Variability of the response of human vaginal Lactobacillus crispatus to 17ß-estradiol.

We previously showed that the physiological concentration of 17ß-estradiol in the vaginal environment is sufficient to affect the membrane dynamics and adhesion phenotype of the Lactobacillus crispatus strain CIP104459. However, L. crispatus is a heterogeneous species. Here, we investigated the effect of 17ß-estradiol on the recently isolated L. crispatus vaginal strain V4, related to a cluster distant from CIP104459 and at the limit of being a different subspecies. Grown in the same medium, the two strains expressed a highly similar pool of proteins. However, in contrast to CIP104459, L. crispatus V4 showed high aggregation potential and 17ß-estradiol promoted this phenotype. This effect was associated with large changes in cell-surface polarity and Lewis acid/base properties. In addition, we observed no effect on the membrane dynamics, contrary to CIP104459. These results can be explained by differences in the properties and organization of the S layer between the two strains. However, as for CIP104459, 17ß-estradiol increased biosurfactant production of L. crispatus V4 and their adhesion to vaginal cells. This suggests that 17ß-estradiol agonists would be valuable tools to favor a stable re-implantation of L. crispatus in the vaginal mucosa.

Epinephrine affects motility, and increases adhesion, biofilm and virulence of Pseudomonas aeruginosa H103.

Microbial endocrinology has demonstrated for more than two decades, that eukaryotic substances (hormones, neurotransmitters, molecules of the immune system) can modulate the physiological behavior of bacteria. Among them, the hormones/neurotransmitters, epinephrine (Epi) and norepinephrine (NE), released in case of stress, physical effort or used in medical treatment, were shown to be able to modify biofilm formation in various bacterial species. In the present study, we have evaluated the effect of Epi on motility, adhesion, biofilm formation and virulence of Pseudomonas aeruginosa, a bacterium linked to many hospital-acquired infections, and responsible for chronic infection in immunocompromised patients including persons suffering from cystic fibrosis. The results showed that Epi increased adhesion and biofilm formation of P. aeruginosa, as well as its virulence towards the Galleria mellonella larvae in vivo model. Deciphering the sensor of this molecule in P. aeruginosa and the molecular mechanisms involved may help to find new strategies of treatment to fight against this bacterium.

A versatile and recyclable molecularly imprinted polymer as an oxidative catalyst of sulfur derivatives: a new possible method for mustard gas and V nerve agent decontamination.

A molecularly imprinted polymer containing a porphyrin unit was developed as a biomimetic heterogenous catalyst for the oxidation of sulfur derivatives. Its catalytic efficiency under mild conditions and its easy recovery represent a great asset for the design of new decontamination tools for yperite and VX.

Extracellular DNA release, quorum sensing, and PrrF1/F2 small RNAs are key players in Pseudomonas aeruginosa tobramycin-enhanced biofilm formation.

Biofilms are structured microbial communities that are the leading cause of numerous chronic infections which are difficult to eradicate. Within the lungs of individuals with cystic fibrosis (CF), Pseudomonas aeruginosa causes persistent biofilm infection that is commonly treated with aminoglycoside antibiotics such as tobramycin. However, sublethal concentrations of this aminoglycoside were previously shown to increase biofilm formation by P. aeruginosa, but the underlying adaptive mechanisms still remain elusive. Herein, we combined confocal laser scanning microscope analyses, proteomics profiling, gene expression assays and phenotypic studies to unravel P. aeruginosa potential adaptive mechanisms in response to tobramycin exposure during biofilm growth. Under this condition, we show that the modified biofilm architecture is related at least in part to increased extracellular DNA (eDNA) release, most likely as a result of biofilm cell death. Furthermore, the activity of quorum sensing (QS) systems was increased, leading to higher production of QS signaling molecules. We also demonstrate upon tobramycin exposure an increase in expression of the PrrF small regulatory RNAs, as well as expression of iron uptake systems. Remarkably, biofilm biovolumes and eDNA relative abundances in pqs and prrF mutant strains decrease in the presence of tobramycin. Overall, our findings offer experimental evidences for a potential adaptive mechanism linking PrrF sRNAs, QS signaling, biofilm cell death, eDNA release, and tobramycin-enhanced biofilm formation in P. aeruginosa. These specific adaptive mechanisms should be considered to improve treatment strategies against P. aeruginosa biofilm establishment in CF patients' lungs.

A Peptidomic Approach to Characterize Peptides Involved in Cerebellar Cortex Development Leads to the Identification of the Neurotrophic Effects of Nociceptin.

The cerebellum is a brain structure involved in motor and cognitive functions. The development of the cerebellar cortex (the external part of the cerebellum) is under the control of numerous factors. Among these factors, neuropeptides including PACAP or somatostatin modulate the survival, migration and/or differentiation of cerebellar granule cells. Interestingly, such peptides contributing to cerebellar ontogenesis usually exhibit a specific transient expression profile with a low abundance at birth, a high expression level during the developmental processes, which take place within the first two postnatal weeks in rodents, and a gradual decline toward adulthood. Thus, to identify new peptides transiently expressed in the cerebellum during development, rat cerebella were sampled from birth to adulthood, and analyzed by a semi-quantitative peptidomic approach. A total of 33 peptides were found to be expressed in the cerebellum. Among these 33 peptides, 8 had a clear differential expression pattern during development, 4 of them i.e. cerebellin 2, nociceptin, somatostatin and VGF [353-372], exhibiting a high expression level during the first two postnatal weeks followed by a significative decrease at adulthood. A focus by a genomic approach on nociceptin, confirmed that its precursor mRNA is transiently expressed during the first week of life in granule neurons within the internal granule cell layer of the cerebellum, and showed that the nociceptin receptor is also actively expressed between P8 and P16 by the same neurons. Finally, functional studies revealed a new role for nociceptin, acting as a neurotrophic peptide able to promote the survival and differentiation of developing cerebellar granule neurons.

Brain proteomic modifications associated to protective effect of grape extract in a murine model of obesity.

In recent years, the obesity epidemic has developed into a major health crisis worldwide. With current treatments limited to expensive, high-risk surgery and minimally efficacious pharmacotherapy, new therapeutic options are urgently needed to fight against this alarming trend. Though brain dysfunction has been studied linked to high fat diet (HFD) and grape seed and skin extract (GSSE) correction, the proteomic modifications linking the two effects on brain lipotoxicity are not well understood. To this end rats were exposed for 8 weeks to HFD treatment, to GSSE (500mg/kg BW) and to binary mixture of HFD and GSSE to gain insight into the potential pathways altered with metabolic disease and the protection afforded by GSSE. Significant modifications of brain proteins were detected using mass spectrometry-based differential proteomics. These proteins were mainly related to oxidative stress, glycolysis and calcium signaling. Additionally, proteins involved in the cytoskeleton were also affected by HFD treatment. Interestingly, whether up- or down regulated protein abundances, GSSE corrected most of the disturbances of HFD treatment. These findings provide impetus for future therapeutic investigation on GSSE against other metabolic disorders.

Two novel peptides with angiotensin I converting enzyme inhibitory and antioxidative activities from Scorpaena notata muscle protein hydrolysate.

Fish protein hydrolysate was prepared from muscle of small red scorpionfish (Scorpaena notata) by treatment with a protease from the fungus Penicillium digitatum. Protein hydrolysate was found to strongly inhibit the angiotensin I converting enzyme and exhibited high antioxidative activity through 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assay. After ultrafiltration, peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high-performance liquid chromatography with a high purification yield of 2.5 mg of peptide per gram of initial protein. Two major peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS), corresponding to the following sequences: Leu-Val-Thr-Gly-Asp-Asp-Lys-Thr-Asn-Leu-Lys (1,204.665 Da) and Asp-Thr-Gly-Ser-Asp-Lys-Lys-Gln-Leu (992.511 Da). These peptides, mainly composed of hydrophilic amino acids, showed high antioxidative and angiotensin I converting enzyme inhibitory activities. These data suggest that the two novel peptides isolated from the muscle hydrolysate of small red scorpionfish can be a beneficial ingredient for functional foods or pharmaceuticals against hypertension and oxidative stress.

A Novel Three Domains Glycoside Hydrolase Family 3 from Sclerotinia sclerotiorum Exhibits ß-Glucosidase and Exoglucanase Activities: Molecular, Biochemical, and Transglycosylation Potential Analysis.

The filamentous fungus Sclerotinia sclerotiorum produces a complete set of cellulolytic enzymes. We report here the purification and the biochemical characterization of a new ß-glucosidase from S. sclerotiorum which belongs to the family 3 of glycoside hydrolases and that was named as SsBgl3. After two size-exclusion chromatography steps, purified protein bands of 80 and 90 kDa from SDS-PAGE were subjected to a mass spectrometry analysis. The results displayed four peptides from the upper band belonging to a polypeptide of 777 amino acids having a calculated molecular weight of 83.7 kDa. Biochemical analysis has been carried out to determine some properties. We showed that this SsBgl3 protein displayed both ß-glucosidase and exoglucanase activities with optimal activity at 55 °C and at pH 5. The transglycosylation activity was investigated using gluco-oligosaccharides TLC analysis. The molecular modeling and comparison with different crystal structures of ß-glucosidases showed that SsBgl3 putative protein present three domains. They correspond to an (α/ß)8 domain TIM barrel, a five-stranded α/ß sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain. Enzyme engineering will be soon investigated to identify the key residues for the catalytic reactions.

The interaction of heparan sulfate proteoglycans with endothelial transglutaminase-2 limits VEGF165-induced angiogenesis.

Sprouting angiogenesis is stimulated by vascular endothelial growth factor (VEGF165) that is localized in the extracellular matrix (ECM) and binds to heparan sulfate (HS)-bearing proteins known as heparan sulfate proteoglycans (HSPGs). VEGF165 presentation by HSPGs enhances VEGF receptor-2 (VEGFR2) signaling. We investigated the effect of TG2, which binds to HSPGs, on the interaction between VEGF165 and HS and angiogenesis. Mice with tg2 deficiency showed transiently enhanced retina vessel formation and increased vascularization of VEGF165-containing Matrigel implants. In addition, endothelial cells in which TG2 was knocked down exhibited enhanced VEGF165-induced sprouting and migration, which was associated with increased phosphorylation of VEGFR2 at Tyr(951) and its targets Src and Akt. TG2 knockdown did not affect the phosphorylation of VEGFR2 at Tyr(1175) or cell proliferation in response to VEGF165 and sprouting or signaling in response to VEGF121. Decreased phosphorylation of VEGFR2 at Tyr(951) was due to ECM-localized TG2, which reduced the binding of VEGF165 to endothelial ECM in a manner that required its ability to bind to HS but not its catalytic activity. Surface plasmon resonance assays demonstrated that TG2 impeded the interaction between VEGF165 and HS. These results show that TG2 controls the formation of VEGF165-HSPG complexes and suggest that this regulation could be pharmacologically targeted to modulate developmental and therapeutic angiogenesis.

Characterization of new outer membrane proteins of Pseudomonas aeruginosa using a combinatorial peptide ligand library.

Most often, the use of ProteoMiner beads has been restricted to human serum proteins for the normalization of major proteins, such as albumin. However, there are other situations of interest in which the presence of major proteins would quench the signals of low abundance polypeptides. We propose the use of these beads for investigating the envelope of the gram-negative bacterium Pseudomonas aeruginosa. Initially, we performed comparative 2D electrophoresis to qualitatively evaluate the incidence of the normalization stage. This demonstrated a significant reduction of the major membrane proteins. Thereafter, using shotgun analysis, the same protein extract was targeted by using combinatorial peptide ligand library capture. This treatment yielded 154 additional outer membrane proteins (OMPs) uncovered by the study of the crude sample.

Identification of S100A9 as biomarker of responsiveness to the methotrexate/etanercept combination in rheumatoid arthritis using a proteomic approach.

OBJECTIVES: One way to optimize the drug prescription in rheumatoid arthritis (RA) is to identify predictive biomarkers of drug responsiveness. Here, we investigated the potential "theranostic" value of proteins of the S100 family by monitoring levels of both S100A8 and S100A9 in blood samples from RA patients. DESIGN: For proteomic analysis, peripheral blood mononuclear cells (PBMC) and serum samples were collected in patients prior to initiation of the methotrexate/etanercept (MTX/ETA) combination. Firstly, relative mass spectrometry (MS) quantification focusing on S100A8 and S100A9 proteins was carried out from PBMCs samples to identify potential biomarkers. The same approach was also performed from serum samples from responder (R) and non responder (NR) patients. Finally, to confirm these results, an absolute quantification of S100A8, S100A9 proteins and calprotectin (heterodimer of S100A8/S100A9) was carried out on the serum samples using ELISA. RESULTS: MS analyses revealed that both S100A8 and S100A9 proteins were significantly accumulated in PBMC from responders. In contrast to PBMC, only the S100A9 protein was significantly overexpressed in the serum of R patients. Absolute quantification by ELISA confirmed this result and pointed out a similar expression level of S100A8 protein and calprotectin in sera from both R and NR groups. Thus, the S100A9 protein revealed to be predictive of MTX/ETA responsiveness, contrarily to parameters of inflammation and auto-antibodies which did not allow significant discrimination. CONCLUSION: This is the first report of an overexpression of S100A9 protein in both PBMCs and serum of patients with subsequent response to the MTX/ETA combination. This protein thus represents an interesting biomarker candidate of therapeutic response in RA.

Biochemical characterization, molecular cloning, and structural modeling of an interesting ß-1,4-glucanase from Sclerotinia sclerotiorum.

The filamentous fungus Sclerotinia sclerotiorum produces a complete set of cellulolytic enzymes needed for efficient solubilization of native cellulose, the major component of plants. In this work, we reported the molecular characterization of an important glycosyl-hydrolase enzyme classified as endo-ß-1,4-glucanase. The importance of this enzyme was revealed with the in-gel activity staining, showing a high degradation capacity of cellulose. When purified from native gel and ran in denaturing polyacrylamide gel, the polypeptide has an apparent molecular mass of about 34 kDa called Endo2. For further characterization of this protein, a mass spectrometry approach was carried out. The LC-MS/MS analysis revealed two peptides belonging to this enzyme. The genomic DNA and cDNA sequences were resolved by PCR amplification and sequencing, revealing a gene with two intron sequences. The open reading frame of 987 bp encoded a putative polypeptide of 328 amino acids having a calculated molecular mass of 33,297 Da. Yet, the molecular modeling and comparative investigation of different 3D cellulase structures showed that this endoglucanase isoform has probably two domains. A core domain having a high similarity with endoglucanases family 5 and a cellulose-binding domain having similarities with those of exo-type cellulases of family 1, linked together by a serine-threonine-rich region. These results are with great interests and show new characteristics of S. sclerotiorum glucanase.

The first "ready-to-use" benzene-based heterotrifunctional cross-linker for multiple bioconjugation.

The synthesis and applications of the first water-soluble benzene derivative bearing a set of three different and orthogonal bioconjugatable groups (aminooxy, azido and thiol) are described. The combined use of a 5-amino isophthalic acid scaffold and unusual acid-labile protecting groups for temporarily masking aminooxy and thiol moieties has enabled the development of a highly convergent approach towards the synthesis of such a trivalent bioconjugation platform in good yields. The potential utility of this "ready-to-use" cross-linking reagent for creating complex and fragile tri-component (bio)molecular systems was illustrated through (1) the rapid preparation of a three-colour FRET cascade with valuable spectral properties and (2) the luminescent/fluorescent labelling of peptides and peptide-oligonucleotide conjugates. Thus, such (bio)molecular assemblies were readily obtained via a three-step process or in a "one-pot" manner, both involving oxime ligation, thiol-alkylation (S(N)2 or Michael addition) and copper-catalysed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reactions.

Cancer immunomics: from serological proteome analysis to multiple affinity protein profiling.

Cancer remains one of the leading causes of death worldwide. Thus, to identify any useful biomarkers is still a need. We performed "cancer immunomics" to identify autoantibody signatures produced in response to the presence of either breast or colorectal cancer. SERological proteome analysis (SERPA) was performed by two-dimensional (2-D) electrophoresis separation, immunoblotting, image analysis, and mass spectrometry. Alternatively, to identify the antigens recognized by the autoantibodies of cancer patients, we developed an approach combining 2-D immunoaffinity chromatography, enzymatic digestion of the isolated antigens, nano flow separation of the resulting peptides, and identification: MAPPing (multiple affinity protein profiling). By these approaches we identified both proteins recognized by autoantibodies independently of a cancer status, and a limited number of proteins reacting preferentially with cancer sera.

Usefulness of autoantigens depletion to detect autoantibody signatures by multiple affinity protein profiling.

Patients with cancer produce specific autoantibodies against protein antigens present in limited amount among a large background of immunoglobulins (Igs), nonrelevant as biomarkers, including natural antibodies. Multiple affinity protein profiling (MAPPing) that combines 2-D immunoaffinity chromatography, enzymatic digestion of the isolated proteins, and identification by MS/MS, may facilitate the identification of these so far unknown patient antibodies. The first immunoaffinity chromatography is crucial, as it is used for selectively removing proteins (autoantigens) recognized by natural antibodies. Application of this depletion step to colon cancer cell proteins is specifically described along with the identification of the natural autoantigens, as well as the coupling of this depletion step with the next steps. By enabling to separate antibody-binding proteins recognized by either natural autoantibodies or patient-specific antibodies this approach may contribute significantly towards the definition of autoantibody signatures.

A proteomic approach to investigate potential biomarkers directed against membrane-associated breast cancer proteins.

The identification of specific protein markers for breast cancer would provide the basis for early diagnosis. Particularly, membrane and membrane-associated proteins are rich in targets for antibodies that may constitute suitable biomarkers of carcinogenesis. However, membrane proteins separation using 2-DE remains difficult. In this work, the breast cancer cell line MCF7 was used as source of proteins for the screening of potential cell membrane-associated antigens recognized by autoantibodies in patients with breast cancer and healthy volunteers. The protein extract obtained using trifluoroethanol (TFE) as cosolvent was compared to a total cell lysate protein extract prepared by a current technique. After 2-DE separation of the two extracts, their protein patterns clearly differed. About 63% of the proteins identified in the TFE-extract were predicted to possess at least one transmembrane domain. 2-D blots probed with sera from cancer patients or from healthy volunteers showed that, as expected, additional antigens were provided in the TFE-extract. Thus, the method described here appeared well suited for proteomic investigation of potential biomarkers undetected by current techniques.

Proteomic Analysis of the MCF7 Breast Cancer Cell Line.

BACKGROUND: The MCF7 breast cancer cell line is a cellular model for breast cancer studies and marker discovery. Therefore, a better knowledge of its proteome is a prerequisite for a more efficient use of this model. MATERIALS AND METHODS: Proteins expressed during the exponential growth phase of MCF7 cells were analyzed and mapped using two-dimensional gel electrophoresis and mass spectrometry. RESULTS: From the spots excised from preparative gels of whole-cell extracts, a subset of 368 different polypeptides, corresponding to 249 different proteins, was identified. These polypeptides were positioned on a silver-stained gel to construct a reference map. CONCLUSION: The data allowed the construction of the most extensive reference map for MCF7 published to date, with 189 novel proteins, which had not been previously listed on maps, and are now accessible on World 2D-PAGE database, providing a basis for further studies on MCF7.