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1.
Front Physiol ; 13: 920289, 2022.
Article in English | MEDLINE | ID: mdl-35846007

ABSTRACT

The sustainable development of modern aquaculture must rely on a significant reduction of the fish meal (FM) used in aquafeed formulations. However, FM substitution with alternative ingredients in diets for carnivorous fish species often showed reduced nutrient absorption, significantly perturbed metabolisms, and histological changes at both hepatic and intestinal levels. In the present study, rainbow trout (Oncorhynchus mykiss) were fed three different experimental aquafeeds. A control diet with higher FM content (27.3%) than two test formulations in which FM was substituted with two more sustainable and promising alternatives: insect meal (Hermetia illucens larvae = 10.1%, FM = 11.6%) and poultry by-products meal (PBM = 14.8%; FM = 11.7%). Combined metabolomics and proteomics analyses of fish liver, together with histological examination of liver and intestine demonstrated that a well-balanced formulation of nutrients in the three diets allowed high metabolic compatibility of either substitution, paving the way for a deeper understanding of the impact of novel raw materials for the fish feed industry. Results show that the main metabolic pathways of nutrient absorption and catabolism were essentially unaltered by alternative feed ingredients, and also histological alterations were negligible. It is demonstrated that the substitution of FM with sustainable alternatives does not have a negative impact on fish metabolism, as long as the nutritional requirements of rainbow trout are fulfilled.

2.
Acta Trop ; 226: 106253, 2022 Feb.
Article in English | MEDLINE | ID: mdl-34822852

ABSTRACT

Cystic echinococcosis (CE) diagnosis by means of serological assays is hampered by the presence of parasites closely related to Echinococcus granulosus sensu lato (s.l.), responsible of the zoonotic disease and with which share cross-reacting antigens. Thus, improvements on the characterization of Echinococcus specific antigens expressed in the larval stage are required, in order to provide useful information for the development of immunological assays for the serodiagnosis of CE in sheep. Here, the proteome of the hydatid cyst fluids (HFs) of Echinococcus granulosus (hydatid fluid, EgHF) and other ovine parasites cyst fluids (CFs), Taenia hydatigena (ThCF) and Taenia multiceps (TmCF) were analyzed by a shotgun proteomic approach. Parasite and host protein profiles in the three types of cyst fluids were characterized and compared. Among the identified proteins, differential parasitic markers with serodiagnostic potential, due to their well-known immunoreactivity in human, included Ag5, AgB proteins, 8-kDa glycoproteins, hydatid disease diagnostic antigen P29 and major egg antigen P40. In particular, seven proteoforms of AgB and 8-kDa glycoprotein resulted to be the most promising diagnostic biomarkers, as they might predict CE in ovine and discriminate between different types of parasites.


Subject(s)
Echinococcus granulosus , Echinococcus , Taenia , Animals , Cyst Fluid , Proteomics , Sheep
3.
PLoS One ; 12(6): e0179338, 2017.
Article in English | MEDLINE | ID: mdl-28594957

ABSTRACT

In plants and protists, dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are part of a bifunctional enzyme (DRTS) that allows efficient recycling of the dihydrofolate resulting from TS activity. Arabidopsis thaliana possesses three DRTS genes, called AtDRTS1, AtDRTS2 and AtDRTS3, that are located downstream of three members of the sec14-like SFH gene family. In this study, a characterization of the AtDRTS genes identified alternatively spliced transcripts coding for AtDRTS isoforms which may account for monofunctional DHFR enzymes supporting pathways unrelated to DNA synthesis. Moreover, we discovered a complex differential regulation of the AtDRTS genes that confirms the expected involvement of the AtDRTS genes in cell proliferation and endoreduplication, but indicates also functions related to other cellular activities. AtDRTS1 is widely expressed in both meristematic and differentiated tissues, whereas AtDRTS2 expression is almost exclusively limited to the apical meristems and AtDRTS3 is preferentially expressed in the shoot apex, in stipules and in root cap cells. The differential regulation of the AtDRTS genes is associated to distinctive promoter architectures and the expression of AtDRTS1 in the apical meristems is strictly dependent on the presence of an intragenic region that includes the second intron of the gene. Upon activation of cell proliferation in germinating seeds, the activity of the AtDRTS1 and AtDRTS2 promoters in meristematic cells appears to be maximal at the G1/S phase of the cell cycle. In addition, the promoters of AtDRTS2 and AtDRTS3 are negatively regulated through E2F cis-acting elements and both genes, but not AtDRTS1, are downregulated in plants overexpressing the AtE2Fa factor. Our study provides new information concerning the function and the regulation of plant DRTS genes and opens the way to further investigations addressing the importance of folate synthesis with respect to specific cellular activities.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/enzymology , Arabidopsis/genetics , Gene Expression Regulation, Plant , Genes, Plant , Amino Acid Sequence , Arabidopsis/cytology , Arabidopsis/drug effects , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Computer Simulation , Cytokinins/pharmacology , Down-Regulation/drug effects , Down-Regulation/genetics , Gene Expression Profiling , Gene Expression Regulation, Plant/drug effects , Germination/drug effects , Germination/genetics , Glucuronidase/metabolism , Indoleacetic Acids/pharmacology , Introns/genetics , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Meristem/drug effects , Meristem/genetics , Open Reading Frames/genetics , Plants, Genetically Modified , Promoter Regions, Genetic , Seeds/drug effects , Seeds/genetics , Seeds/growth & development , Subcellular Fractions/drug effects , Subcellular Fractions/enzymology , Transformation, Genetic/drug effects
4.
Data Brief ; 8: 824-7, 2016 Sep.
Article in English | MEDLINE | ID: mdl-27508229

ABSTRACT

We report the proteomic dataset of gonads from wild Paracentrotus lividus related to the research article entitled "Proteomic changes occurring along gonad maturation in the edible sea urchin Paracentrotus lividus" [1]. Gonads of three individuals per sex in the recovery, pre-mature, mature, and spent stages were analyzed using a shotgun proteomics approach based on filter-aided sample preparation followed by tandem mass spectrometry, protein identification carried out using Sequest-HT as the search engine within the Proteome Discoverer informatics platform, and label-free differential analysis. The dataset has been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD004200.

5.
J Proteomics ; 144: 63-72, 2016 07 20.
Article in English | MEDLINE | ID: mdl-27265320

ABSTRACT

UNLABELLED: The reproductive stage of Paracentrotus lividus strongly influences product quality that, in turn, impacts significantly on the market price. Large, compact and sweet gonads are preferred, and sensory attributes are positively related to the ratio of nutritive phagocytes to gametes. Gonads at advanced maturation stages, although larger, have less desirable attributes, being more watery and bitter especially in females. Therefore, the best compromise among size, texture, and taste needs to be reached. In this study, wild P. lividus were collected along coastal Sardinia, and gonads in the recovery, pre-mature, mature, and spent stages were analyzed by gel-based and by shotgun proteomics. A detailed characterization of the proteome changes occurring in gonads of both sexes along maturation was achieved, and significant modifications were seen in numerous proteins involved in nutrient accumulation in nutritive phagocytes, as well as in gamete biology and maturation. Adding to an improved understanding of the P. lividus reproductive cycle in its natural environment, the results described in this work may form the basis for defining novel protein markers and procedures for an easier sexing and staging, and for monitoring sea urchin gonad maturation cycles in aquaculture plants. Mass spectrometry data are deposited in ProteomeXchange (PXD004200). SIGNIFICANCE: The sensory quality of P. lividus gonads is strongly influenced by the reproductive cycle, with significant changes in flavor, texture, and size. A better knowledge of the protein profiles, patterns, and markers associated with gonad sex and maturation stage can have useful implications for understanding and monitoring these changes. One of these is the ability to identify protein profiles specifically associated with a given stage and, in perspective, to identify maturation and sex markers. The comprehensive proteomic evaluation achieved in this work was made possible by the application of combined gel-based and shotgun approaches. As a result, this study generated the largest proteomic dataset available in the literature for P. lividus, as well as a general picture of protein abundance changes occurring along maturation.


Subject(s)
Paracentrotus , Animals , Sea Urchins
6.
Proteome Sci ; 12(1): 44, 2014.
Article in English | MEDLINE | ID: mdl-25342931

ABSTRACT

BACKGROUND: The zootechnical performance of three different commercial feeds and their impact on liver and serum proteins of gilthead sea bream (Sparus aurata, L.) were assessed in a 12 week feeding trial. The three feeds, named A, B, and C, were subjected to lipid and protein characterization by gas chromatography (GC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. RESULTS: Feed B was higher in fish-derived lipids and proteins, while feeds C and A were higher in vegetable components, although the largest proportion of feed C proteins was represented by pig hemoglobin. According to biometric measurements, the feeds had significantly different impacts on fish growth, producing a higher average weight gain and a lower liver somatic index in feed B over feeds A and C, respectively. 2D DIGE/MS analysis of liver tissue and Ingenuity pathways analysis (IPA) highlighted differential changes in proteins involved in key metabolic pathways of liver, spanning carbohydrate, lipid, protein, and oxidative metabolism. In addition, serum proteomics revealed interesting changes in apolipoproteins, transferrin, warm temperature acclimation-related 65 kDa protein (Wap65), fibrinogen, F-type lectin, and alpha-1-antitrypsin. CONCLUSIONS: This study highlights the contribution of proteomics for understanding and improving the metabolic compatibility of feeds for marine aquaculture, and opens new perspectives for its monitoring with serological tests.

7.
Plant Physiol ; 162(1): 157-79, 2013 May.
Article in English | MEDLINE | ID: mdl-23548781

ABSTRACT

The plastid ClpPRT protease consists of two heptameric rings of ClpP1/ClpR1/ClpR2/ClpR3/ClpR4 (the R-ring) and ClpP3/ClpP4/ClpP5/ClpP6 (the P-ring) and peripherally associated ClpT1/ClpT2 subunits. Here, we address the contributions of ClpP3 and ClpP4 to ClpPRT core organization and function in Arabidopsis (Arabidopsis thaliana). ClpP4 is strictly required for embryogenesis, similar to ClpP5. In contrast, loss of ClpP3 (clpp3-1) leads to arrest at the hypocotyl stage; this developmental arrest can be removed by supplementation with sucrose or glucose. Heterotrophically grown clpp3-1 can be transferred to soil and generate viable seed, which is surprising, since we previously showed that CLPR2 and CLPR4 null alleles are always sterile and die on soil. Based on native gels and mass spectrometry-based quantification, we show that despite the loss of ClpP3, modified ClpPR core(s) could be formed, albeit at strongly reduced levels. A large portion of ClpPR subunits accumulated in heptameric rings, with overaccumulation of ClpP1/ClpP5/ClpP6 and ClpR3. Remarkably, the association of ClpT1 to the modified Clp core was unchanged. Large-scale quantitative proteomics assays of clpp3-1 showed a 50% loss of photosynthetic capacity and the up-regulation of plastoglobules and all chloroplast stromal chaperone systems. Specific chloroplast proteases were significantly up-regulated, whereas the major thylakoid protease (FtsH1/FtsH2/FtsH5/FtsH8) was clearly unchanged, indicating a controlled protease network response. clpp3-1 showed a systematic decrease of chloroplast-encoded proteins that are part of the photosynthetic apparatus but not of chloroplast-encoded proteins with other functions. Candidate substrates and an explanation for the differential phenotypes between the CLPP3, CLPP4, and CLPP5 null mutants are discussed.


Subject(s)
Arabidopsis/enzymology , Arabidopsis/genetics , Chloroplasts/physiology , Endopeptidase Clp/genetics , Gene Expression Regulation, Plant , Proteomics , Alleles , Arabidopsis/growth & development , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Down-Regulation , Endopeptidase Clp/metabolism , Flowers/enzymology , Flowers/genetics , Flowers/growth & development , Flowers/physiology , Gene Expression Regulation, Developmental , Genotype , Homeostasis , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Phenotype , Photosynthesis , Plant Leaves/enzymology , Plant Leaves/genetics , Plant Leaves/growth & development , Plant Leaves/physiology , Seedlings/enzymology , Seedlings/genetics , Seedlings/growth & development , Seedlings/physiology , Seeds/enzymology , Seeds/genetics , Seeds/growth & development , Seeds/physiology , Sequence Deletion , Up-Regulation
8.
Infect Immun ; 79(9): 3833-45, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21690237

ABSTRACT

Milk fat globules (MFGs) are vesicles released in milk as fat droplets surrounded by the endoplasmic reticulum and apical cell membranes. During formation and apocrine secretion by lactocytes, various amounts of cytoplasmic crescents remain trapped within the released vesicle, making MFGs a natural sampling mechanism of the lactating cell contents. With the aim of investigating the events occurring in the mammary epithelium during bacterial infection, the MFG proteome was characterized by two-dimensional difference gel electrophoresis (2-D DIGE), SDS-PAGE followed by shotgun liquid chromatography-tandem mass spectrometry (GeLC-MS/MS), label-free quantification by the normalized spectral abundance factor (NSAF) approach, Western blotting, and pathway analysis, using sheep naturally infected by Mycoplasma agalactiae. A number of protein classes were found to increase in MFGs upon infection, including proteins involved in inflammation and host defense, cortical cytoskeleton proteins, heat shock proteins, and proteins related to oxidative stress. Conversely, a strikingly lower abundance was observed for proteins devoted to MFG metabolism and secretion. To our knowledge, this is the first report describing proteomic changes occurring in MFGs during sheep infectious mastitis. The results presented here offer new insights into the in vivo response of mammary epithelial cells to bacterial infection and open the way to the discovery of protein biomarkers for monitoring clinical and subclinical mastitis.


Subject(s)
Glycolipids/chemistry , Glycolipids/metabolism , Glycoproteins/chemistry , Glycoproteins/metabolism , Mastitis/veterinary , Mycoplasma Infections/veterinary , Mycoplasma agalactiae/metabolism , Sheep Diseases/immunology , Animals , Blotting, Western , Chromatography, Liquid , Cytoskeleton , Electrophoresis, Polyacrylamide Gel , Epithelium/microbiology , Epithelium/pathology , Female , Heat-Shock Proteins/biosynthesis , Lactation , Lipid Droplets , Mastitis/immunology , Mastitis/metabolism , Mastitis/microbiology , Milk/chemistry , Milk Proteins/analysis , Mycoplasma Infections/immunology , Mycoplasma Infections/metabolism , Mycoplasma Infections/microbiology , Mycoplasma agalactiae/genetics , Oxidative Stress , Proteomics , Sheep , Sheep Diseases/metabolism , Sheep Diseases/microbiology , Tandem Mass Spectrometry , Virulence Factors/metabolism
9.
J Proteomics ; 74(3): 350-8, 2011 Mar 01.
Article in English | MEDLINE | ID: mdl-21147282

ABSTRACT

Milk fat globule membranes (MFGM) are three-layered structures that enclose fat droplets, and are composed by an internal monolayer of endoplasmic reticulum origin, surrounded by a bilayer derived from the apical membrane of the lactating cell. In this work, an optimized protein extraction method was applied to sheep MFGM, and extracts were subjected to SDS-PAGE separation followed by shotgun LC tandem mass spectrometry (GeLC-MS/MS) for identification and characterization. In total, 140 unique sheep MFGM proteins (MFGMPs) were identified. All protein identification data were subjected to Gene Ontology (GO) classification for localization and function. Moreover, the relative abundance of all identified MFGMPs was estimated by means of the normalized spectral abundance factor (NSAF) approach, and GO abundance classes were obtained. The data gathered in this work provide a detailed picture of the proteome expressed in healthy sheep MFGs, and lay the foundations for future studies on sheep lactation physiology and on its alterations in pathological conditions.


Subject(s)
Cell Membrane/metabolism , Glycolipids/metabolism , Glycoproteins/metabolism , Membrane Proteins/chemistry , Proteomics/methods , Animals , Cattle , Chromatography, Gel/methods , Chromatography, Liquid/methods , Electrophoresis, Polyacrylamide Gel , Endoplasmic Reticulum/metabolism , Lipid Droplets , Mass Spectrometry/methods , Proteome , Sheep
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