[Show abstract][Hide abstract] ABSTRACT: Prolonged enteral or parenteral nutrition in neonatal periods sometimes results in eating difficulties persisting for years, with reduced food intake through the oral route and thereby reduced stimulation of the oral cavity. Aiming at describing the consequences on oral physiology, saliva of 21 children with eating difficulties (ED) was compared to that of 23 healthy controls, using various omics and targeted methods. Overall, despite heterogeneity within the groups (age, medication etc.), the three spectral methods (MALDI-TOF, SELDI-TOF, (1)H NMR) allowed discriminating ED and controls, confirming that oral stimulation by food intake plays a role in shaping the composition of saliva. Saliva of ED patients exhibited a lower antioxidant status and lower levels of the salivary protease inhibitors cystatins. Other discriminant features (IgA1, dimethylamine) may relate to modified oral and/or intestinal microbial ecology. Finally, salivary profiles of ED patients were partly comparable to those of subjects with exacerbated gustatory sensitivities, in particular with reduced abundance of cystatin SN and higher abundance of zinc-alpha-2-glycoprotein. Whether this translates taste hypersensitivity and contributes to the eating difficulties deserves further attention.
Full-text · Article · Jul 2015 · Journal of proteomics
[Show abstract][Hide abstract] ABSTRACT: Stomata remain abnormally opened and unresponsive to abscisic acid in grapevine leaves infected by downy mildew. This deregulation occurs from 3 days post inoculation and increases concomitantly with leaf colonization by the pathogen. Using epidermal peels, we demonstrated that the active compound involved in this deregulation is located in the apoplast. Biochemical assays showed that the active compound present in the apoplastic fluids isolated from Plasmopara viticola infected grapevine leaves (IAF) is a CysCys bridge-independent, thermostable and glycosylated protein. Fractionation guided assays based on chromatography / stomatal response and proteomic analysis allowed the identification of both plant and pathogen proteins in the active fraction obtained from IAF. Further in silico analysis and discriminant filtrations based on the comparison between predictions and experimental indications lead to the identification of two V. vinifera proteins as candidates for the observed stomatal deregulation.
No preview · Article · Jun 2015 · Molecular Plant-Microbe Interactions
[Show abstract][Hide abstract] ABSTRACT: Abstract Saliva has different functions in the mouth and is involved, for example, in taste perception. Saliva composition can also be modified rapidly by taste stimulation. It remains unclear, however, whether the perceived intensity of a tastant may modulate this response. Based on increasing evidence that fat can be perceived by the taste system and that fat taste perception may be associated with fat intake, the aim of this work was to study if stimulation by a fatty acid (oleic acid) modifies saliva composition differently in subjects highly (sensitive+) or weakly (sensitive-) sensitive to that taste. For that purpose, saliva of two groups of subjects was collected after stimulation by either a control emulsion or an emulsion containing 5.61 mM oleic acid. Saliva was analyzed by 2D electrophoresis and (1)H NMR spectroscopy. The results show that sensitive+ and sensitive- subjects differ in their salivary response in terms of proteome and metabolome composition. Oppositely to sensitive- subjects, sensitive+ subjects responded to oleic acid by increased abundance of polymeric immunoglobulin receptor, rab GDP dissociation inhibitor beta, and organic acids, and decreased abundance of metabolites characteristic of mucins. The results highlight that modification of saliva composition by taste stimulation may be modulated by taste perception.
No preview · Article · Oct 2014 · Omics A Journal of Integrative Biology
[Show abstract][Hide abstract] ABSTRACT: High plasma concentrations of non-esterified fatty acids (NEFAs), transported bound to serum albumin, are associated with type 2 diabetes (T2D). The effects of albumin on platelet function were investigated in vitro. Modifications of albumin, such as those due to glycoxidation, were found in patients with T2D, and the consequences of these modifications on biological mechanisms related to NEFA handling were investigated. Mass spectrometry profiles of albumin from patients with T2D differed from those from healthy controls. Diabetic albumin showed impaired NEFA binding capacity, and both structural and functional alterations could be reproduced in vitro by incubating native albumin with glucose and methylglyoxal. Platelets incubated with albumin isolated from patients with T2D aggregated approximately twice as much as platelets incubated with albumin isolated from healthy controls. Accordingly, platelets incubated with modified albumin produced significantly higher amounts of arachidonate metabolites than did platelets incubated with control albumin. It is concluded that higher amounts of free arachidonate are made available for the generation of active metabolites in platelets when the NEFA binding capacity of albumin is blunted by glycoxidation. This newly described mechanism, in addition to hypoalbuminaemia may contribute to platelet hyperactivity and increased thrombosis, known to occur in patients with T2D.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Based on recent studies in adult subjects, saliva composition is increasingly considered as a physiological factor contributing to taste sensitivity or acceptance. In order to evaluate a possible link between salivary protein composition and taste acceptance in infants, 73 infants participated longitudinally in taste acceptance tests and donated saliva at the age at 3 and 6 months. Intake ratios, reflecting acceptance of a taste solution relative to water were calculated for the five basic tastes. Salivary proteins were separated by one-dimensional electrophoresis and bands were semi-quantified by image analysis. Partial least square (PLS) regression analyses were performed for each taste at both ages to explain intake ratios by band intensities. Bitterness acceptance in the younger infants was unique in the sense that salivary protein profiles could partly predict bitter taste acceptance. At that age, infants were on average indifferent to the 0.18-M urea solution, but great variability in acceptance was observed. The six bands considered as the best predictors for bitterness acceptance were identified by MALDI-TOF mass spectrometry. Higher abundance of bands containing secretory component, zinc-α-2-glycoprotein and carbonic anhydrase 6 was associated to a lower bitterness acceptance, while higher abundance of bands containing lactoperoxidase, prolactin-inducible protein and S-type cystatins was associated to a higher bitterness acceptance. In a second stage, S-type cystatin abundance was measured by Western blotting in order to tentatively confirm this particular finding in an independent group of 22 infants. Although not reaching statistical significance, probably due to a relatively small sample size, it was again observed that cystatin abundance was higher in infants accepting more readily the bitter solution over water.
saliva protein composition may contribute to bitter taste acceptance in the younger infants.
Full-text · Article · Nov 2013 · European Journal of Pediatrics
[Show abstract][Hide abstract] ABSTRACT: MOZ and MLL encoding a histone acetyltransferase and a histone methyltransferase, respectively, are targets for recurrent chromosomal translocations found in acute myeloblastic or lymphoblastic leukemia. We have previously shown that MOZ and MLL cooperate to activate HOXA9 gene expression in hematopoietic stem/progenitors cells. To dissect the mechanism of action of this complex, we decided to identify new proteins interacting with MOZ. We found that the scaffold protein Symplekin that supports the assembly of polyadenylation machinery was identified by mass spectrometry. Symplekin interacts and co-localizes with both MOZ and MLL in immature hematopoietic cells. Its inhibition leads to a decrease of HOXA9 protein level but not of Hoxa9 mRNA and to an over-recruitment of MOZ and MLL onto HOXA9 promoter. Altogether, our results highlight the role of Symplekin in transcription repression involving a regulatory network between MOZ, MLL and Symplekin.
Full-text · Article · Aug 2013 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: Saliva is a biological fluid playing numerous roles in the oral cavity and increasingly considered as a source of markers. The role of saliva in sensory perception has been known for years but it is only recently that its potential role in oral fatty acids (FA) perception has been suggested. The aim of the present work was to study the relationships between taste sensitivity to oleic acid and the salivary proteome (2D electrophoresis) and metabolome (1H NMR). This was achieved by comparing saliva from two groups of subjects, highly (sensitive+) and weakly sensitive (sensitive−) to the taste of oleic acid. Partial least squares-discriminant analyses (PLS-DA) were used to model the relationship between sensitivity to C18:1, and the proteome and metabolome data. The two groups could be discriminated by ten spots. In particular, cystatin SN, cystatin D, zinc-alpha-2-glycoprotein and carbonic anhydrase 6 were overexpressed in the sensitive+ group. The latter result was confirmed by ELISA. The overexpression of these proteins, which have been associated to taste perception, supports the argument that C18:1 is perceived by the taste system. The two groups could also be discriminated on the basis of eight metabolites, with FA, FA/proline, lysine and FA/pyruvate overexpressed in the sensitive+ group and acetate, leucine/isoleucine and butyrate overexpressed in the sensitive− group. The overexpression of these metabolites suggests a higher bacterial load in this group which could be implicated in perception of FAs.
[Show abstract][Hide abstract] ABSTRACT: L’identification conventionnelle (IC) des levures est fondée sur l’utilisation des tests morphologiques, immunologiques et biochimiques (galerie API® 32 C, bioMérieux). La spectrométrie de masse MALDI-TOF (SM) a été proposée récemment comme nouvelle approche pour l’identification des microorganismes. L’objectif de notre étude était de comparer prospectivement les performances analytiques de la SM et de l’IC pour l’identification des isolats cliniques de levures. En cas de discordance d’identification, le séquençage des régions ITS de l’ADN ribosomal était utilisé comme méthode d’identification de référence. Un total de 1 207 souches a été analysé. Une concordance entre SM et IC a été observée pour 1 105 souches (91,5 %) alors que la proportion de souches différemment identifiées par IC et SM n’était que de 6,1 % (74 souches). Parmi ces 74 identifications discordantes entre SM, l’identification par biologie moléculaire confirmait l’identification obtenue par SM pour 73 isolats (6 %) et celle obtenue par IC pour 1 isolat (0,1 %). Pour les principales espèces d’intérêt médical, les concordances d’identification entre les deux techniques étaient excellentes (entre 98 et 100 %) y compris pour les espèces phylogénétiquement proches (Candida albicans/Candida dubliniensis; Candida inconspicua/Candida norvegensis; Candida parapsilosis/Candida metapsilosis/Candida orthopsilosis). La SM n’a été mise en défaut que pour 2,3 % des souches appar tenant aux espèces Candida famata, Candida lambica, Candida magnoliae et aux genres Geotrichum sp. et Trichosporon sp. Nos investigations soulignent le potentiel de la SM pour l’identification des levures et comme alternative fiable, rapide et de moindre coût par rapport aux méthodes conventionnelles.
No preview · Article · Dec 2012 · Bio Tribune Magazine
[Show abstract][Hide abstract] ABSTRACT: Immuno-SPR-MS is the combination of immuno-sensors in biochip format with mass spectrometry. This association of instrumentation allows the detection and the quantification of proteins of interest by SPR and their molecular characterization by additional MS analysis. However, two major bottlenecks must be overcome for a wide diffusion of the SPR-MS analytical platform: (i) To warrant all the potentialities of MS, an enzymatic digestion step must be developed taking into account the spot formats on the biochip and (ii) the biological relevancy of such an analytical solution requires that biosensing must be performed in complex media. In this study, we developed a procedure for the detection and the characterization at ~1 µg/mL of the LAG3 protein spiked in human plasma. The analytical performances of this new method was established, particularly its specificity (S/N > 9) and sensitivity (100% of LAG3 identification with high significant mascot score >68 at the femtomole level). The collective and automated on-chip MALDI-MS imaging and analysis based on peptidic fragments opens numerous applications in the fields of proteomics and diagnosis.
[Show abstract][Hide abstract] ABSTRACT: The combination of Surface Plasmon Resonance technology with Mass Spectrometry becomes a key method for the characterization of targeted proteins in the fields of diagnosis and functional proteomics. We demonstrated in this work the ability of our SPRi-chip to capture targeted protein in biological fluids and in situ analyze by MS and MS/MS modes through automated procedure to go beyond classical immunoassays. Here, we established a proof of concept of SUPRA-MS for the detection, the identification and the characterization of a potential breast cancer marker.
[Show abstract][Hide abstract] ABSTRACT: In the aim to realize an efficient resonant biosensor, gallium arsenide (GaAs) presents many advantages. In addition to its properties of transduction, GaAs is a crystal for which microfabrication processes were developed, conferring the possibility to miniaturize the device and integrate electronic circuit. Moreover, the biofunctionalization could be realized on the crystalline surface without layer deposition, constituting a real advantage to perform reusable sensor. The functionalization of GaAs surface was engaged in order to immobilize a protein monolayer on this substrate. Functionalization was done using a mixed self assembled monolayer of thiolate molecules. Characterizations at micro and nanoscale were performed to control the surface state, the establishment of thiolates self-assembled monolayer, the surface atomic composition and the topography of the GaAs substrate at the different steps of the process. Protein immobilization on thiolates modified GaAs was revealed through a detailed AFM study and in situ MALDI-TOF MS and MS/MS modified surface interrogations.
No preview · Article · Aug 2012 · International Journal of Nanoscience
[Show abstract][Hide abstract] ABSTRACT: We report here that modifications of the preanalytical steps of matrix-assisted laser desorption ionization–time of flight
mass spectrometry (MALDI-TOF MS) identification of yeasts, with regard to the original protocol provided by the manufacturers,
appear to be efficient for the reliable routine identification of clinical yeast isolates in medical laboratories. Indeed,
when one colony was sampled instead of five and the protein extraction protocol was modified, the performance of MALDI-TOF
MS was superior to that of the API ID 32C method (discrepancies were confirmed by using molecular identification), allowing
the correct identification of 94% of the 335 clinical isolates prospectively tested. We then demonstrated that the time for
which the primary cultures were preincubated on CHROMagar did not impact the identification of yeasts by MALDI-TOF MS, since
95.1 and 96.2% of the 183 clinical yeast isolates prospectively tested were correctly identified after 48 and 72 h of preincubation,
Full-text · Article · Jun 2012 · Journal of clinical microbiology
[Show abstract][Hide abstract] ABSTRACT: Conventional identification (CI) of yeasts is based on morphological, biochemical and/or immunological methods. Matrix-assisted
laser desorption/ionization – time of flight (MALDI-TOF or MT-MS) mass spectrometry has been proposed as a new method for
the identification of microorganisms. This prospective study compared the performance of MT-MS and CI for the identification
of yeasts isolated from clinical samples. Sequencing of the internal transcribed spacer (ITS) regions of ribosomal DNA was
used as the reference method in the analysis of a total of 1207 yeast isolates. Concordance between MT-MS and CI was observed
for 1105 isolates (91.5%), while 74 isolates (6.1%) were misidentified. Molecular identification revealed that 73 of these
74 isolates were identified correctly by MT-MS and CI correctly identified the last one. Concordance between the two techniques
was excellent for the medically-important species (98–100%), including the identification of closely-related species (Candida albicans/C. dubliniensis; C. inconspicua/C. norvegensis; C. parapsilosis/C. metapsilosis/C. orthopsilosis). Only 2.3% of isolates belonging to C. famata, C. lambica and C. magnoliae or to Geotrichum spp. and Trichosporon spp. were not identified by MT-MS. This investigation highlights the potential of MT-MS-based yeast identification as a reliable,
time and cost-efficient alternative to CI.
Full-text · Article · Jun 2012 · Medical mycology: official publication of the International Society for Human and Animal Mycology
[Show abstract][Hide abstract] ABSTRACT: In order to describe developmental changes in human salivary peptidome, whole saliva was obtained from 98 infants followed longitudinally at 3 and 6months of age. Data on teeth eruption and diet at the age of 6months were also recorded. Salivary peptide extracts were characterised by label-free MALDI-MS. Peptides differentially expressed between the two ages, and those significantly affected by teeth eruption or introduction of solid foods were identified by MALDI TOF-TOF and LC ESI MS-MS. Out of 81 peaks retained for statistical analysis, 26 were overexpressed at the age of 6months. Exposure to solid foods had a more pronounced effect on profiles (overexpression of nine peaks) than teeth eruption (overexpression of one peak). Differential peaks corresponded to fragments of acidic and basic PRPs, statherin and histatin. Comparison with existing knowledge on adult saliva peptidome revealed that proteolytic processing of salivary proteins is qualitatively quite comparable in infants and in adults. However, age and diet are modulators of salivary peptidome in human infants.
No preview · Article · Apr 2012 · Journal of proteomics
[Show abstract][Hide abstract] ABSTRACT: GABARAPL1 belongs to the small family of GABARAP proteins (including GABARAP, GABARAPL1 and GABARAPL2/GATE-16), one of the two subfamilies of the yeast Atg8 orthologue. GABARAPL1 is involved in the intracellular transport of receptors, via an interaction with tubulin and GABA(A) or kappa opioid receptors, and also participates in autophagy and cell proliferation. In the present study, we identify the HSP90 protein as a novel interaction partner for GABARAPL1 using GST pull-down, mass spectrometry and coimmunoprecipitation experiments. GABARAPL1 and HSP90 partially colocalize in MCF-7 breast cancer cells overexpressed Dsred-GABARAPL1 and in rat brain. Moreover, treatment of MCF-7 cells overexpressed FLAG-GABARAPL1-6HIS with the HSP90 inhibitor 17-AAG promotes the GABARAPL1 degradation, a process that is blocked by proteasome inhibitors such as MG132, bortezomib and lactacystin. Accordingly, we demonstrate that HSP90 interacts and protects GABARAPL1 from its degradation by the proteasome.
[Show abstract][Hide abstract] ABSTRACT: The interindividual variation in the sensitivity to bitterness is attributed in part to genetic polymorphism at the taste receptor level, but other factors, such as saliva composition, might be involved. In order to investigate this, 2 groups of subjects (hyposensitive, hypersensitive) were selected from 29 healthy male volunteers based on their detection thresholds for caffeine, and their salivary proteome composition was compared. Abundance of 26 of the 255 spots detected on saliva electrophoretic patterns was significantly different between hypo- and hypersensitive subjects. Saliva of hypersensitive subjects contained higher levels of amylase fragments, immunoglobulins, and serum albumin and/or serum albumin fragments. It also contained lower levels of cystatin SN, an inhibitor of protease. The results suggest that proteolysis occurring within the oral cavity is an important perireceptor factor associated to the sensitivity to the bitter taste of caffeine.
[Show abstract][Hide abstract] ABSTRACT: Peroxisome proliferators have been extensively studied in rodents and are known to induce liver tumors, whereas the effects of these compounds are not very clearly identified in humans when they are widely exposed to herbicides, plasticizers, solvents or drugs such as the lipid-lowering fibrate bezafibrate (BEZA). We assessed the effect of BEZA on human hepatocyte proteome. Hepatocyte proteins, including those membrane-associated, were successfully extracted and separated using 2D-liquid chromatography (PF2D, Beckman coulter). Proteins that were regulated by ≥ 1.5 fold compared to controls were identified by mass spectrometry (MALDI-TOF, Bruker Daltonics) and SwissProt bank search. BEZA modified the expression of proteins involved in various metabolic pathways as well as in cell homeostasis. No marker of peroxisome proliferation was obtained but surprisingly the expression of proteins involved in liver carcinogenicity was modulated. The co-treatment of cultures with N-acetylcysteine modified the set of proteins regulated by BEZA, either by a potentiation or an inhibition of the effects. Our study points out that the hepatocellular redox environment has to be taken into account when using fibrates in therapeutics.
No preview · Article · Mar 2011 · Toxicology Letters
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to describe the changes in salivary protein profiles in infants between the ages of 3 and 6 months, and to evaluate the impact of teeth eruption and introduction of solid foods on such profiles.
73 infants were followed longitudinally at 3 and 6 months of age. Their whole saliva proteins were separated by SDS-PAGE electrophoresis and semi-quantified by image analysis. Amylase activity was also measured on a sub-sample of the population (n=42 infants). Bands which abundance was significantly different between the two ages according to paired comparisons were identified by mass spectrometry techniques.
Out of 21 bands, 13 were significantly different between 3 and 6 months of age. Two short variants of amylase increased in abundance with age, as did amylase activity. Other changes possibly translated developmental physiological events, for example maturation of the adaptive immune system. The balance between S-type cystatins and cystatins A and B was modified, in favour of S-type cystatins at 6 months of age. Teeth eruption resulted in an increase in albumin abundance, whilst introduction of solid foods was associated with higher levels of β-2 microglobulin and S-type cystatins.
Salivary profiles were modified substantially between the ages of 3 and 6 months. Both teeth eruption and diet had an impact on abundance changes for some proteins, revealing dynamic interactions between saliva proteome, oral physiology and diet.
No preview · Article · Mar 2011 · Archives of oral biology