Utilisation des termites comme source de microorganismes dans la filière de production du bioéthanol de seconde génération

Poster (2012, November 14)

Les termites abritent une microflore symbiotique qui intervient dans la dégradation des fibres constitutives du bois, synthétisant des enzymes capables d’hydrolyser ses composants. Les sucres ... [more ▼]

Les termites abritent une microflore symbiotique qui intervient dans la dégradation des fibres constitutives du bois, synthétisant des enzymes capables d’hydrolyser ses composants. Les sucres fermentescibles libérés suite à cette hydrolyse sont utilisables dans le cadre de la production du bioéthanol de seconde génération. [less ▲]

Determination of the molecular players of adaptation to anti-angiogenic therapy in breast cancer by quantitative proteomic and high molecular MALDI Imaging.

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 ▲]

Development of a quantitative approach to measure phospholipids in dried drops by Raman spectroscopy

Poster (2012, October)

We present here the results obtained during our tentative to analyse quantitatively dried drops of phospholipidic solutions by Raman spectroscopy. Drops of different solutions of phospholipid were deposed ... [more ▼]

We present here the results obtained during our tentative to analyse quantitatively dried drops of phospholipidic solutions by Raman spectroscopy. Drops of different solutions of phospholipid were deposed onto different material supports. The spots were then analyses by confocal Raman microspectroscopy. Experimental settings have been optimised and the analysis of the intensity profile of the Raman signal inside the spot allows the establishment of a calibration curve for the determination of the phospholipids amount within a 1 µL solution. [less ▲]

Distribution and identification of molecular interactions between tomato roots and bacterial biofilms

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 ▲]

Study  of  breast  cancer  adaptation  to  anti-­angiogenic  therapies  by   molecular  imaging  on  tissue  slides

Conference (2012, September 04)

Breast   carcinoma   is   the   most   common   and   second   leading   cause   of   cancer   mortality   in   women1.   The   ␣␣␣␣␣␣␣␣␣␣␣␣ ␣␣␣ ␣␣␣␣ ␣␣␣␣␣␣␣␣␣␣␣␣ ␣␣␣␣␣␣␣␣ ␣␣␣ ␣␣ ␣␣␣␣-­‐limiting   ... [more ▼]

Breast   carcinoma   is   the   most   common   and   second   leading   cause   of   cancer   mortality   in   women1.   The   ␣␣␣␣␣␣␣␣␣␣␣␣ ␣␣␣ ␣␣␣␣ ␣␣␣␣␣␣␣␣␣␣␣␣ ␣␣␣␣␣␣␣␣ ␣␣␣ ␣␣ ␣␣␣␣-­‐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   agents2.   Unfortunately,   like   the   cytotoxic   therapies,   these   drugs   do   not   produce   lasting   effects   and   resistance   to   treatment   appeared   clinically3.   Recently,   independent   laboratories   have   reported   experimental   data   demonstrating   that   anti-­‐ angiogenic   treatments   inhibit   tumor   growth,   but   also   stimulate   the   formation   of   lung   metastases   after   treatment   discontinuation4.   The   field   of   imaging   mass   spectrometry   provides   new   tools   to   visualize   and   study  the  profiles  of  proteins  and  small  molecules  associated  with  biomedical  problems5.   To  this  aim,  we  conducted  a  series  of  experiments  to  setup  a  reproductible  model  of  resistance  to  sunitinib.   The   cells   MDA-­‐MB-­‐231   triple   negative,   from   human   breast   cancer   and   expressing   luciferase   are   injected   subcutaneously  into  mice  RAG1-­‐/-­‐.  The  mice  were  divided  into  four  experimental  groups  including,  on  the   one  hand,  control  mice  treated  with  placebo  (Carboxymethyl  cellulose,  CMC)  sacrificed  on  day  30  (group  1)   or  when  the  tumor  reached  a  volume  of  300  mm3  (group  2).    On  the  other  hand,  Sunitinib-­‐treated  mice  (LC   Laboratories,   40mg/kg/day),   sacrificed   at   day   30   (group   3),   or   when   the   tumor   reached   a   volume   of   300   mm3  (group  4).  MALDI  mass  spectrometry  imaging  was  performed  on  tissue  sections  of  tumors  and  organs   subsequently   colonized   by   metastases.   Matrix   sublimation   was   used   to   coat   tumor   sections   (14   μm-­‐tick)   with   1.5   Diaminonaphthalene   (1.5   DAN)   for   lipids   analysis   and   Sinapinic   acid   (SA)   for   entire   proteins   analysis.   Ion   cartographies   were   recorded   with   a   Solarix9.4T   FTMS   instrument   for   lipids   and   with   an   Ultraflex   II   TOF-­‐TOF   instrument   for   entire   proteins   (BrukerDaltonics,   Bremen,   Germany)   with   a   spatial   resolution  of  100  μm.     The  analysis  of  differential  protein/lipid  profiles  with  high  mass  accuracy  and  broadband  resolution  allows   detection   of   intense   signals   from   lipid   families   such   as   Phosphatidylcholine   (PC),   Triglyceride   (TAG),   Sphingomyelin   (SM)   and   precise   lipid   droplets   or   tumor   cells   differentiated   location   in   the   Sunitinib   resistant   tumor   cells   compared   to   control   cells.The   protein   profiles   of   the   4   groups   of   mice   show   differences   in   intensity   and   location,   enabling   a   correlation   to   inflammatory   (highlighted   by   histological   staining)  and  angiogenic  phenomenon.   [less ▲]

Quantification of Acrylamide in Various Belgian Potato Products Using Solid Phase Extraction and Liquid Chromatography Tandem Mass Spectrometry Detection

in Food and Public Health (2012), 2(5), 137-141

Acrylamide (CH2=CHCONH2), a neurotoxic and potentially carcinogenic substance for human health, is in the glare of the spotlights for a few years. This is mostly due to the fact that acrylamide was found ... [more ▼]

Acrylamide (CH2=CHCONH2), a neurotoxic and potentially carcinogenic substance for human health, is in the glare of the spotlights for a few years. This is mostly due to the fact that acrylamide was found worldwide in various heated foodstuffs. Levels reported in the literature vary from 25 to 2000 ìg/kg and potato products are considered as the most contaminated. A possible pathway of synthesis of acrylamide is the Maillard reaction between reducing sugars and the amino acid asparagine. The aim of this study was to develop a liquid chromatography/mass spectrometry method to analyse as quickly as possible acrylamide in a variety of Belgian food samples such as potatoes, French fries, crisp bread, coffee, corn-flakes, etc. The sample preparation consisted in a liquid/liquid extraction, a centrifugation, followed by purification with Solid Phase Extraction (SPE). The instruments used were a Waters 2690 Alliance HPLC system coupled to a Micro-mass Quattro Ultima Platinum triple-quadrupole mass spectrometer. The analysis was performed in MS/MS mode using isotopic dilution technique for quantification. An internal 13C3 labelled standard was added prior to extraction. Quantifica-tion in MS/MS mode was calculated by reconstructing the ion current with the most abundant daughter ions for native and 13C labelled standard (ions of m/z 55 and 58). [less ▲]

Identification and Relative-quantification of Glycans by Matrix-assisted Laser Desorption/Ionization In-Source Decay with Hydrogen Abstraction

in Analytical Chemistry (2012)

The use of specific matrices allows enhancing the scope of in-source decay (ISD) applications in matrix-assisted laser desorption ionization (MALDI) thanks to the specificity of analyte-matrix chemistry ... [more ▼]

The use of specific matrices allows enhancing the scope of in-source decay (ISD) applications in matrix-assisted laser desorption ionization (MALDI) thanks to the specificity of analyte-matrix chemistry. The use of an oxidizing matrix, 5-nitrosalicylic acid (5-NSA) for MALDI-ISD of glycans is shown to promote fragmentation pathways involving radical precursors. Both glycosidic and cross-ring cleavages are promoted by hydrogen abstraction from hydroxyl group of glycans by 5-NSA molecules. Cross-ring cleavage ions are potentially useful in linkage analysis, one of the most critical steps of glycan characterization. Moreover, we show here that isobaric glycans could be distinguished by structure specific ISD ions, and that the molar ratio of glycan isomers in the mixture can be estimated from their fragment ions abundance. The use of 5-NSA also opens the possibility to perform pseudo-MS3 analysis of glycans. Therefore, MALDI-ISD with 5-NSA is a useful method for identification of glycans and semi-quantitative analysis of mixture of glycan isomers. [less ▲]

Risk assessment for furan contamination through the food chain in Belgian children

in Food Additives & Contaminants (2012), 29(8), 1219-1229

Young, old, pregnant and immuno-compromised persons are of great concern for risk assessors as they represent the sub-populations most at risk. The present paper focuses on risk assessment linked to furan ... [more ▼]

Young, old, pregnant and immuno-compromised persons are of great concern for risk assessors as they represent the sub-populations most at risk. The present paper focuses on risk assessment linked to furan exposure in children. Only the Belgian population was considered because individual contamination and consumption data that are required for accurate risk assessment were available for Belgian children only. Two risk assessment approaches, so called deterministic and probabilistic, were applied and their results were compared for the estimation of the daily intake. A significant difference between the average Estimated Daily Intake (EDI) was underlined between the deterministic (419 ng * (kgb.w. * day)-1) and the probabilistic (583 ng * (kgb.w. * day)-1) approaches, which results from the mathematical treatment of the null consumption and contamination data. The risk was characterized by two ways: (1) the classical approach by comparison of the EDI to a reference dose (RfDchronic-oral) and (2) the most recent approach, namely the Margin of Exposure (MoE) approach. Both reached similar conclusions: the risk level is not of a major concern, but is neither negligible. In the first approach, only 2.7% or 6.6% (respectively in the deterministic and in the probabilistic way) of the studied population presented an EDI above the RfDchronic-oral. In the second approach, the percentage of children displaying a MoE above 10,000 and below 100 is 3% - 0% and 20% - 0.01% in the deterministic and probabilistic modes respectively. In addition, children were compared to adults and significant differences between the contamination patterns were highlighted. Whilst major contamination was linked to coffee consumption in adults (55%), no item predominantly contributed to the contamination in children. The most important were soups (19%), dairy products (17%), pasta and rice (11%), fruit and potatoes (9% each). [less ▲]

Termites artificially-fed on unusual diet and resulting enzymatic switches

Poster (2012, August)

Wood-feeding termites as Reticulitermes santonensis generally feed on cellulose, hemicelluloses and lignin. However, these opportunistic insects are also able to degrade other carbohydrates, such as ... [more ▼]

Wood-feeding termites as Reticulitermes santonensis generally feed on cellulose, hemicelluloses and lignin. However, these opportunistic insects are also able to degrade other carbohydrates, such as starch. The production of putative endogenous α - amylase has been previously shown in R. flavipes, as the disappearance of the major symbiotic flagellates from the hindgut. Here, we compared enzymatic activities (CMCase, MCCase, xylanase, amylase, α- and β-glucosidase) between different fractions of the digestive tract of starch-, cellulose-, and wood-fed termites. Main compounds of the artificial diets, namely starch or MCC, resulted in differential enzymatic activity. Even the substitution of wood by artificial diets itself seemed to induce changes in enzymatic activities, regardless of the main substrate in the diet, as we observed strong midgut α-glucosidase activity only for artificially-fed termites. Preliminary assays to isolate and characterize enzymes were performed using proteomic methods. [less ▲]

Selection and cultivation of hydrolytic microorganisms extracted from the digestive tract of the termite Reticulitermes santonensis (3DV.1.55)

Poster (2012, June 21)

Biofuel production can be based on the use of fermentable substrates issued from the hydrolysis of lignocellulosic biomass stemming from agricultural residues and by-products. However, such substrates are ... [more ▼]

Biofuel production can be based on the use of fermentable substrates issued from the hydrolysis of lignocellulosic biomass stemming from agricultural residues and by-products. However, such substrates are not easy to degrade. Enzymes (cellulases, xylanases, etc.) can be used for this purpose and pre-treatments can increase their action by providing more available extremities. The digestive tract of the termite Reticulitermes santonensis contains various microorganisms (bacteria, molds, protists) able to degrade the wood components. These microorganisms act as consortia, leading to a better hydrolysis than in the cow rumen. Our purpose is the isolation of microorganisms from termite guts in order to evaluate their potential for hydrolysis of lignocellulosic materials. This approach led us to isolate and to study a bacteria (Bacillus sp.) displaying a xylanase activity, a mold (Aspergillus sp.) displaying a cellulase activity and a chrysophyte (protist) displaying an amylase activity. [less ▲]