Abbreviations: AFP, antifreeze proteins; APOA1, Apolipoprotein A1; ASC, apoptosisassociated speck-like protein containing a caspase recruitment domain; C3, complement component 3; CALM, calmodulin; CALR, calreticulin; CASP, caspase; cDNA, complementary DNA; ERp57,endoplasmic reticulum protein 57; FBL, fucose-binding lectin; H1/4, histone H1/H4; IL1B, interleukin 1-beta; LEI, leukocyte elastase inhibitor; LYZ, lysozyme; qPCR, real-time PCR; PRDX, peroxiredoxin; SOD superoxide dismutase; TF, transferrin; WAP65, warm temperature acclimation related 65 kDa protein; www.proteomics-journal.com Page 2 ProteomicsThis article is protected by copyright. All rights reserved.Keywords: European sea bass (Dicentrarchus labrax), mucosal immunity, proteome, skin mucus, transcript profile. AbstractSkin mucus is the first barrier of fish defence. Proteins from skin mucus of European sea bass (Dicentrarchus labrax) were identified by 2DE followed by LC-MS/MS. From all the identified proteins in the proteome map, we focus on the proteins associated with several immune pathways in fish. Furthermore, the qPCR transcript levels in skin are shown. Proteins found include apolipoprotein A1, calmodulin, complement C3, fucose-binding lectin, lysozyme and several caspases. To our knowledge, this is the first skin mucus proteome study and further transcriptional profiling of the identified proteins done on this bony fish species.This not only contributes knowledge on the routes involved in mucosal innate immunity, but also establishes a non-invasive technique based on locating immune markers with a potential use for prevention and/or diagnosis of fish diseases.www.proteomics-journal.com Page 3 ProteomicsThis article is protected by copyright. All rights reserved. Statement of significance of the studyThe skin mucus is the first defence barrier of teleost fish; together with the skin, it protects the animal from pathogens, potential harmful chemicals and physical factors in the water where it is constantly submerged. This study identifies for the first time the main proteins in the skin mucus of European sea bass (Dicentrarchus labrax). In particular, we identify immune relevant proteins. The presence of RNA of the immune relevant genes in skin indicates that the proteins could be synthesised in the skin itself.

Infection with Burkholderia pseudomallei or B. thailandensis triggers activation of the NLRP3 and NLRC4 inflammasomes leading to release of IL-1β and IL-18 and death of infected macrophages by pyroptosis, respectively. The non-canonical inflammasome composed of caspase-11 is also activated by these bacteria and provides protection through induction of pyroptosis. The recent generation of bona fide caspase-1-deficient mice allowed us to reexamine in a mouse model of pneumonic melioidosis the role of caspase-1 independently of caspase-11 (that was also absent in previously generated Casp1-/- mice). Mice lacking either caspase-1 or caspase-11 were significantly more susceptible than wild type mice to intranasal infection with B. thailandensis. Absence of caspase-1 completely abolished production of IL-1β and IL-18 as well as pyroptosis of infected macrophages. In contrast, in mice lacking caspase-11 IL-1β and IL-18 were produced at normal level and macrophages pyroptosis was only marginally affected. Adoptive transfer of bone marrow indicated that caspase-11 exerted its protective action both in myeloid cells and in radio-resistant cell types. B. thailandensis was shown to readily infect mouse lung epithelial cells triggering pyroptosis in a caspase-11-dependent way in vitro and in vivo. Importantly, we show that lung epithelial cells do not express inflammasomes components or caspase-1 suggesting that this cell type relies exclusively on caspase-11 for undergoing cell death in response to bacterial infection. Finally, we show that IL-18’s protective action in melioidosis was completely dependent on its ability to induce IFNγ production. In turn, protection conferred by IFNγ against melioidosis was dependent on generation of ROS through the NADPH oxidase but independent of induction of caspase-11. Altogether, our results identify two non-redundant protective roles for caspase-1 and caspase-11 in melioidosis: Caspase-1 primarily controls pyroptosis of infected macrophages and production of IL-18. In contrast, caspase-11 mediates pyroptosis of infected lung epithelial cells.

Differential proteome profile of skin mucus of gilthead seabream (sparus aurata) after probiotic intake and/or overcrowding stress, Journal of Proteomics (2015Proteomics ( ), doi: 10.1016Proteomics ( /j.jprot.2015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E DM A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 AbstractGilthead seabream (Sparus aurata L.) is the major cultured fish species in the Mediterranean area. High density stocking causes stress and increases the impact of diseases leading to economic losses. Probiotics could represent a solution to prevent diseases through several mechanisms such as improving the immune status and/or mucosal microbiota or competing with pathogens. The probiotic Shewanella putrefaciens, also known as Pdp11, was firstly isolated from the skin of healthy gilthead seabream. Our study focuses on the skin mucus proteome after dietary probiotic Pdp11 intake in fish maintained under normal or overcrowding conditions. 2-DE of skin mucus followed by LC-MS/MS analysis was done for each experimental group and differentially expressed proteins were identified. The results showed differentially expressed proteins especially involved in immune processes, such as lysozyme, complement C3, natural killer cell enhancing factor and nonspecific cytotoxic cell receptor protein 1, whose transcript profiles were studied by qPCR. A consistency between lysozyme protein levels in the mucus and lysozyme mRNA levels in skin were found. Further research is necessary to unravel the implications of skin mucosal immunity on fish welfare and disease. Biological significanceThe present work reveals the proteomic changes, which are taking place in the skin mucus of stressed and non-stressed gilthead seabream after Pdp11 probiotic intake. The study contributes to improve the knowledge on skin mucosal immunology of this relevant farmed fish species.Furthermore, the paper shows for the first time how a suitable proteomic methodology, in this case 2-DE followed by LC-MS/MS is useful to perform a comparative study with a noninvasive technique of skin mucus of gilthead seabream.

Multiple myeloma (MM) is a hematological malignancy of clonal plasma cells in the bone marrow (BM). The microenvironment plays a key role in MM cell survival and drug resistance through release of soluble factors, expression of adhesion molecules and release of extracellular vesicles (EV). The aim of this manuscript is to use proteomic profiling of EVs as a tool to identify circulating tumor associated markers in MM patients. First, we characterized the EV protein content obtained from different MM cell lines. Then, we established differences in protein abundance among EVs isolated from MM patient serum and BM and the serum of healthy donors. These data show the Major Histocompatibility Complex Class I is highly enriched in EVs of MM cell lines and MM patient's serum. Next, we show that CD44 is highly expressed in the EVs isolated from the corticosteroid resistant MM cell line, MM.1R. Furthermore, CD44 was found to be differentially expressed in EVs isolated from newly diagnosed MM patients. Finally through ELISA analysis, we establish the potential of serum CD44 as a predictive biomarker of overall survival. These results support the analysis of EVs as an easily accessible source for MM biomarkers.

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