References of "Mazzucchelli, Gabriel"
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See detailPotential Role of Epithelial Protein Disulphide Isomerases in Crohn’s Disease Fibrosis
VIEUJEAN, Sophie ULiege; Hu, Shurong; Bequet, Emeline ULiege et al

Poster (2021, July)

Background and aims: Intestinal fibrosis is a common complication of Crohn’s disease (CD) characterized by an accumulation of fibroblasts differentiating into activated myofibroblasts secreting excessive ... [more ▼]

Background and aims: 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. Conclusions: 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. [less ▲]

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See detailPotential Role of Epithelial Endoplasmic Reticulum Stress and Anterior Gradient Protein 2 Homolog in Crohn’s Disease Fibrosis
VIEUJEAN, Sophie ULiege; Hu, Shurong; Bequet, Emeline ULiege et al

in Journal of Crohn's and Colitis (2021)

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 ... [more ▼]

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 5 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 homolog (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. [less ▲]

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See detailPotential Role of Epithelial Protein Disulphide Isomerases in Crohn’s Disease Fibrosis
Vieujean, Sophie ULiege; Hu, Shurong; Bequet, Emeline ULiege et al

in Acta Gastro-Enterologica Belgica (2021, March 03)

Background and aims: Intestinal fibrosis is a common complication of Crohn’s disease (CD) characterized by an accumulation of fibroblasts differentiating into activated myofibroblasts secreting excessive ... [more ▼]

Background and aims: 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. Conclusions: 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. [less ▲]

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See detailTumor 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.
Luis, Géraldine; Godfroid, Adrien; Nishiumi, Shin et al

in Redox Biology (2021), 43

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 ... [more ▼]

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. [less ▲]

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See detailWobble tRNA modification and hydrophilic amino acid patterns dictate protein fate.
Rapino, Francesca ULiege; ZHOU, ZHAOLI; RONCERO SANCHEZ, Ana Maria et al

in Nature Communications (2021), 12(1), 2170

Regulation of mRNA translation elongation impacts nascent protein synthesis and integrity and plays a critical role in disease establishment. Here, we investigate features linking regulation of codon ... [more ▼]

Regulation of mRNA translation elongation impacts nascent protein synthesis and integrity and plays a critical role in disease establishment. Here, we investigate features linking regulation of codon-dependent translation elongation to protein expression and homeostasis. Using knockdown models of enzymes that catalyze the mcm(5)s(2) wobble uridine tRNA modification (U(34)-enzymes), we show that gene codon content is necessary but not sufficient to predict protein fate. While translation defects upon perturbation of U(34)-enzymes are strictly dependent on codon content, the consequences on protein output are determined by other features. Specific hydrophilic motifs cause protein aggregation and degradation upon codon-dependent translation elongation defects. Accordingly, the combination of codon content and the presence of hydrophilic motifs define the proteome whose maintenance relies on U(34)-tRNA modification. Together, these results uncover the mechanism linking wobble tRNA modification to mRNA translation and aggregation to maintain proteome homeostasis. [less ▲]

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See detailIn vivo N-Terminomics Highlights Novel Functions of ADAMTS2 and ADAMTS14 in Skin Collagen Matrix Building
Leduc, Cédric ULiege; Dupont, Laura ULiege; Joannes, Loïc ULiege et al

in Frontiers in Molecular Biosciences (2021)

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 ... [more ▼]

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. [less ▲]

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See detailHuman Liver-Derived Extracellular Matrix for the Culture of Distinct Human Primary Liver Cells
Alevra Sarika, Niki; Payen, Valéry; Fleron, Maximilien ULiege et al

in Cells (2020), 9(6)

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 ... [more ▼]

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. [less ▲]

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See detailIncreased Endoplasmic Reticulum stress specific chaperones characterise CD fibrosis epithelium tissues and participates to in vitro induction of intestinal fibroblasts differentiation
Vieujean, Sophie ULiege; Hu, Shurong; Bequet, Emeline ULiege et al

Poster (2020, March 05)

Background: Intestinal fibrosis is a complication of Crohn’s disease (CD) characterized by myofibroblasts and extracellular matrix accumulation within the submucosa and smooth muscles, leading to bowel ... [more ▼]

Background: Intestinal fibrosis is a complication of Crohn’s disease (CD) characterized 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 to 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 78kDA 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. Conclusions: 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. [less ▲]

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See detailSLC12A2 as a potential histological marker of ulcerative colitis associated colorectal dysplasia
Merli, Angela-Maria ULiege; Vieujean, Sophie ULiege; MASSOT, Charlotte ULiege et al

Conference (2020, March 04)

Introduction: Patients suffering from ulcerative colitis (UC) are at increased risk of developing dysplasia (DAI) and colorectal cancer (CAC). Differentiating DAI from inflammation remains difficult for ... [more ▼]

Introduction: Patients suffering from ulcerative colitis (UC) are at increased risk of developing dysplasia (DAI) and colorectal cancer (CAC). Differentiating DAI from inflammation remains difficult for both endoscopists and anatomopathologists due to macro and microscopic features shared by these lesions. Aim: The aim of our work was to confirm, by histological evaluation, a potential proteomic biomarker discriminating early DAI lesions from chronic inflamed and normal tissues in UC. Methods: We included 15 paired tissues from UC patients (n=5) presenting low-grade DAI. Epithelial cells were isolated by laser capture microdissection and analyzed by label-free proteomics. We selected one protein differentially distributed between DAI, inflamed (I) and normal (N) tissues for confirmation by immunochemistry (IHC). IHC characterization was performed using both the staining intensity score (0 to 4) and the staining pattern: “gradient” (staining intensity increasing from the epithelium lumen to the bottom of the crypts) or “no gradient” (homogenous staining). UC patients with DAI (n=28), dysplastic lesion in non-inflammatory colon (DSp) (n=9), CAC (n=14) and at high risk of CAC (>10 years of UC duration) but free of dysplasia or cancer (n=23) were included. We further studied this potential marker tissue distribution in the mouse model of CAC (AOM/DSS treated mice) to trace its presentation at different evolution stages and assessed low (n=51), high-grade DAI (n=35) and CAC (n=38), as well as relevant paired control tissues. This potential tissue marker was finally evaluated in sporadic precancerous colorectal lesions of UC-free patients with low (n=19) and highgrade (n=16) adenomas and cancerous lesions (CRC): pT1 to pT4 (n=82) and compared to paired normal tissues when available. Results: Proteomics identified 1070 proteins among which 19 showed a differential distribution between DAI and I or N. The sodium chloride co-transporter SLC12A2 was only identified in DAI. SLC12A2 IHC “no gradient” staining pattern was associated to DAI and DSp compared to I or N (with p <0.0001 and 0.0002 respectively). The IHC score was also higher for DAI, DSp and CAC compared to paired I and N (p<0.0001 and 0.0084 respectively). These results were confirmed from low-grade dysplasia to more advanced lesions in the AOM/DSS mice model. The “no gradient” pattern was also significantly associated to low and high-grade adenomas, and CRC of UC-free patients compared to normal control tissues. The sensitivity and specificity of SLC12A2 histological pattern reached 89% and 95% for DAI versusI; 90% and 93% for CAC and/or DAI versus I. In addition, the sensitivity and specificity reached 99% and 87% for all precancerous and cancerous lesions (DAI, DSp, CAC and CRC) versus N and I (including also non-progressing UC patients). Conclusions: A specific histological pattern for SLC12A2 is associated to precancerous and cancerous colorectal lesions, and is able to be discriminate these lesions from inflammation and normal tissue in UC. The continuous upregulation of SLC12A2 in advanced colorectal lesionsin the CAC mice model also suggests a role of this protein in the pathophysiology of inflammation-associated colon neoplasia. [less ▲]

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See detailIncreased Endoplasmic Reticulum stress specific chaperones characterise CD fibrosis epithelium tissues and participate to in vitro induction of intestinal fibroblasts differentiation
Vieujean, Sophie ULiege; Hu, Shurong; Bequet, Emeline ULiege et al

Poster (2020, February 14)

Background: Intestinal fibrosis is a complication of Crohn’s disease (CD) characterized by myofibroblasts and extracellular matrix accumulation within the submucosa and smooth muscles, leading to bowel ... [more ▼]

Background: Intestinal fibrosis is a complication of Crohn’s disease (CD) characterized 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 to 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 78kDA 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. Conclusions: 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. [less ▲]

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See detailOn the Risks of Phylogeny-Based Strain Prioritization for Drug Discovery: Streptomyces lunaelactis as a Case Study
Martinet, Loïc ULiege; Naômé, A.; Baiwir, Dominique ULiege et al

in Biomolecules (2020), 10(7),

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 ... [more ▼]

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. [less ▲]

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See detailSolute carrier family 12 member 2 as a proteomic and histological biomarker of dysplasia and neoplasia in ulcerative colitis.
Merli, Angela-Maria ULiege; Vieujean, Sophie ULiege; MASSOT, Charlotte ULiege et al

in Journal of Crohn's & colitis (2020)

BACKGROUND AND AIMS: Ulcerative colitis (UC) patients have a greater risk of developing colorectal cancer through inflammation-dysplasia-carcinoma sequence of transformation. The histopathological ... [more ▼]

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 5 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 AOM/DSS mouse model lesions, 37 UC dysplasia, 14 UC cancers, 23 longstanding 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 to inflammation, including Solute carrier family 12 member 2 (SLC12A2) which was confidently identified with 8 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, 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 κfree of 0.90; IC95%: 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. [less ▲]

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See detailDiscovery 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.
Pierre, Nicolas ULiege; Baiwir, Dominique ULiege; Huynh-Thu, Vân Anh ULiege et al

in Gut (2020)

OBJECTIVE: A subset of Crohn's disease (CD) patients experiences mid/long-term remission after infliximab withdrawal. Biomarkers are needed to identify those patients. DESIGN: New biomarkers of relapse ... [more ▼]

OBJECTIVE: A subset of Crohn's disease (CD) patients experiences mid/long-term remission after infliximab withdrawal. Biomarkers are needed to identify those patients. DESIGN: New 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) or mid/long-term relapse (>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. RESULTS: Distinct biomarker candidates were associated with the risk (HR) of short-term (15 proteins, 2.9<HR<16.1, p<0.05) and mid/long-term (17 proteins, 2.1<HR<4.7, p<0.05) relapse, they reflect different pathophysiological processes. In stratified and non-stratified datasets, novel marker combinations exhibited a high predicting capacity as shown by their higher Z-scores (false discovery rate <0.001) than C reactive protein and faecal calprotectin (current references in predicting relapse). CONCLUSION: We 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. [less ▲]

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See detailGlycosylation deficiency of lipopolysaccharide-binding protein and corticosteroid-binding globulin associated with activity and response to treatment for rheumatoid arthritis
Ciregia, Federica ULiege; Baiwir, Dominique ULiege; COBRAIVILLE, Gaël ULiege et al

in Journal of Translational Medicine (2020), 18(1),

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 ... [more ▼]

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. © 2020 The Author(s). [less ▲]

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See detailPrecise co-registration of mass spectrometry imaging, histology, and laser microdissection-based omics
Dewez, Frédéric ULiege; Martin-Lorenzo, Marta; Herfs, Michael ULiege et al

in Analytical and Bioanalytical Chemistry (2019), 411

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 annotation of ... [more ▼]

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 annotation 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 was performed on a breast cancer tissue and segmentation on the lipid data was performed 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 micrometers. 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. [less ▲]

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See detailNew methodology to monitor the oxidation of MET due to LC separation
Baumans, France ULiege; Baiwir, Dominique ULiege; Colombo, Maria et al

Poster (2019, April)

In the context of biomarker discovery and their absolute quantification in complex samples, we are developing a standardization strategy aiming to control the entire sample preparation process before LC ... [more ▼]

In the context of biomarker discovery and their absolute quantification in complex samples, we are developing a standardization strategy aiming to control the entire sample preparation process before LC-MS analysis. The strategy involves the use of a chimeric protein and different levels of its heavy peptides spiked at opportune moment in the sample during sample processing. Among these peptides, some containing a methionine are inserted. Methionine oxidation is a well-known protein modification occurring in vivo but also in vitro and may result in structural and biological changes of the protein. Controlling these artefactual modifications is then of great interest and will be possible thanks to our strategy. To address the origin of methionine oxidation, we sought differential oxidation levels comparing direct infusion, LC-MS using new columns and LC-MS using old ones. For the last two, an Acquity UPLC M-Class system with a nanoEase MZ symmetry C18 trap column and a NanoEase MZ HSS T3 analytical column has been used. The direct infusion of pure synthetic peptides in a Q ExactiveTM Hybrid Quadrupole-OrbitrapTM showed a low percentage of oxidized peptide (less than 2%). Once injected onto the M-Class system coupled online with the Q Exactive, this percentage jumps to higher value (50-60% dependant of the peptide injected). The same peptides have been injected on the same M-Class system on which both trapping and analytical columns were replaced by new ones. Methionine oxidation in this case, was as low as in the direct infusion, meaning that the previous use of columns has a huge influence on the oxidation of methionine in pure synthetic peptides. This effect is certainly due to the presence of metal ions in the columns that promote methionine oxidation during chromatography separation. Furthermore, when looking at the chromatogram, one can see a difference between the shape of the oxidized peak from new columns and old columns. When using new columns, the shape of the oxidized peptide peak is sharp whereas on old columns its shape is broadened and extended. In both cases, the peak of the native peptide stays sharp, proving that this modification occurs during the chromatographic separation. The same effect was seen on peptides coming from the injection of a complex sample (i.e. commercial HeLa protein digest). Indeed, the percentage of oxidized peptides varies from 1 to 8% when using the new columns and increases to 20% to 80% when injecting the same HeLa digest on the old columns. The use history of a column greatly impacts the oxidation degree of peptides and has to be taken into account when studying methionine oxidation. Future works will be dedicated to the implementation of strategies that could stabilize the oxidation during chromatography separation. [less ▲]

Detailed reference viewed: 35 (1 ULiège)
See detailNew methodology to monitor the oxidation of Met due to LC separation
Baumans, France ULiege; Baiwir, Dominique ULiege; Colombo, Maria et al

Conference (2019, April)

In the context of biomarker discovery and their absolute quantification in complex samples, we are developing a standardization strategy aiming to control the entire sample preparation process before LC ... [more ▼]

In the context of biomarker discovery and their absolute quantification in complex samples, we are developing a standardization strategy aiming to control the entire sample preparation process before LC-MS analysis. The strategy involves the use of a chimeric protein and different levels of its heavy peptides spiked at opportune moment in the sample during sample processing. Among these peptides, some containing a methionine are inserted. Methionine oxidation is a well-known protein modification occurring in vivo but also in vitro and may result in structural and biological changes of the protein. Controlling these artefactual modifications is then of great interest and will be possible thanks to our strategy. To address the origin of methionine oxidation, we sought differential oxidation levels comparing direct infusion, LC-MS using new columns and LC-MS using old ones. For the last two, an Acquity UPLC M-Class system with a nanoEase MZ symmetry C18 trap column and a NanoEase MZ HSS T3 analytical column has been used. The direct infusion of pure synthetic peptides in a Q ExactiveTM Hybrid Quadrupole-OrbitrapTM showed a low percentage of oxidized peptide (less than 2%). Once injected onto the M-Class system coupled online with the Q Exactive, this percentage jumps to higher value (50-60% dependant of the peptide injected). The same peptides have been injected on the same M-Class system on which both trapping and analytical columns were replaced by new ones. Methionine oxidation in this case, was as low as in the direct infusion, meaning that the previous use of columns has a huge influence on the oxidation of methionine in pure synthetic peptides. This effect is certainly due to the presence of metal ions in the columns that promote methionine oxidation during chromatography separation. Furthermore, when looking at the chromatogram, one can see a difference between the shape of the oxidized peak from new columns and old columns. When using new columns, the shape of the oxidized peptide peak is sharp whereas on old columns its shape is broadened and extended. In both cases, the peak of the native peptide stays sharp, proving that this modification occurs during the chromatographic separation. The same effect was seen on peptides coming from the injection of a complex sample (i.e. commercial HeLa protein digest). Indeed, the percentage of oxidized peptides varies from 1 to 8% when using the new columns and increases to 20% to 80% when injecting the same HeLa digest on the old columns. The use history of a column greatly impacts the oxidation degree of peptides and has to be taken into account when studying methionine oxidation. Future works will be dedicated to the implementation of strategies that could stabilize the oxidation during chromatography separation. [less ▲]

Detailed reference viewed: 35 (1 ULiège)
See detailKIT QUANTA -standardization kit for absolute protein quantitation: monitoring of methionine oxidation induced by liquid chromatography separation
Baumans, France ULiege; Baiwir, Dominique ULiege; Colombo, Maria et al

Conference (2019, March)

In the context of biomarker discovery and their absolute quantification in complex samples, a standardization strategy aiming to control the entire sample preparation process before LC-MS analysis would ... [more ▼]

In the context of biomarker discovery and their absolute quantification in complex samples, a standardization strategy aiming to control the entire sample preparation process before LC-MS analysis would be extremely valuable. Our approach involves the use of a chimeric protein and different levels of its heavy peptides spiked at opportune moment in the sample during sample processing. Among these peptides, some containing a methionine are inserted. Methionine oxidation is a well-known protein modification occurring in vivo but also in vitro and may result in structural and functional protein alteration. Controlling this artefactual modification is then of great interest and will be possible thanks to our strategy. To address the origin of methionine oxidation, we sought differential oxidation levels comparing direct infusion, LC-MS using new columns and LC-MS using old ones. The direct infusion of pure synthetic peptides in a Q ExactiveTM Hybrid Quadrupole-OrbitrapTM showed a low percentage of oxidized peptide (less than 2%). Once injected onto the LC system coupled online with the Q Exactive, this percentage jumps to higher value (50-60% dependant of the peptide injected). The same peptides have been injected on the same LC system on which both trapping and analytical columns were replaced by new ones. Methionine oxidation in this case, was as low as in the direct infusion, meaning that the previous use of columns has a huge influence on the oxidation of methionine in pure synthetic peptides. This effect is certainly due to the presence of metal ions in the columns that promote methionine oxidation during chromatography separation. The same effect was observed on complex samples ((i.e. commercial HeLa protein digest and plasma digest). Indeed, the percentage of oxidized peptides varies from 1 to 8% when using the new columns and increases to 20% to 80% when the same samples were injected on the old columns. The use history of a column greatly impacts the oxidation degree of peptides and has to be taken into account when studying methionine oxidation. Future works will be dedicated to the implementation of strategies that could stabilize the oxidation during chromatography separation. [less ▲]

Detailed reference viewed: 28 (3 ULiège)
Full Text
See detailResolution of the proteome, transcript and ionome dynamics upon Zn re-supply in Zn-deficient Arabidopsis
Arsova, Borjana; Amini, Sahand ULiege; Scheepers, Maxime ULiege et al

E-print/Working paper (2019)

Detailed reference viewed: 34 (4 ULiège)