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Introduction. Septic shock is a systemic inflammatory response syndrome associated with organ failures. Earlier clinical diagnosis would be of benefit to a decrease in the mortality rate. However, there is currently a lack of predictive biomarkers. The secretome is the set of proteins secreted by a cell, tissue, or organism at a given time and under certain conditions. The plasma secretome is easily accessible from biological fluids and represents a good opportunity to discover new biomarkers that can be studied with nontargeted “omic” strategies. Aims. To identify relevant deregulated proteins (DEP) in the secretome of a rat endotoxemic shock model. Methods. Endotoxemic shock was induced in rats by intravenous injection of lipopolysaccharides (LPS, S. enterica typhi, 0.5 mg/kg) and compared to controls (Ringer Lactate, iv). Under isoflurane anesthesia, carotid cannulation allowed mean arterial blood pressure (MAP) and heart rate (HR) monitoring and blood sampling at different time points (T0 and T50 or T0 and T90, with EDTA and protease inhibitor). Samples were prepared for large-scale tandem mass spectrometry (MS-MS) based on a label-free quantification to allow identification of the proteins deregulated upon endotoxemic conditions. A Gene Ontology (GO) analysis defined several clusters of biological processes (BP) in which the DEP are involved. Results. Ninety minutes after shock induction, the LPS group presents a reduction in MAP (-45%, p < 0.05 ) and increased lactate levels (+27.5%, p < 0.05 ) compared to the control group. Proteomic analyses revealed 10 and 33 DEP in the LPS group, respectively, at 50 and 90 minutes after LPS injection. At these time points, GO-BP showed alterations in pathways involved in oxidative stress response and coagulation. Conclusion. This study proposes an approach to identify relevant DEP in septic shock and brings new insights into the understanding of the secretome adaptations upon sepsis.

Mistletoe (Viscum album L.) is used in German-speaking European countries in the field of integrative oncology linking conventional and complementary medicine therapies to improve quality of life. Various companies sell extracts, fermented or not, for injection by subcutaneous or intra-tumoral route with a regulatory status of anthroposophic medicinal products (European Medicinal Agency (EMA) assessment status). These companies as well as anthroposophical physicians argue that complex matrices composed of many molecules in mixture are necessary for activity and that the host tree of the mistletoe parasitic plant is the main determining factor for this matrix composition. The critical point is that parenteral devices of European mistletoe extracts do not have a standard chemical composition regulated by EMA quality guidelines, because they are not drugs, regulatory speaking. However, the mechanism of mistletoe’s anticancer activity and its effectiveness in treating and supporting cancer patients are not fully understood. Because of this lack of transparency and knowledge regarding the matrix chemical composition, we undertook an untargeted metabolomics study of several mistletoe extracts to explore and compare their fingerprints by LC-(HR)MS(/MS) and 1H-NMR. Unexpectedly, we showed that the composition was primarily driven by the manufacturer/preparation method rather than the different host trees. This differential composition may cause differences in immunostimulating and anti-cancer activities of the different commercially available mistletoe extracts as illustrated by structure–activity relationships based on LC–MS/MS and 1H-NMR identifications completed by docking experiments. In conclusion, in order to move towards an evidence-based medicine use of mistletoe, it is a priority to bring rigor and quality, chemically speaking.

Animal venoms are small natural mixtures highly enriched in bioactive components. They are known to target at least two important pharmacological classes of cell surface receptors: ion channels and G protein coupled receptors. Since sperm cells express a wide variety of ion channels and membrane receptors, required for the control of cell motility and acrosome reaction, two functions that are defective in infertility issues, animal venoms should contain interesting compounds capable of modulating these two essential physiological functions. Herein, we screened for bioactive compounds from the venom of the Egyptian black snake Walterinnesia aegyptia (Wa) that possess the property to activate sperm motility in vitro from male mice OF1. Using RP-HPLC and cation exchange chromatography, we identified a new toxin of 6389.89 Da (termed walterospermin) that activates sperm motility. Walterospermin was de novo sequenced using a combination of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF MS/MS) following reduction, alkylation, and enzymatic proteolytic digestion with trypsin, chymotrypsin or V8 protease. The peptide is 57 amino acid residues long and contains three disulfide bridges and was found to be identical to the previously cloned Wa Kunitz-type protease inhibitor II (Wa Kln-II) sequence. Moreover, it has strong homology with several other hitherto cloned Elapidae and Viperidae snake toxins suggesting that it belongs to a family of compounds able to regulate sperm function. The synthetic peptide shows promising activation of sperm motility from a variety of species, including humans. Its fluorescently-labelled analog predominantly marks the flagellum, a localization in agreement with a receptor that controls motility function.

International audience; Acetylation is a widely occurring post-translational modification (PTM) of proteins that plays a crucial role in many cellular physiological and pathological processes. Over the last decade, acetylation analyses required the development of multiple methods to target individual acetylated proteins, as well as to cover a broader description of acetylated proteins that comprise the acetylome. Areas covered: This review discusses the different types of acetylation (N-ter/K-/O-acetylation) and then describes some major strategies that have been reported in the literature to detect, enrich, identify and quantify protein acetylation. The review highlights the advantages and limitations of these strategies, to guide researchers in designing their experimental investigations and analysis of protein acetylation. Finally, this review highlights the main applications of acetylomics (proteomics based on mass spectrometry) for understanding physiological and pathological conditions. Expert opinion: Recent advances in acetylomics have enhanced knowledge of the biological and pathological roles of protein acetylation and the acetylome. Besides, radiolabeling and western blotting remain also techniques-of-choice for targeted protein acetylation. Future challenges in acetylomics to analyze the N-ter and K-acetylome will most likely require enrichment/fractionation, MS instrumentation and bioinformatics. Challenges also remain to identify the potential biological roles of O-acetylation and cross-talk with other PTMs.

High-throughput mass spectrometry-based proteomic analysis requires peptide fractionation to simplify complex biological samples and increase proteome coverage. OFFGEL fractionation technology became a common method to separate peptides or proteins using isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing process may be further optimized and controlled in terms of separation time and pI resolution. Here we evaluated OFFGEL technology to separate peptides from different samples in the presence of low-molecular-weight (LMW) color pI markers to visualize the focusing process. LMW color pI markers covering a large pH range were added to the peptide mixture before OFFGEL fractionation using a 24-wells device encompassing the pH range 3-10. We also explored the impact of LMW color pI markers on peptide fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by RP_HPLC prior to MS analysis using MALDI-TOF/TOF mass spectrometry in MS and MS/MS modes. Here we report the performance of the peptide focusing process in the presence of LMW color pI markers as on-line trackers during the OFFGEL process and the possibility to use them as pI controls for peptide focusing. This method improves the workflow for peptide fractionation in a bottom-up proteomic approach with or without iTRAQ labeling.

Ucar, Evren Onay/0000-0003-1152-4881; Akdeniz, Nilgun/0000-0002-6208-3193; ARDA, NAZLI/0000-0002-1043-5652; KARLIOGLU KILIC, Nurgul/0000-0002-6255-6819; Bourgoin-Voillard, Sandrine/0000-0001-6511-0612 WOS:000506666400003 PubMed ID: 30447650 Background: Tree pollens are well-known aeroallergens all over the world. Little is known about the allergenicity of Morus alba (white mulberry) pollen. Objective: We aimed to explore the potential allergens of this pollen and its clinical relevance in tree pollen allergic patients living in Istanbul, Turkey. Methods: Twenty three seasonal allergic rhinitis patients with a confirmed tree pollen allergy and 5 healthy control subjects underwent skin prick and nasal provocation tests with M.alba pollen extract. The pollen extract was then resolved by gel electrophoresis, and immunoblotted with sera from patients/control individuals to detect the potential allergenic proteins. The prevalent IgE binding proteins from ID-gel were analyzed by MALDI-TOF/TOE Results: Eleven out of 23 patients were reactive to the extract with skin prick tests. Seven of those patients also reacted positively to the nasal provocation tests. The most common IgE-binding pollen proteins were detected between 55-100 kDa, and also at molecular weights lower than 30 kDa for some patients. Mass spectrometry analyses revealed that the principal IgE-binding protein was methionine synthase (5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase), which is then proposed as a novel allergen in M.alba pollen. Conclusion: This study provides the first detailed information for the potential allergens of Morus alba pollen of Istanbul. Methionine synthase with an apparent molecular weight of 80 to 85 kDa has been recognized as one of the allergens in Morus alba pollen for the first time. Research Fund of Istanbul UniversityIstanbul University [3063, 30217]; Bio-Health Computing Erasmus Mundus program [512383-1-2010-1-FR] This study was supported by the Research Fund of Istanbul University (Project no. 3063 and Project no. 30217) and the Bio-Health Computing Erasmus Mundus program (512383-1-2010-1-FR).

Introduction The secretome is a set of proteins secreted by a cell at a given time and under certain conditions. The secretome, easily accessible from plasma and analyzable by a proteomic approach, represents a good opportunity to identify biomarkers in septic shock. This pathology corresponds to a systemic inflammatory response syndrome related to an infection and associated with cardiocirculatory failure leading to organ dysfunction with a mortality rate of 40%. The identification of diagnostic/prognostic biomarkers is crucial to improve patient outcome. Objective Study the secretome of a rat model of endotoxemic shock to identify relevant deregulated proteins (DEP). Methods Endotoxemic shock was induced by lipopolysaccharides injection (LPS, S. enterica typhi, 0.5 mg/kg, iv) and compared to controls (Ringer lactate, iv). Under isoflurane anesthesia, carotid cannulation allowed evaluation of mean arterial blood pressure (MAP), heart rate and blood sampling (T0, and T50 or T90 min on EDTA and protease inhibitor). Plasma was analyzed with tandem MS based on a label free quantification. Gene ontology (GO) analyses defined several groups of biological process (BP) in which the DEP were involved. Results Ninety minutes after shock induction, the LPS group present a reduction in MAP (−45%, P Conclusion This study proposes an approach to identify DEP in septic shock. DEP need to be tested in cellular models in order to understand their role in the physiopathology of shock and to improve understanding of adaptations of the secretome during sepsis. The link between our results and patient prognosis will need to be studied for clinical use.

Introduction Septic shock is a systemic inflammatory response syndrome associated with circulation failure leading to organ failure with a mortality rate of 40%. Early diagnosis and prognosis of septic shock are necessary for timely and specific treatment but no predictive biomarkers are available. The plasma proteome is easily accessible and may contain biomarkers that can be studied with non-targeted “omic” strategies. Aims We aim to decipher a relevant proteome signature through a non-targeted mass spectrometry (MS) analysis in a rat septic shock model. Methods In 10-week-old Wistar rats (n = 11/group), 30 min post-buprenorphine (0.05 mg/kg, s) endotoxemic shock was induced by lipopolysaccharides injection (LPS, S. enterica typhi, 0.5 mg/kg, iv) and compared to controls (NaCl 0.9%, iv). Under isoflurane anesthesia, carotid cannulation allowed mean arterial blood pressure (MAP), heart rate (HR) monitoring and blood sampling (T0, 50 and 150 min-EDTA with protease inhibitor). Each plasma was prepared for RPHPLC/MS by performing Proteominer, dialysis and trypsin digestion. RPHPLC-MS analysis was performed on a ESI-Q-Exactive mass spectrometer (ThermoFisher) according to a non-targeted label-free MS analysis. Data were analyzed with PEAKS and R software. Results LPS group present circulation failure (−40% MAP, +20% of HR vs Ctrl, P Conclusion This preliminary study revealed propose a new approach to identify relevant deregulated proteins in septic shock. The investigation of the link between our results and patients’ clinical features is in progress.

International audience; KDAC inhibitors (KDACi) overcome gefitinib primary resistance in non-small cell lung cancer (NSCLC) including mutant-KRAS lung adenocarcinoma. To identify which proteins are involved in the restoration of this sensitivity and to provide new therapeutic targets for mutant-KRAS lung adenocarcinoma, we performed an iTRAQ quantitative proteomic analysis after subcellular fractionation of H358-NSCLC treated with gefitinib and KDACi (TSA/NAM) versus gefitinib alone. The 86 proteins found to have been significantly dysregulated between the two conditions, were mainly involved in cellular metabolism and cell transcription processes. As expected, the pathway related to histone modifications was affected by the KDACi. Pathways known for controlling tumor development and (chemo)-resistance (miRNA biogenesis/glutathione metabolism) were affected by the KDACi/gefitinib treatment. Moreover, 57 dysregulated proteins were upstream of apoptosis (such as eEF1A2 and STAT1) and hence provide potential therapeutic targets. The inhibition by siRNA of eEF1A2 expression resulted in a slight decrease in H358-NSCLC viability. In addition, eEF1A2 and STAT1 siRNA transfections suggested that both STAT1 and eEF1A2 prevent AKT phosphorylation known for enhancing gefitinib resistance in NSCLC. Therefore, altogether our data provide new insights into proteome regulations in the context of overcoming the NSCLC resistance to gefitinib through KDACi in H358 KRAS mutated and amphiregulin-overexpressing NSCLC cells.

Tree pollens are well-known aeroallergens all over the world. Little is known about the allergenicity of Morus alba (white mulberry) pollen.We aimed to explore the potential allergens of this pollen and its clinical relevance in tree pollen allergic patients living in Istanbul, Turkey.Twenty three seasonal allergic rhinitis patients with a confirmed tree pollen allergy and 5 healthy control subjects underwent skin prick and nasal provocation tests with M.alba pollen extract. The pollen extract was then resolved by gel electrophoresis, and immunoblotted with sera from patients/control individuals to detect the potential allergenic proteins. The prevalent IgE binding proteins from 1D-gel were analyzed by MALDI-TOF/TOF.Eleven out of 23 patients were reactive to the extract with skin prick tests. Seven of those patients also reacted positively to the nasal provocation tests. The most common IgE-binding pollen proteins were detected between 55-100 kDa, and also at molecular weights lower than 30 kDa for some patients. Mass spectrometry analyses revealed that the principal IgE-binding protein was methionine synthase (5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase), which is then proposed as a novel allergen in M.alba pollen.This study provides the first detailed information for the potential allergens of Morus alba pollen of Istanbul. Methionine synthase with an apparent molecular weight of 80 to 85 kDa has been recognized as one of the allergens in Morus alba pollen for the first time.

High-throughput mass spectrometry-based proteomic analysis requires peptide fractionation to simplify complex biological samples and increase proteome coverage. OFFGEL fractionation technology became a common method to separate peptides or proteins using isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing process may be further optimized and controlled in terms of separation time and pI resolution. Here we evaluated OFFGEL technology to separate peptides from different samples in the presence of low-molecular-weight (LMW) color pI markers to visualize the focusing process. LMW color pI markers covering a large pH range were added to the peptide mixture before OFFGEL fractionation using a 24-wells device encompassing the pH range 3-10. We also explored the impact of LMW color pI markers on peptide fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by RP_HPLC prior to MS analysis using MALDI-TOF/TOF mass spectrometry in MS and MS/MS modes. Here we report the performance of the peptide focusing process in the presence of LMW color pI markers as on-line trackers during the OFFGEL process and the possibility to use them as pI controls for peptide focusing. This method improves the workflow for peptide fractionation in a bottom-up proteomic approach with or without iTRAQ labeling.

Background: Leishmaniasis represents a serious public health problem in Algeria. The parasite, in the promastigote form, upon transfer by the infected vector to the vertebrate host, invades mononuclear macrophages and causes substantial human morbidity and mortality. Investigating new antimicrobial and antiparasitic components from Viperidae venoms represents an alternative therapeutic strategy for leishmaniasis treatment. Objective: In the present study, we report the characterization of a disintegrin, isolated from Cerastes cerastes venom, exhibiting antiparasitic activity on Leishmania infantum promastigotes. Methods: The active biomolecule, referred to as Disintegrin_Cc, was isolated by RP-HPLC chromatography. The in vitro antiparasitic activity of this molecule was evaluated on Leishmania infantum promastigotes. The isolated disintegrin was analyzed by SDS-PAGE for homogeneity and molecular weight determination and then subjected to MALDI-TOF MS/MS analysis. A 3D structure was predicted in silico using SWISS-Model. Results: Isolated Disintegrin_Cc induced deep morphological alterations on the parasites and a strong anti-leishmanial activity estimated to 84.75% of mortality. SDS-PAGE analysis indicated that this disintegrin was homogenous. This dimeric disintegrin of 14,193.97 Da contains an RGD domain and four intramolecular disulfide bridges. This biomolecule presents a high percentage of identity with other related types of snake disintegrin. Predicted 3D structure indicated that this peptide shares partial homology with well-known active antimicrobial peptides. Conclusion: This study demonstrated the leishmanicidal activity of Disintegrin_Cc. This molecule may be useful to design a new therapy against leishmaniasis.

Malignant processes such as metastasis, invasion, or angiogenesis are tightly dependent on the composition of the extracellular medium, which is itself affected by the release of proteins by the tumor cells. p53, a major tumor suppressor protein very frequently mutated and/or inactivated in cancer cells, is known to modulate the release of proteins by the tumor cells; however, while p53-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. Here, we characterized the p53-dependent secretome of a lung tumor model in vitro (H358 human nonsmall cell lung adenocarcinoma cell line with a homozygous deletion of p53) and demonstrate that the modulation of exported proteins can also be detected in vivo in the plasma of tumor-bearing mice. We used a clone of H358, stably transfected with a tetracycline-inducible wild-type p53-expressing vector. With the use of iTRAQ labeling and LC-MALDI-MS/MS analysis, we identified 909 proteins released in vitro by the cells, among which 91 are p53-modulated. Three proteins (GDF-15, FGF-19, and VEGF) were also investigated in H358/TetOn/p53 xenograft mice. The ELISA dosage on total tumor protein extracts confirmed the influence of p53 on the release of these proteins in vivo. Moreover, the GDF-15 concentration was measured in the plasma and its p53-dependent modulation was confirmed. To our knowledge, this is the first report establishing that the in vitro cell line secretome is reliable and reflects the extracellular release of proteins from tumor cells in vivo and could be used to identify putative tumor markers.

Resume Introduction Le terme « secretome » ou « secretomique » est utilise pour decrire l’etude globale de l’ensemble des proteines secretees par une cellule, un tissu ou un organisme. Exegese Le secretome represente une classe importante de proteines controlant une multitude de processus biologiques et physiologiques. Differentes voies de secretion sont impliquees dans le relargage de ces proteines. Conclusion Le secretome est une source potentielle appropriee pour la recherche de nouvelles cibles therapeutiques ou de candidats biomarqueurs.

International audience; De manière générale, les entreprises de bio-informatique sont de petites ou moyennes entreprises, très jeunes qui sont entrées sur le marché par le biais des biotechnologies. Avec l'arrivée sur le marché d'entreprises comme Hewlett Packard et IBM, et le portefeuille de propriété intellectuelle acheté par Motorola et Kodak, la suprématie des start-up de biotechnologie pourrait bien se terminer, surtout si l'on considère le pouvoir des intrants basés sur les technologies de l'information. Bien qu'il ne soit pas nécessaire d'éliminer les start-up basées sur les biotechnologies, il serait souhaitable de créer avec les entreprises spécialisées dans les technologies de l'information, un schéma d'alliance similaire à celui qui existe entre les entreprises pharmaceutiques et les start-up de biotechnologie dans les sciences de la vie.

Background Currently, the best characterized genetic aberration in neuroblastoma (NB) is MYCN amplification, which has been clearly related to prognosis. In the present study, we investigated whether specific epigenetic alterations are associated with stage of disease. Procedure Sixty-two NBs (45 primary tumors and 17 NBs at relapse) were studied in terms of the methylation status of 19 genes (p15INK4a, p16INK4a, p14ARF, APC, RB1, RASSF1A, BLU, FHIT, RARβ, INI1, TIMP3, NF2, MGMT, DAPK, FLIP, ECAD, CASP8, and the receptors DcR1 and DcR2). Results At diagnosis, we found hypermethylation of RASSF1A in 93% of these tumors, hypermethylation of TIMP3 in 51%, of CASP8 in 38%, of BLU in 34%, of DcR2 in 25%, and of DcR1 in 11%. All 17 tumors tested at relapse showed hypermethylation of RASSF1A (100%), while 10 showed hypermethylation of TIMP3 (59%), six of CASP8 (35%), five of DcR2 (29%), four of BLU (24%), and three of DcR1 (18%). Hypermethylation was related to clinical stage; NBs at stages 1, 2, and 4s were less frequently methylated than stages 3 and 4 disease (P = 0.002). Conclusion These results from our series indicate that hypermethylation of tumor-suppressor genes may be important in the development and evolution of NB. These epigenetic alterations could be used as a marker of the disease and genes regulating methylation should be considered as possible therapeutic targets in NB. Pediatr Blood Cancer 2008;50:29–32. © 2007 Wiley-Liss, Inc.

Ependymomas (EP) represent the third most frequent type of central nervous system (CNS) tumor of childhood, after astrocytomas and medulloblastomas. No prognostic biological markers are available, and differentiation from choroid plexus papilloma (CPP) is difficult. The present objective was, for a sample of 27 children with intracranial EP and 7 with CPP, to describe and compare the methylation status of 19 genes (with current HUGO symbol, if any): p15INK4a (CDKN2B), p16INK4a and p14ARF (both CDKN2A), APC, RB1, RASSF1A (RASSF1), BLU (ZMYND10) FHIT, RARB, MGMT, DAPK (DAPK1), ECAD (CDH1), CASP8, TNFRSF10C, TNFRSF10D, FLIP (CFLAR), INI1 (SMARCB1), TIMP3, and NF2. Three adult corteses were used as a control. We detected a similar percentage of methylated tumors in both groups (71% in CPP and 77% in EP). No gene was methylated in that control group. RASSF1A was the most frequently methylated gene in both benign tumors (66%) and EP (56%). The genes associated with apoptosis were methylated in both groups of tumors. The percentages of TRAIL pathway genes (CASP8, TFRSF10C, and TFRSF10D) methylated were 30, 9.5, and 36.4%, respectively, in ependymomas and 50, 50, and 16.7%, respectively, in choroid plexus papillomas. No other gene was methylated in the benign tumors, whereas FHIT was methylated in 22%, RARB in 14.8%, BLU in 13.6%, p16INK4a in 11.1%, TNFRSF10C in 9.5%, and DAPK in 7.4% of ependymomas. Although we did not observe a statistical relationship between methylation and clinical outcome, the methylation pattern does not appear to be randomly distributed in ependymoma and may represent a mechanism of tumor development and evolution.

A prospective screening program, including CT, autofluorescent bronchoscopy, biopsies and bronchial lavage (BL) collection, was initiated with the specific goal of identifying biomarkers for the early detection of non-small cell lung cancer. We report and discuss the results of p16, DAPK, MGMT, FHIT and APC methylation analysis in the 126 first patients: 77 at high risk of cancer and 49 followed up after primary cancer resection. Positive results were found in 49% of BLs, 53% in current smokers and 43% in former smokers. In presence of peripheral tumours, only 38% of BLs were abnormal versus 73% in presence of central tumours, 50% in presence of preneoplasic lesions and 47% in absence of lesions. FHIT methylation was an early event, observed in one-third of the BLs from patients with or without lesions as well as in tumours. APC methylation was a late event observed in 33% of tumours but rarely in BLs. p16 was methylated in 17% of BLs but in 48% of tumours; DAPK in 15% of BL and 22% of tumours. MGMT methylation was rare. Among patients followed up after cancer surgery, 14 were in remission with normalised BL, whereas three had positive BLs and relapsed with a central tumour. Thus, gene methylation in BL might help to detect central tumours but a CT is crucial for peripheral cancer detection.

International audience; Lung carcinomas are represented by non-small-cell lung carcinomas (NSCLC) and neuroendocrine carcinomas (NE) which differ in their clinical presentation and prognosis. We used comparative genomic hybridization (CGH) to characterize and compare the chromosomal pattern of 11 NSCLC and 11 high-grade NE lung carcinomas. Overall, the total number of aberrations was higher in NSCLC than in high-grade NE lung tumors (p < 0.05) and gains predominated over losses in NSCLC (p < 0.0003). Gains common to both lung tumor phenotypes were detected in 1p, 1q, 3q, 5p, 6p, 8q, 12, 17q, 19p, 19q, 20p, 20q, and X, whereas common losses were found in 2q, 3p, 4p, 4q, 5q, 8p, 9p, 10p, 11p, 11q, 13q, and 17p. Major gains on 18q and losses on 2p and 16q were exclusively detected in high-grade NE lung tumors. On the other hand, major gains on 2p and 15q and losses on 21q were found only in NSCLC. Furthermore, gains within 22q11-q12 and 7p12-p15 were associated with NSCLC (p < 0.05). The differences in the pattern and distribution of genetic changes observed in NSCLC as opposed to high-grade NE lung carcinomas suggest the existence of distinct tumorigenic pathways between these two major classes of lung tumors.

International audience; AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK β-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions.

Background The proteomes of mammalian biological fluids, cells and tissues are complex and composed of proteins with a wide dynamic range. The effective way to overcome the complexity of these proteomes is to combine several fractionation steps. OFFGEL fractionation, recently developed by Agilent Technologies, provides the ability to pre-fractionate peptides into discrete liquid fractions and demonstrated high efficiency and repeatability necessary for the analysis of such complex proteomes. Results We evaluated OFFGEL fractionator technology to separate peptides from two complex proteomes, human secretome and human plasma, using a 24-wells device encompassing the pH range 3–10. In combination with reverse phase liquid chromatography, peptides from these two samples were separated and identified by MALDI TOF-TOF. The repartition profiles of the peptides in the different fractions were analyzed and explained by their content in charged amino acids using an algorithmic model based on the possible combinations of amino acids. We also demonstrated for the first time the compatibility of OFFGEL separation technology with the quantitative proteomic labeling technique iTRAQ allowing inclusion of this technique in complex samples comparative proteomic workflow. Conclusion The reported data showed that OFFGEL system provides a highly valuable tool to fractionate peptides from complex eukaryotic proteomes (plasma and secretome) and is compatible with iTRAQ labeling quantitative studies. We therefore consider peptides OFFGEL fractionation as an effective addition to our strategy and an important system for quantitative proteomics studies.