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See detailMetabolomic, proteomic and preclinical imaging of patient-derived tumor xenografts for improving treatment of liver metastases patients
Perez Palacios, A; Blomme, A; Boutry, S et al

in Acta Gastro-Enterologica Belgica (2015, March), 78(1), 134

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See detailPlant polysaccharides initiate underground crosstalk with bacilli by inducing synthesis of the immunogenic lipopeptide surfactin
Debois, Delphine ULg; Fernandez, O.; Franzil, Laurent ULg et al

in Environmental Microbiology Reports (2015), 7(3), 570-582

Some plant-associated bacteria such as Bacillus sp. can protect their host from pathogen ingress and this biocontrol activity correlates with their potential to form multiple antibiotics upon in vitro ... [more ▼]

Some plant-associated bacteria such as Bacillus sp. can protect their host from pathogen ingress and this biocontrol activity correlates with their potential to form multiple antibiotics upon in vitro growth. However, our knowledge on antibiotic production by soil bacilli evolving on roots in natural conditions is still limited. In this work, antibiome imaging first revealed that the lipopeptide surfactin is the main bacterial ingredient produced in planta within the first hours of interaction with root tissues. We further demonstrated that surfactin synthesis is specifically stimulated upon perception of plant cell wall polymers such as xylan or arabinogalactan, leading to fast accumulation of micromolar amounts in the root environment. At such concentrations, the lipopeptide may not only favour the ecological fitness of the producing strain in term of root colonization, but also triggers systemic resistance in the host plant. This surfactin-induced immunity primes the plant to better resist further pathogen ingress, and involves only limited expression of defence-related molecular events and does not provoke seedling growth inhibition. By contrast with the strong response mounted upon perception of pathogens, this strongly attenuated defensive reaction induced by surfactin in plant tissues should help Bacillus to be tolerated as saprophytic partner by its host. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd. [less ▲]

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See detailLipopeptides as main ingredients for inhibition of fungal phytopathogens by Bacillus subtilis/amyloliquefaciens
Cawoy, H.; Debois, Delphine ULg; Franzil, Laurent ULg et al

in Microbial Biotechnology (2015), 8(2), 281-295

Summary: Some isolates of the Bacillus subtilis/amyloliquefaciens species are known for their plant protective activity against fungal phytopathogens. It is notably due to their genetic potential to form ... [more ▼]

Summary: Some isolates of the Bacillus subtilis/amyloliquefaciens species are known for their plant protective activity against fungal phytopathogens. It is notably due to their genetic potential to form an impressive array of antibiotics including non-ribosomal lipopeptides (LPs). In the work presented here, we wanted to gain further insights into the relative role of these LPs in the global antifungal activity of B.subtilis/amyloliquefaciens. To that end, a comparative study was conducted involving multiple strains that were tested against four different phytopathogens. We combined various approaches to further exemplify that secretion of those LPs is a crucial trait in direct pathogen ward off and this can actually be generalized to all members of these species. Our data illustrate that for each LP family, the fungitoxic activity varies in function of the target species and that the production of iturins and fengycins is modulated by the presence of pathogens. Our data on the relative involvement of these LPs in the biocontrol activity and modulation of their production are discussed in the context of natural conditions in the rhizosphere. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. [less ▲]

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See detailMALDI Mass Spectrometry Imaging: a new tool to decipher the antibiome of Bacillus amyloliquefaciens
Debois, Delphine ULg; Jourdan, Emmanuel; Cawoy, Hélène et al

Conference (2014, June 05)

Soil Bacillus isolates may devote up to 8% of their genome to nonribosomal synthesis of lipopeptide (LP)- and polyketide (PK)-type antibiotics. LPs from surfactin, iturin and fengycin families are known ... [more ▼]

Soil Bacillus isolates may devote up to 8% of their genome to nonribosomal synthesis of lipopeptide (LP)- and polyketide (PK)-type antibiotics. LPs from surfactin, iturin and fengycin families are known to exert different actions on the wellness of the producing strain such as fungitoxicity (iturin, fengycin) or motility, root colonization and immune stimulating agent (surfactin). Nevertheless, few is reported about the actual antibiome secreted in situ by Bacillus cells during confrontation with phytopathogens or plant root colonization. We developed a method mimicking the conditions prevailing in the rhizosphere and, taking advantage of the versatility of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging, we were able to localize and identify antibiotics produced in situ by bacterial cells. First, we applied this new methodology to bioassays in which Bacillus amyloliquefaciens 98S were grown together with Fusarium oxysporum, with the aim of deciphering the role of the different LP families during the phytopathogen growth inhibition. Our results showed that the three LP families were readily produced in different proportions. Especially, images of surfactins, iturins and fengycins demonstrated that iturins are the antibiotic family actually involved in the antagonism against Fusarium oxysporum. In a second approach, we used a “in planta” model in which Bacillus amyloliquefaciens S499 was simultaneously grown with Tomato and Arabidopsis thaliana roots. Imaging results, obtained during a time course analysis, showed that surfactin is always the major lipopeptide detected. In further experiments involving a refined time-window, we observed that surfactin is actually produced as soon as 24h post inoculation. These results were the starting point of a wider study showing that the early accumulation of surfactin is a complex phenomenon involving, among other mechanisms, cell-well components recognition by bacteria, and that this interaction is a win-win association for both plant and bacterial cells. [less ▲]

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See detailSpatiotemporal monitoring of the antibiome secreted by Bacillus biofilms on plant roots using MALDI mass spectrometry imaging
Debois, Delphine ULg; Jourdan, Emmanuel; Smargiasso, Nicolas ULg et al

in Analytical Chemistry (2014), 86(9), 4431-4438

Some soil Bacilli living in association with plant roots can protect their host from infection by pathogenic microbes and are therefore being developed as biological agents to control plant diseases. The ... [more ▼]

Some soil Bacilli living in association with plant roots can protect their host from infection by pathogenic microbes and are therefore being developed as biological agents to control plant diseases. The plant protective activity of these bacteria has been correlated with the potential to secrete a wide array of antibiotic compounds upon growth as planktonic cells in isolated cultures under laboratory conditions. However, in situ expression of these antibiotics in the rhizosphere where bacterial cells naturally colonize root tissues is still poorly understood. In this work, we used Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) to examine spatio-temporal changes in the secreted antibiome of B. amyloliquefaciens developing as biofilms on roots. Non-ribosomal lipopeptides such as the plant immunity elicitor surfactin or the highly fungitoxic iturins and fengycins were readily produced albeit in different time-frames and quantities in the surrounding medium. Interestingly, MS/MS experiments performed directly from the gelified culture medium, also allowed to identify a new variant of surfactins released at later time points. However, no other bioactive compounds such as polyketides were detected at any time, strongly suggesting that the antibiome expressed in planta by B. amyloliquefaciens does not reflect the vast genetic arsenal devoted to the formation of such compounds. This first dynamic study reveals the power of MALDI MSI as tool to identify and map antibiotics synthesized by root-associated bacteria and more generally, to investigate plant-microbe interactions at the molecular level. [less ▲]

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See detailImaging MS: strategies for the identification of analytes
Debois, Delphine ULg; Smargiasso, Nicolas ULg; Jourdan, Emmanuel et al

Scientific conference (2014, April 04)

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See detailOrganized proteomic heterogeneity in colorectal liver metastases and implications for therapies
Turtoi, Andrei ULg; Blomme, Arnaud ULg; Debois, Delphine ULg et al

in Acta Gastro-Enterologica Belgica (2014, March), 77(1), 07

Introduction : Tumor heterogeneity is a major obstacle for developing effective anti-cancer treatments. Recent studies have pointed at large stochastic genetic heterogeneity within cancer lesions, where ... [more ▼]

Introduction : Tumor heterogeneity is a major obstacle for developing effective anti-cancer treatments. Recent studies have pointed at large stochastic genetic heterogeneity within cancer lesions, where no pattern seems to exist that would enable a more structured targeted therapy approach. Aim : Because to date no similar information is available at the protein (phenotype) level, we aimed at characterising the proteomic heterogeneity in human colorectal carcinoma (CRC) liver metastases. Methods & Results : We employed MALDI imaging-guided proteomics and explored the heterogeneity of extracellular distribution of over 1000 proteins we found unexpectedly that all liver metastasis lesions displayed a reproducible, zon- ally delineated, pattern of functional and therapeutic biomarker heterogeneity. Peritumoral region featured elevated lipid metabolism and protein synthesis, the rim of the metastasis displayed increased cellular growth, movement and drug metabolism whereas the center of the lesion was characterized by elevated carbohydrate metabolism and DNA- repair activity. From the aspect of therapeutic targeting zonal expression of known and novel biomarkers was evident, reinforcing the need to select several targets in order to achieve optimal coverage of the lesion. Finally we highlight two novel antigens, LTBP2 and TGFBI, whose expression is a consistent feature of CRC liver metastasis. Conclusions : proteome heterogeneity has a distinct, organized, pattern. This particular hallmark can now be used as a part of the strategy for developing rational therapies based on multiple sets of targetable antigens. [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 detailTandem MS of -new- antibiotics from Bacillus guided by MALDI Mass Spectrometry Imaging
Debois, Delphine ULg; Jourdan, Emmanuel; Cawoy, Hélène ULg et al

Conference (2013, December 05)

Generally, an antibiotic is thought to have a role in antagonism simply because the producing strain is known to exhibit a potential for pathogen growth inhibition. Some genetic approaches such as PCR ... [more ▼]

Generally, an antibiotic is thought to have a role in antagonism simply because the producing strain is known to exhibit a potential for pathogen growth inhibition. Some genetic approaches such as PCR using specific primers or genome mining using known sequence data of close relatives are also used. Nevertheless, none of these methods allows stating for a link between a specific compound and the observed antagonism. Yet MALDI Mass Spectrometry Imaging (MSI) is a powerful tool to decipher the chemical messengers exchanged by two protagonists [1,2,3;]. Tandem mass spectrometry (MS/MS) may be also used, either on extracts [2,3] or directly on the microbial colonies [4]. The presentation will thus be focused on two examples of application of MALDI MSI combined to in situ tandem mass spectrometry. The first presented case will be the antagonism between soilborne strain Paenibacillus polymyxa Pp56 and the fungal phytopathogen Fusarium oxysporum. Using MALDI MSI, we were able to precisely localize each detected antibiotic, allowing discriminating which LI-F lipopeptides (fusaricidin) were really active against the pathogen progression. Besides, the use of in situ MS/MS allowed us to sequence the peptide moiety of several LI-F lipopeptides, showing that some of them are actually a mixture of several forms. The second example concerns the metabolites that are released by Bacillus amyloliquefaciens S499 cells following their inoculation on 7 days old tomato roots. We developed specific bioassays for time-course monitoring by MALDI MSI. First analyses revealed an efficient secretion of surfactin by Bacillus cells after 3 days when colonization as biofilm-structured populations is well established. Even if the composition of antibiotic mixture does not greatly evolve over time, after long incubation periods (32 or 35 days post inoculation), new series of compounds are detected in the tomato root -surrounding medium. Structural analysis based on exact mass measurements and MS/MS experiments, performed directly on the semi-solid agar medium, allowed us to identify these compounds as new variants of surfactins. [1] Barger, S., et al., Anton Leeuw Int J G, 2012, 102, 435-445. [2] Hoefler, B. C., et al,. Natl Acad Sci USA, 2012, 109, 13082-13087. [3] Moree, W. J., et al., Natl Acad Sci USA, 2012, 109, 13811-13816. [4] Debois, D., et al., J Am Soc Mass Spectrom. 2013, 24, 1202-1213 [less ▲]

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See detailUse of 1,5-diaminonaphthalene to combine matrix-assisted laser desorption/ionization in-source decay fragmentation with hydrogen/deuterium exchange
Lemaire, Pascale; Debois, Delphine ULg; Smargiasso, Nicolas ULg et al

in Rapid Communications in Mass Spectrometry [=RCM] (2013), 27(16), 1837-1846

In-Source Decay (ISD) in Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry is a fast and easy top-down activation method. Our objective is to find a suitable matrix to locate the ... [more ▼]

In-Source Decay (ISD) in Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry is a fast and easy top-down activation method. Our objective is to find a suitable matrix to locate the deuterons following in-solution hydrogen/deuterium exchange (HDX). This matrix must circumvent the commonly encountered undesired back-exchange reactions, in order to preserve the regioselective deuteration pattern. The 1,5-diaminonaphthalene (1,5-DAN) matrix is known to be suitable for MALDI-ISD fragmentation. MALDI Mass Spectrometry Imaging (MSI) was employed to compare 1,5-DAN and other commonly used MALDI matrices with respect to the extent of back-exchange and the uniformity of the H/D exchange profiles within the MALDI spots. We tested the back-exchange on the highly sensitive amyloid-beta peptide (1-40), and proved the regioselectivity on ubiquitin and b-endorphin. MALDI-MSI results show that 1,5-DAN leads to the least back-exchange over all the spot. MALDI-ISD fragmentation combined with H/D exchange using 1,5-DAN matrix was validated by localizing deuterons in native ubiquitin. Results agree with previous data obtained by Nuclear Magnetic Resonance (NMR) and Electron Transfer Dissociation (ETD). Moreover, 1,5-DAN matrix was used to study the H/D exchange profile of the methanol-induced helical structure of b-endorphin, and the relative protection can be explained by the polarity of residues involved in hydrogen bond formation. We found that controlling crystallization is the most important parameter when combining H/D exchange with MALDI. The 1,5-DAN matrix is characterized by a fast crystallization kinetics, and therefore gives robust and reliable H/D exchange profiles using MALDI-ISD. [less ▲]

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See detailMALDI-FTICR MS Imaging as a Powerful Tool to Identify Paenibacillus Antibiotics Involved in the Inhibition of Plant Pathogens
Debois, Delphine ULg; Ongena, Marc ULg; Cawoy, Hélène ULg et al

in Journal of the American Society for Mass Spectrometry (2013), 24(8), 1202-1213

Nowadays, microorganisms are more and more often used as biocontrol agents for crop protection against diseases. Among them, bacteria of Bacillus and Paenibacillus genders are already used as commercial ... [more ▼]

Nowadays, microorganisms are more and more often used as biocontrol agents for crop protection against diseases. Among them, bacteria of Bacillus and Paenibacillus genders are already used as commercial biocontrol agents. Their mode of action is supposed to be related to their production of antibiotics, such as cyclic lipopeptides, which exhibit great antimicrobial activities. We chose to work with a Paenibacillus polymyxa strain (Pp56) very resistant to various microorganisms. The bacteria were grown simultaneously with Fusarium oxysporum and we applied matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry to identify the antibiotics compounds present in the fungus growth inhibition area. We, therefore, identified fusaricidins A, B, and C and numerous members of the LI-F antibiotics family. MALDIFTICR mass spectrometry imaging was then used to follow the diffusion of lipopeptides involved in the inhibitory activity over time. We analyzed the molecular content of the inhibitory area at different Pp56 and Fusarium incubation durations and concluded that some lipopeptides such as fusaricidin B and a mixture of LI-F05b/06b/08a were mainly involved in the defense mechanism of Pp56. Our study confirms that MALDI imaging may be a powerful tool to quickly determine which molecular species is involved in an antagonism with another microorganism, avoiding time-consuming steps of extraction, purification, and activity tests, which are still commonly used in microbiology. [less ▲]

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See detailDistribution and identification of molecular interactions between tomato roots and bacterial biofilms
Debois, Delphine ULg; Jourdan, Emmanuel; Smargiasso, Nicolas ULg et al

Poster (2013, June 12)

Some non-pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in ... [more ▼]

Some non-pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in soil (1). To initiate both phenomena leading to biocontrol activity, microorganisms use plant exudates to grow on roots and to produce in-situ active compounds. In Bacilli, cyclic lipopeptides of the surfactin, iturin and fengycin families represent important antibiotics involved in biocontrol (2). Recent studies in microbiology allowed a better understanding of plant microorganism interactions but few has been done at the molecular level. In this study, MALDI MS imaging has been used to study the nature of the secreted lipopeptide molecules, their relative quantity and their distribution in the root’s environment.Disinfected tomato seeds were first germinated at 28°C in sterile conditions for germination. Seedlings were then placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution containing 1,75% of agar and treated with freshly-grown cells of Bacillus amyloliquefaciens S499. Petri dishes were incubated at 28°C with a 16h photoperiod. Different growth / incubation durations were studied: 10/3; 13/7; 21/14 and 39/32. For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. (HCCA, 5mg/mL in ACN/0.2% TFA 70:30) was used as matrix. UltraFlex II TOF/TOF and Solarix FT-ICR mass spectrometers were used to record molecular cartographies and perform MS/MS experiments for structural analysis purposes. The average mass spectra recorded around the tomato root (2-3 mm on both sides of the root) showed that lipopeptides were major compounds detected on the agar. The relative intensity of lipopeptides families varied with respect to the age of the root/biofilm system. In the 10/3 system, 3 homologues of surfactins were essentially detected (C13, C14 and C15), with very few iturins and fengycins. Their localizations were identical, whatever the considered homologue. Then the production of iturin and fengycin families increases in older systems (13/7 and 21/14) and a novel homologue of surfactin is detected (C12). Some variations in localizations within families may be observed (around the root or at the close vicinity of it in function of the considered homologue or alkali adduct). Then for the oldest system we studied, iturins and fengycins are not detected anymore and the localization of surfactins is less precise. In the 39/32 system, we also detected unknown compounds at 986.6, 1000.6, 1014.7 and 1028.7 m/z. The mass range of these compounds allied to the mass difference between two consecutive ion peaks let us think that these unknown compounds could be a new lipopeptide family. Tandem mass spectrometry experiments, performed on the dried culture medium, allowed to partially sequence these new lipopeptides. MS/MS results allied to exact mass measurements and isotopic pattern simulation give good confidence in the chemical structure we suggest. Nevertheless, to fully identify these new variants of surfactin, micro-extractions followed by (LC)-nano-ESI-MS/MS using a LESA module are in progress. MALDI Mass Spectrometry Imaging becomes a tool to decipher inter-species molecular communication. [less ▲]

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See detailMALDI In-Source Decay, from sequencing to imaging
Debois, Delphine ULg; Smargiasso, Nicolas ULg; Demeure, Kevin ULg et al

in Topics in Current Chemistry (2013), 331

MALDI is now a mature method allowing the identification and, more challenging, the quantification of biopolymers (proteins, nucleic acids, glycans…). MALDI spectra show mostly intact singly charged ions ... [more ▼]

MALDI is now a mature method allowing the identification and, more challenging, the quantification of biopolymers (proteins, nucleic acids, glycans…). MALDI spectra show mostly intact singly charged ions. To obtain fragments, the activation of singly charged precursors is necessary, but not efficient above 3.5 kDa thus making MALDI MS/MS difficult for large species. In-source decay (ISD) is a prompt fragmentation reaction that can be induced thermally or by radicals. As fragments are formed in the source, precursor ions cannot be selected; however, the technique is not limited by the mass of the analyzed compounds and pseudo MS/MS can be performed on intense fragments. The discovery of new matrices that enhance the ISD yield, combined with the high sensitivity of MALDI mass spectrometers, and software development, opens new perspectives. We first review the mechanisms involved in the ISD processes, then discuss ISD applications like top-down sequencing and post-translational modifications studies, and finally review MALDI-ISD tissue imaging applications. [less ▲]

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See detailTowards Lipidomics of Low-Abundant Species for Exploring Tumor Heterogeneity Guided by High-Resolution Mass Spectrometry Imaging
Cimino, Jonathan ULg; Calligaris, David; Far, Johann ULg et al

in International Journal of Molecular Sciences (2013), 14

Many studies have evidenced the main role of lipids in physiological and also pathological processes such as cancer, diabetes or neurodegenerative diseases. The identification and the in situ localization ... [more ▼]

Many studies have evidenced the main role of lipids in physiological and also pathological processes such as cancer, diabetes or neurodegenerative diseases. The identification and the in situ localization of specific low-abundant lipid species involved in cancer biology are still challenging for both fundamental studies and lipid marker discovery. In this paper, we report the identification and the localization of specific isobaric minor phospholipids in human breast cancer xenografts by FTICR MALDI imaging supported by histochemistry. These potential candidates can be further confirmed by liquid chromatography coupled with electrospray mass spectrometry (LC-ESI-MS) after extraction from the region of interest defined by MALDI imaging. Finally, this study highlights the importance of characterizing the heterogeneous distribution of low-abundant lipid species, relevant in complex histological samples for biological purposes. [less ▲]

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See detailSelected Protein Monitoring in Histological Sections by Targeted MALDI-FTICR in-source decay Imaging.
Calligaris, David ULg; Longuespée, Rémi ULg; Debois, Delphine ULg et al

in Analytical Chemistry (2013), 85(4), 2117-26

MALDI mass spectrometry imaging (MALDI MSI) is a rapidly growing method in biomedical research allowing molecular mapping of proteins on histological sections. The images can be analyzed in terms of ... [more ▼]

MALDI mass spectrometry imaging (MALDI MSI) is a rapidly growing method in biomedical research allowing molecular mapping of proteins on histological sections. The images can be analyzed in terms of spectral pattern to define regions of interest. However, the identification and the differential quantitative analysis of proteins require off line or in situ proteomic methods using enzymatic digestion. The rapid identification of biomarkers holds great promise for diagnostic research but the major obstacle is the absence of rapid and direct method to detect and identify with a sufficient dynamic range a set of specific biomarkers. In the current work, we present a proof of concept for a method allowing identifying simultaneously a set of selected biomarkers on histological slices with minimal sample treatment using in-source decay (ISD) MSI and MALDI-Fourier transform ion cyclotron resonance (FTICR). In the proposed method, known biomarkers are spotted next to the tissue of interest, the whole MALDI plate being coated with 1,5-DAN matrix. The latter enhances MALDI radical-induced ISD, providing large tags of the amino acid sequences. Comparative analysis of ISD fragments between the reference spots and the specimen in imaging mode allows for unambiguous identification of the selected biomarker while preserving full spatial resolution. Moreover, the high resolution/high mass accuracy provided by FTICR mass spectrometry allows the identification of proteins. Well-resolved peaks and precise measurements of masses and mass differences allow the construction of reliable sequence tags for proteins identification. The method will allow the use MALDI-FTICR MSI as method for rapid targeted biomarker detection in complement to classical histology. [less ▲]

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See detailSéminaire des chercheurs Télévie 2013
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, December 10)

Séminaire des chercheurs Télévie 2013

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See detailNew Advances for In Situ Protein Identification by MALDI In-Source Decay FTMS Imaging
Calligaris, David; Longuespée, Rémi ULg; Zimmerman, Tyler et al

Poster (2012, November)

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See detailDetermination of the molecular players of adaptation to anti-angiogenic therapy in breast cancer by quantitative proteomic and high molecular MALDI Imaging.
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, October 13)

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive ... [more ▼]

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing antibodies (bevacizumab) and VEGF receptor tyrosine kinase inhibitors (RTKs:Sunitinib). The Sunitinib has been used clinically in patients with breast cancer refractory to other therapeutic agents. Unfortunately, like the cytotoxic therapies, these drugs do not produce lasting effects and resistance to treatment appeared clinically. Questions have emerged about the failure of anti-angiogenic therapy in clinic and the limitations of predictive preclinical models, and also about the molecular assessment of all stages of tumor adaptation and me<x>tastatic disease. To this end, we applied quantitative proteomics and imaging mass spectrometry tools to visualize and study the profiles of proteins and small molecules associated with tumor treated or not with Sunitinib using a novel preclinical model of breast carcinoma cells. In this project, we first developed a reproducible model of resistance to Sunitinib of human triple negative breast cancer MDA-MB-231 cells expressing luciferase gene. Cells were subcutaneously injected into mice RAG1-/- and divided into four experimental groups including, control mice treated with vehicle or Sunitinib for 30 days and sacrificed 1 days after treatment withdrawal or when tumor reached a volume of 300 mm3. In the second step. Tumors were analyzed using a nanoAcquity UPLC Synapt TM HDMS TM G1 (Waters, Manchester,UK) and Mass Spectrometry Imaging. For quantitative proteomic analyses of tumors, a bioinformatics analysis was used with the Protein lynx global server 2.2.5 software. Imaging mass spectrometry was performed on tissue sections of tumors and organs subsequently colonized by me<x>tastases. Matrix sublimation was used to coat tumor sections (14 µm-tick) with 1.5 Diaminonaphthalene for lipids analysis and Sinapinic acid for entire proteins analysis. Ion cartographies were recorded with a Solarix 9.4T FTMS instrument for lipids and with an Ultraflex II TOF-TOF instrument for entire proteins (Bruker Daltonics, Germany) with a spatial resolution of 100 µm. Global protemic revealed different protein profiles between tumor treated or not with Sunitinib. The Mass Spectrometry Imaging detected differences in intensity and location of some proteins and lipids are also associated with some histological features including inflammatory, necrotic and angiogenic areas. Bioinformatics analysis will be applied to ensure the integration of all data in order to provide the basis for identifying molecular pathways activated during the acquisition of refractoriness to drug treatments. [less ▲]

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See detailDistribution and identification of molecular interactions between tomato roots and bacterial biofilms
Debois, Delphine ULg; Jourdan, Emmanuel ULg; Ongena, Marc ULg et al

Conference (2012, September 05)

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in ... [more ▼]

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in soil (1). To initiate both phenomena leading to biocontrol activity, microorganisms use plant exudates to grow on roots and to produce in-situ active compounds. In Bacilli, cyclic lipopeptides of the surfactin, iturin and fengycin families represent important antibiotics involved in biocontrol (2). Recent studies in microbiology allowed a better understanding of plant microorganism interactions but few has been done at the molecular level. In this study, MALDI MS imaging has been used to study the nature of the secreted lipopeptide molecules, their relative quantity and their distribution in the root’s environment. Disinfected tomato seeds were first germinated at 28°C in sterile conditions for germination. Seedlings were then placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution (Hoagland) containing 1,75% of agar and treated with freshly-grown cells of Bacillus amyloliquefaciens S499. Petri dishes were finally incubated vertically in phytotron at 28°C with a 16h photoperiod. Different root age / time of incubation were studied: 13 / 3; 13 / 7; 21 / 14 and 39 / 32. Control tomato root (without bacterial treatment) of the same ages were also analyzed (13 / 0; 21 / 0 and 42 / 0. For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. The matrix solution (α-cyano-hydroxycinnamic acid, 5mg/mL in ACN/0.2% TFA 70/30) was applied with an ImagePrep automated sprayer (Bruker Daltonics). An UltraFlex II TOF/TOF and a Solarix FT-ICR mass spectrometers were used to record molecular cartographies. The average mass spectra recorded around the tomato root (2-3 mm on both sides of the root) showed that lipopeptides were major compounds detected on the agar. The relative intensity of lipopeptides families varied with respect to the age of the root/biofilm system. In the 13/3 system, 3 homologues of surfactins were essentially detected (C13, C14 and C15), with very few iturins and fengycins. Their localizations were identical, whatever the considered homologue. Then the production of iturin and fengycin families increases in older systems (13/7 and 21/14) and a novel homologue of surfactin is detected (C12). Some variations in localizations within families may be observed (around the root or at the close vicinity of it in function of the considered homologue or alkali adduct). Then for the oldest system we studied, iturins and fengycins are not detected anymore and the localization of surfactins is less precise. In the 39/32 system, we also detected unknown compounds at 986.6, 1000.6, 1014.7 and 1028.7 m/z. The mass range of these compounds allied to the mass difference between two consecutive ion peaks let us think that these unknown compounds could be a new lipopeptide family. Investigations are in progress to identify these new secondary metabolites of Bacillus amyloliquefaciens. [less ▲]

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