency of occurrence, by larger windows of aa, by the local spectrum-derived confidence, and by combinations of these factors. Preliminary Data The MELD protocol was first validated by applying a traditional database search workflow on several commercial proteins with an inter-day and inter-individual procedure. These experiments showed 100% sequence coverage for each protein analyzed, involving information on peptide identity and modifications localization. Strong confident identification was obtained due to multiple overlapping peptides matches with the given sequence and with a high number of overlapping peptides assignments. The analysis of the four proteins provided the following results: HSA, Myoglobin and Lysozyme were identified with 100% sequence coverage with respectively 890, 300 and 120 unique peptides (CV<10%, peptide FDR<0.1%). The variable region of each heavy and light chains of Adalimumab antibody were identified with 100% sequence coverage. With the MELD, an average of 10 different peptides covering each sequence stretch of the protein could be obtained. The combinatory effect of the multiple enzymes used and the limited digestions leads to an increased robustness, very high confidence identifications and allows clear localization of PTMs. The MELD protocol as presented here and tested on several pure proteins digested in solution, certainly improves the general "bottom-up" strategy applied for highly confident protein identification and would allow better protein characterization, even for those having PTMs. In addition, in our analysis, each fragment position of the entire protein sequence was evidenced either by a "y" or a "b" fragment ion. This high and confident amount of information enables extensive de novo sequencing using PEAKS software, followed by application of our “sequence assembly” algorithm. The first version of our assembling tool on MELD experimental data generated long sequence tags, up to 90 amino acids long. [less ▲]

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See detailProteome variations in Macrobrachium rosenbergii exposed to chlordecone: a gel-free proteomic approach
Lafontaine, Anne ULg; De Pauw, Edwin ULg; Forget-Leray, Joelle et al

Poster (2015, May 06)

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See detailComparison of early stages of colorectal cancer by label free proteomics
QUESADA CALVO, Florence ULg; MEUWIS, Marie-Alice ULg; Bertrand, Virginie ULg et al

in Acta Gastroenterologica (2015, February 27)

Introduction and objectives: Colorectal cancer (CRC) is the second most frequent cancer in women and the third in men. Identification of the mechanisms of progression in these early CRC stages is ... [more ▼]

Introduction and objectives: Colorectal cancer (CRC) is the second most frequent cancer in women and the third in men. Identification of the mechanisms of progression in these early CRC stages is important to develop new diagnostic and therapeutic tools. Formalin-Fixed Paraffin-Embedded (FFPE) specimens are materials that enable proteomic clinical research. Hence our aim was to address the comparison of FFPE samples from early CRC stages patients using shotgun proteomic analysis. Methods: We performed a retrospective study on 36 CRC tissue samples (pT1N0M0, n=16 and pT2N0M0, n=20) compared together and with 40 control tissue samples (20 patients with diverticulitis, using paired inflamed (DI) and healthy tissue (DH)). Each tissue slice was macrodissected to enrich in epithelial cells. We used FFPE-FASP kit (Expedeon) for sample preparation and protein digests were analyzed using 2D-nanoAquity UPLC separation online with Q-Tof Synapt HDMSTM G2 using ion mobility as additional separation. We performed protein identification and differential analysis using Progenesis QI for proteomics (Nonlinear Dynamics). Results and discussion: We selected 149 proteins differentially distributed between T1 and T2 CRC stages which were not significantly different between CRC and DH or DI. Only 30 proteins were significantly more abundant in T1 versus T2 and 119 were distributed inversely, with a minimum fold ratio of 2. Among those, ATP synthase subunit beta, Aspartate-tRNA ligase, Haptoglobin and Kininogen were identified. . Moreover, we validated Kininogen and 3 others proteins with a significant differential distribution between pT1N0M0 and pT2N0M0 stages by immunohistochemistry. Conclusion: This FFPE retrospective study comparing T1 and T2 CRC highlighted proteins already previously identified as potential CRC biomarkers. These proteins may reflect important early changes in cancer development and may help understanding early tumor progression. [less ▲]

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See detailPotential Proteomic Biomarkers Associated with Mucosal Healing and Relapse Prediction after IFX Withdrawals in Crohn’s Disease
MEUWIS, Marie-Alice ULg; QUESADA CALVO, Florence ULg; Baiwir, Dominique ULg et al

in Acta gastroenterologica (2015, February 26)

Introduction and objectives: In Crohn's disease (CD), there is a discrepancy between clinical activity of the disease (symptoms) and intestinal healing. However absence of tissue healing is associated ... [more ▼]

Introduction and objectives: In Crohn's disease (CD), there is a discrepancy between clinical activity of the disease (symptoms) and intestinal healing. However absence of tissue healing is associated with the risk of relapse and tissue damage progression. Endoscopy is costly and invasive. Hence biomarkers correlating with intestinal healing could improve disease management and potentially decrease the number of endoscopy when patients are in clinical remission. Aim: We aimed to identify potential biomarkers associated to CD mucosal healing and relapse after IFX withdrawals by a shotgun label-free proteomic study. Methods: We used the STORI1 clinical trial cohort (n=103) aiming at identifying markers associated to relapse prediction after Infliximab treatment withdrawals. We used serum samples of patients in clinical remission (at base line). We grouped these according to the degree of intestinal healing seen at endoscopy or according to relapse occurrence during the 28 month follow-up and composed pooled samples. We performed depletion of the 20 most abundant plasma proteins on each serum pools and ran a proteomic label-free differential analysis using 2D-nanoUPLC-MSE HDMS Synapt G2 for data acquisition. We performed different statistical analysis. Moreover, a Gene Ontology annotation was also performed for the potential biomarkers highlighted. Results and Discussion: We identified analysing these depleted serum pools 430 different proteins and 188 proteins common to all samples. Among these, 40 were found with a significant differential abundance in the groups compared. We selected some among the most significant one (ratio>1.3) or being by nature consistent with the context of this study (sample origin and clinical question addressed). For example, the C-reactive protein (CRP) was found with a significant Ratio of 2 between Relapsers and Non Relapsers. The other potential biomarkers associated to mucosal healing or to relapse prediction, were selected for further validation by Western Blot analysis (WB), routine laboratory tests and also by a Mass Spectrometry based technology: multiplexed selected reaction monitoring (SRM). Multiplexed SRM will enable quantitative analysis of these candidates in each individual patient as well as WB tests. Conclusions: This research strategy and the validation results on potential biomarkers associated to mucosal healing or relapse after treatment cessation in this cohort of CD patients, as well as tests done on other CD patients, might provide new opportunities for patient follow-up test developments. The next step is to perform SRM validation on the STORI cohort and design signatures using these potential biomarkers SRM data for prognosis power evaluation. 1. Louis E, Mary JY, Vernier-Massouille G, et al. Maintenance of remission among patients with Crohn's disease on antimetabolite therapy after infliximab therapy is stopped. Gastroenterology 2012;142:63-70 e5; quiz e31. [less ▲]

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See detailPotential Proteomic Biomarkers Associated To Mucosal Healing In Crohn’s Disease
MEUWIS, Marie-Alice ULg; Baiwir, Dominique ULg; Mazzucchelli, Gabriel ULg et al

Poster (2014, October 06)

Introduction and objectives: In Crohn's disease (CD), there is a discrepancy between clinical activity of the disease (symptoms) and intestinal healing. However absence of tissue healing is associated ... [more ▼]

Introduction and objectives: In Crohn's disease (CD), there is a discrepancy between clinical activity of the disease (symptoms) and intestinal healing. However absence of tissue healing is associated with the risk of relapse and tissue damage progression. Endoscopy is costly and invasive. Hence biomarkers correlating with intestinal healing could improve disease management. We aimed to identify potential biomarkers associated to CD mucosal healing by a shotgun proteomics label free study. Methods: We used the STORI clinical trial cohort (n=103) aiming at identifying markers associated to relapse prediction after Infliximab treatment withdrawals. We used serum samples of patients in clinical remission, grouped according to the degree of intestinal healing seen at endoscopy. We performed depletion of the 20 most abundant plasma proteins on each serum pools and ran a proteomics label free differential analysis using 2D-nanoUPLC-MSE HDMS Synapt G1 for data acquisition and Protein Lynks Global Server vs 2.4 for data analysis (Waters, Corp., Milford, USA). Results and Discussion: We obtained potential biomarkers and designed a multiplexed -selected reaction monitoring (SRM) method for validation of these candidates in each individual patient. The method may also be tested in an independent set of IBD patients with and without mucosal healing. Conclusions: This research strategy and results of SRM validation of potential biomarkers associated to mucosal healing in this cohort of CD patients as well as the tests done on other CD patients, may provide new opportunities for CD follow-up tests development. [less ▲]

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See detailAphid - symbiont interactions : multitrophic "omic" approaches to investigate multitrophic interactions
Bosquée, Emilie ULg; Bauwens, Julien ULg; Mazzucchelli, Gabriel ULg et al

Poster (2014, August)

« Omics » found recent developments due to significant improvement and availability of both separation and identification methods. For proteomics, functional information’s linked to the studied proteins ... [more ▼]

« Omics » found recent developments due to significant improvement and availability of both separation and identification methods. For proteomics, functional information’s linked to the studied proteins was brought when compared to genomic approach. For these reasons, a panel of tools is available to determine the proteome patterns related to differential adaptation of insects to cope with plant defence mechanisms or to transmit virus. The adaptation and metabolic changes of aphids in relation to host plants focusing on the role of the bacterial endosymbionts was investigated. Use of artificial diet including diverse antibiotics but also the comparison of proteomes related to whole aphid and respective purified bacterial symbionts were studied to identify the respective origin and function of proteins constituting the studied proteomes. Diverse methods including 2D-DIGE, liquid chromatography coupled with mass spectrometry and data bank investigations were developed. From the proteome investigation, characterisation of good and bad virus vectors was also performed in different aphid - plant - virus models. Particular proteins of interest were selected. This broad proteomic approach will be discussed as an interesting and reliable tool to study the biologically involved proteins from aphids in response to several environmental changes [less ▲]

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See detailBlocking lipid synthesis overcomes tumor re-growth and metastasis after anti-angiogenic therapy withdrawal.
Sounni, Nor Eddine ULg; Cimino, Jonathan ULg; BLACHER, Silvia ULg et al

in Cell Metabolism (2014), 20(2), 280-94

The molecular mechanisms responsible for the failure of antiangiogenic therapies and how tumors adapt to these therapies are unclear. Here, we applied transcriptomic, proteomic, and metabolomic approaches ... [more ▼]

The molecular mechanisms responsible for the failure of antiangiogenic therapies and how tumors adapt to these therapies are unclear. Here, we applied transcriptomic, proteomic, and metabolomic approaches to preclinical models and provide evidence for tumor adaptation to vascular endothelial growth factor blockade through a metabolic shift toward carbohydrate and lipid metabolism in tumors. During sunitinib or sorafenib treatment, tumor growth was inhibited and tumors were hypoxic and glycolytic. In sharp contrast, treatment withdrawal led to tumor regrowth, angiogenesis restoration, moderate lactate production, and enhanced lipid synthesis. This metabolic shift was associated with a drastic increase in metastatic dissemination. Interestingly, pharmacological lipogenesis inhibition with orlistat or fatty acid synthase downregulation with shRNA inhibited tumor regrowth and metastases after sunitinib treatment withdrawal. Our data shed light on metabolic alterations that result in cancer adaptation to antiangiogenic treatments and identify key molecules involved in lipid metabolism as putative therapeutic targets. [less ▲]

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See detailComparison of two FFPE preparation methods using label-free shotgun proteomics: Application to tissues of diverticulitis patients.
Quesada-Calvo, Florence; Bertrand, Virginie ULg; Longuespée, Rémi ULg et al

in Journal of proteomics (2014), 112C

Formalin-fixed paraffin-embedded (FFPE) specimens of patients are useful sources of materials for clinical research and have recently gained interest for use in the discovery of clinical proteomic ... [more ▼]

Formalin-fixed paraffin-embedded (FFPE) specimens of patients are useful sources of materials for clinical research and have recently gained interest for use in the discovery of clinical proteomic biomarkers. However, the critical step in this field is the ability to obtain an efficient and repeatable extraction using the limited quantities of material available for research in hospital biobanks. This work describes the evaluation of the peptide/protein extraction using FFPE sections treated by the following two methods before shotgun proteomic analysis: a commercial solution (FFPE-FASP) (filter aided sample preparation) and an antigen retrieval-derived protocol (On Slice AR). Their efficiencies and repeatabilities are compared using data-independent differential quantitative label-free analysis. FFPE-FASP was shown to be globally better both qualitatively and quantitatively than On Slice AR. FFPE-FASP was tested on several samples, and differential analysis was used to compare the tissues of diverticulitis patients (healthy and inflammatory tissues). In this differential proteomic analysis using retrospective clinical FFPE material, FFPE-FASP was reproducible and provided a high number of confident protein identifications, highlighting potential protein biomarkers. BIOLOGICAL SIGNIFICANCE: In clinical proteomics, FFPE is an important resource for retrospective analysis and for the discovery of biomarkers. The challenge for FFPE shotgun proteomic analysis is preparation by an efficient and reproducible protocol, which includes protein extraction and digestion. In this study, we analyzed two different methods and evaluated their repeatabilities and efficiencies. We illustrated the reproducibility of the most efficient method, FFPE-FASP, by a pilot study on diverticulitis tissue and on FFPE samples amount accessible in hospital biobanks. These data showed that FFPE is suitable for use in clinical proteomics, especially when the FFPE-FASP method is combined with label-free shotgun proteomics as described in the workflow presented in this work. [less ▲]

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See detailTissue Proteomics for the Next Decade? Towards a Molecular Dimension in Histology
Longuespée, Rémi ULg; Fléron, Maximilien; Pottier, Charles et al

in OMICS : A Journal of Integrative Biology (2014)

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See detailProteomic Analysis of the Reproductive Organs of the Hermaphroditic Gastropod Lymnea stagnalis Exposed to Different Endocrine Disrupting Chemicals
Giusti, Arnaud ULg; Leprince, Pierre ULg; Mazzucchelli, Gabriel ULg et al

in PLoS ONE (2013), 8(11), 81086

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 ... [more ▼]

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. [less ▲]

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See detailPolymer based intraocular lens adsorbome: a bottom up proteomics study
Bertrand, Virginie ULg; Huang, Yi-Shiang ULg; Mazzucchelli, Gabriel ULg et al

Conference (2013, September 08)

In the present work an optimized sample preparation protocol to identify and quantify the “adsorbomes” of hydrophilic and hydrophobic materials for IOLs known to have a higher or a lower incidence of PCO ... [more ▼]

In the present work an optimized sample preparation protocol to identify and quantify the “adsorbomes” of hydrophilic and hydrophobic materials for IOLs known to have a higher or a lower incidence of PCO, respectively was obtained. [less ▲]

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