Exposure of intestinal explants to NX, but not to DON, enriches the secretome in mitochondrial proteins.

NX is a type A trichothecene produced by Fusarium graminearum with limited information on its toxicity. NX is structurally similar to deoxynivalenol (DON), only differing by the lacking keto group at C8. Because of the structural similarity of the two toxins as well as their potential co-occurrence in food and feed, it is of interest to determine the toxicity of this new compound. In this study, we compared the protein composition of the extracellular media of pig intestinal explants (secretome) exposed to 10 µM of DON or NX for 4 h compared with controls. The combination of two complementary quantitative proteomic approaches (a gel-based and a gel-free approach) identified 18 and 23 differentially abundant proteins (DAPs) for DON and NX, respectively, compared to controls. Functional analysis suggested that, whereas DON toxicity was associated with decreased cell viability and cell destruction, NX toxicity was associated with an enrichment of mitochondrial proteins in the secretome. The presence of these proteins may be associated with the already known ability of NX to induce an intestinal inflammation. Overall, our results indicated that DON- and NX-induced changes in the extracellular proteome of intestinal explants are different. The increased leakage/secretion of mitochondrial proteins by NX may be a feature of NX toxicity.

Toxicity of DDT to the hooded oyster Saccostrea cucullata: Mortality, histopathology and molecular mechanisms as revealed by a proteomic approach.

Dichlorodiphenyltrichloroethane (DDT), a persistent organochlorine pesticide, has been linked to adverse biological effects in organisms. However, there is limited knowledge about its toxic effects on marine organisms and the underlying molecular mechanisms. This study investigated the toxic effects of DDT in the hooded oyster Saccostrea cucullata. The oysters were exposed to DDT at concentrations of 0, 10, 50, 100, 500, 1000 and 2000 µg/L for 96 h and the LC50 (96 h) was 891.25 µg/L. Two sublethal concentrations (10 and 100 µg/L) were used to investigate the histopathological effects and the proteome response. Histopathological results showed that DDT caused the alteration of mantle tissue. This included the induction of mucocytes in the epithelium and the inflammatory effect in the connective tissue indicated by the enlargement of blood sinus and hemocyte aggregation within the sinus. Proteomic results showed that, amongst approximately 500 protein spots that were detected across 2DE gels, 51 protein spots were differentially expressed (P < 0.01; fold change > 1.2). Of these, 29 protein spots were identified by LC-MS/MS. These included 23 up-regulated, 5 down-regulated and 1 fluctuating spots. Thus, we observed that stress response and cytoskeletal proteins are the central targets of DDT action. Furthermore, DDT alters the expression of proteins involved in energy metabolism, calcium homeostasis and other proteins of unknown function. Additionally, proteomic results clearly elucidated the molecular response of the histopathological changes which were driven by the alteration of cytoskeletal proteins. Our results improve the current knowledge of toxicity of the DDT to histology and molecular response of oyster proteome to DDT exposure. In addition, histopathological changes will be beneficial for the development of an appropriate guideline for health assessment of this species in ecotoxicological context.

ECM Characterization Reveals a Massive Activation of Acute Phase Response during FSGS.

The glomerular basement membrane (GBM) and extra-cellular matrix (ECM) are essential to maintain a functional interaction between the glomerular podocytes and the fenestrated endothelial cells in the formation of the slit diaphragm for the filtration of blood. Dysregulation of ECM homeostasis can cause Focal segmental glomerulosclerosis (FSGS). Despite this central role, alterations in ECM composition during FSGS have not been analyzed in detail yet. Here, we characterized the ECM proteome changes in miR-193a-overexpressing mice, which suffer from FSGS due to suppression of Wilms' tumor 1 (WT1). By mass spectrometry we identified a massive activation of the acute phase response, especially the complement and fibrinogen pathways. Several protease inhibitors (ITIH1, SERPINA1, SERPINA3) were also strongly increased. Complementary analysis of RNA expression data from both miR-193a mice and human FSGS patients identified additional candidate genes also mainly involved in the acute phase response. In total, we identified more than 60 dysregulated, ECM-associated genes with potential relevance for FSGS progression. Our comprehensive analysis of a murine FSGS model and translational comparison with human data offers novel targets for FSGS therapy.

Identification of Rabbit Oviductal Fluid Proteins Involved in Pre-Fertilization Processes by Quantitative Proteomics.

Oviductal fluid (ODF) proteins modulate and support reproductive processes in the oviduct. In the present study, proteins involved in the biological events that precede fertilization have been identified in the rabbit ODF proteome, isolated from the ampulla and isthmus of the oviduct at different time points within 8 h after intrauterine insemination. A workflow is used that integrates lectin affinity capture with stable-isotope dimethyl labeling prior to nanoLC-MS/MS analysis. In total, over 400 ODF proteins, including 214 lectin enriched glycoproteins, are identified and quantified. Selected data are validated by Western blot analysis. Spatiotemporal alterations in the abundance of ODF proteins in response to insemination are detected by global analysis. A subset of 63 potentially biologically relevant ODF proteins is identified, including extracellular matrix components, chaperones, oxidoreductases, and immunity proteins. Functional enrichment analysis reveals an altered peptidase regulator activity upon insemination. In addition to protein identification and abundance changes, N-glycopeptide analysis further identifies 281 glycosites on 199 proteins. Taken together, these results show, for the first time, the evolving oviductal milieu early upon insemination. The identified proteins are likely those that modulate in vitro processes, including spermatozoa function.

Pharmacokinetics of harpagoside in horses after intragastric administration of a Devil's claw (Harpagophytum procumbens) extract.

Devil's claw is used for the treatment of inflammatory symptoms and degenerative disorders in horses since many years, but without the substantive pharmacokinetic data. The pharmacokinetic parameters of harpagoside, the main active constituent of Harpagophytum procumbens DC ex Meisn., were evaluated in equine plasma after administration of Harpagophytum extract FB 8858 in an open, single-dose, two-treatment, two-period, randomized cross-over design. Six horses received a single dose of Harpagophytum extract, corresponding to 5 mg/kg BM harpagoside, and after 7 days washout period, 10 mg/kg BM harpagoside via nasogastric tube. Plasma samples at certain time points (before and 0-24 hr after administration) were collected, cleaned up by solid-phase extraction, and harpagoside concentrations were determined by LC-MS/MS using apigenin-7-glucoside as internal standard. Plasma concentration-time data and relevant parameters were described by noncompartmental model through PKSolver software. Harpagoside could be detected up to 9 hr after administration. Cmax was found at 25.59 and 55.46 ng/ml, t1/2 at 2.53 and 2.32 hr, respectively, and tmax at 1 hr in both trials. AUC0-inf was 70.46 and 117.85 ng hr ml-1 , respectively. A proportional relationship between dose, Cmax and AUC was observed. Distribution (Vz /F) was 259.04 and 283.83 L/kg and clearance (CL/F) 70.96 and 84.86 L hr-1  kg-1 , respectively. Treatment of horses with Harpagophytum extract did not cause any clinically detectable side effects.

Alterations in haemolymph proteome of Mytilus galloprovincialis mussel after an induced injury.

A proteomic and biochemical approach was performed to assess the effects of an induced muscle injury on the haemolymph of bivalve molluscs. For this purpose, Mytilus galloprovincialis were exposed to puncture of adductor muscle for three consecutive days, and their haemolymph proteome was then compared to healthy animals using 2-dimensional electrophoresis (2-DE) to identify proteins that differed significantly in abundance. Those proteins were then subjected to tandem mass spectrometry and 6 proteins, namely myosin, tropomyosin, CuZn superoxide dismutase (SOD), triosephosphate isomerase, EP protein and small heat shock protein were identified. SOD and tropomyosin changes were verified by spectrophotometric measurements and western blotting, respectively. As some of the proteins identified are related to muscular damage and oxidative stress, other biomarkers associated with these processes that can be evaluated by automatic biochemical assays were measured including troponin, creatine kinase (CK), and aspartate aminotransferase (AST) for muscle damage, and SOD, trolox equivalent antioxidant capacity (TEAC) and esterase activity (EA) for oxidative stress. Significantly higher concentrations of troponin, CK, AST, and TEAC were observed in mussels after puncture, being also possible biomarkers of non-specific induced damage.

Comprehensive proteomic analysis of Penicillium verrucosum.

Mass spectrometric identification of proteins in species lacking validated sequence information is a major problem in veterinary science. In the present study, we used ochratoxin A producing Penicillium verrucosum to identify and quantitatively analyze proteins of an organism with yet no protein information available. The work presented here aimed to provide a comprehensive protein identification of P. verrucosum using shotgun proteomics. We were able to identify 3631 proteins in an "ab initio" translated database from DNA sequences of P. verrucosum. Additionally, a sequential window acquisition of all theoretical fragment-ion spectra analysis was done to find differentially regulated proteins at two different time points of the growth curve. We compared the proteins at the beginning (day 3) and at the end of the log phase (day 12).

Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths.

The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 1012 /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 1011 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 109 particles.

Protein expression and transcription profiles of three strains of Aeromonas salmonicida ssp. salmonicida under normal and iron-limited culture conditions.

BACKGROUND: Aeromonas salmonicida is an important fish pathogen that produces a wide and varied array of virulence factors. Here we used iron deprivation by addition of the chelator 2'2-dipyridyl to induce the expression of several such virulence factors in three isolates of Aeromonas salmonicida (one avirulent and two virulent). By using SDS-PAGE followed by mass spectrometry, we identified proteins that appeared differentially expressed under these conditions. The differential transcription of the identified gene products were subsequently measured by reverse transcription quantitative real-time PCR (RT-qPCR). RESULTS: Our initial screening using SDS-PAGE identified five proteins that appeared differentially expressed in virulent and avirulent isolates or, within the same isolates, between bacteria cultivated under iron-rich or iron-deprived conditions. The transcription of the genes coding for these proteins were subsequently quantified by RT-qPCR. Results of this analysis demonstrated that the gene coding for alkyl hydroperoxide reductase (AhpC), a protein involved in oxidative stress response, was transcribed at a higher rate in the virulent strain as compared to the avirulent strain. Additionally, it was observed that addition of an iron chelator to the culture medium lead to a reduction of the transcription levels of the regulatory histone-like nucleoid structuring protein (H-NS). This was consistent in all three isolates. On the other hand, the transcription levels of the virulence array protein (VapA) and the protein ATP-synthetase F (ATPF) displayed only limited changes, despite being the dominant component of a protein fraction that displayed changes during the preliminary SDS-PAGE screening. This was true regardless of the culture conditions and of the isolates considered. Finally, transcription of the enzyme enolase was upregulated in the iron-deprived broths in all isolates. CONCLUSIONS: We identified several genes differentially expressed under culture conditions known to lead to the overexpression of virulence factors. In addition, we identified alkyl hydroperoxide as being overexpressed in the virulent isolates compared to the avirulent isolates. The results from this study will contribute to enhance our understanding of the virulence of A. salmonicida and may suggest new directions for further research.

Diet and phytogenic supplementation substantially modulate the salivary proteome in dairy cows.

Phytogenic compounds may influence salivation or salivary properties. However, their effects on the bovine salivary proteome have not been evaluated. We investigated changes in the bovine salivary proteome due to transition from forage to high-concentrate diet, with and without supplementation with a phytogenic feed additive. Eight non-lactating cows were fed forage, then transitioned to a 65% concentrate diet (DM basis) over a week. Cows were control (n = 4, CON) or supplemented with a phytogenic feed additive (n = 4, PHY). Proteomic analysis was conducted using liquid chromatography coupled with mass spectrometry. We identified 1233 proteins; 878 were bovine proteins, 189 corresponded to bacteria, and 166 were plant proteins. Between forage and high-concentrate, 139 proteins were differentially abundant (P < 0.05), with 48 proteins having a log2FC difference > |2|. The salivary proteome reflected shifts in processes involving nutrient utilization, body tissue accretion, and immune response. Between PHY and CON, 195 proteins were differently abundant (P < 0.05), with 37 having a log2FC difference > |2|; 86 proteins were increased by PHY, including proteins involved in smell recognition. Many differentially abundant proteins correlated (r > |0.70|) with salivary bicarbonate, total mucins or pH. Results provide novel insights into the bovine salivary proteome using a non-invasive approach, and the association of specific proteins with major salivary properties influencing rumen homeostasis. SIGNIFICANCE: Phytogenic compounds may stimulate salivation due to their olfactory properties, but their effects on the salivary proteome have not been investigated. We investigated the effect of high-concentrate diets and supplementation with a phytogenic additive on the salivary proteome of cows. We show that analysis of cows' saliva can be a non-invasive approach to detect effects occurring not only in the gut, but also systemically including indications for gut health and immune response. Thus, results provide unique insights into the bovine salivary proteome, and will have a crucial contribution to further understand animal response in terms of nutrient utilization and immune activity due to the change from forage to a high-energy diet. Additionally, our findings reveal changes due to supplementation with a phytogenic feed additive with regard to health and olfactory stimulation. Furthermore, findings suggest an association between salivary proteins and other components like bicarbonate content.

Bos d 13, A Novel Heat-Stable Beef Allergen.

SCOPE: Red meat, a staple food of Western diets, can also induce IgE-mediated allergic reactions. Yet, apart from the heat-labile protein serum albumin and the carbohydrate α-Gal, the molecules causing allergic reactions to red meat remain unknown. METHODS AND RESULTS: IgE reactivity profiles of beef-sensitized individuals are analyzed by IgE-immunoblotting with protein extracts from raw and cooked beef. Two IgE-reactive proteins are identified by peptide mass fingerprinting as myosinlight chain 1 (MYL1) and myosin light chain 3 (MYL3) in cooked beef extract and are designated Bos d 13 isoallergens. MYL1 and MYL3 are produced recombinantly in Escherichia coli. ELISAs proved their IgE reactivity and circular dichroism analysis showed that they represent folded molecules with remarkable thermal stability. In vitro gastrointestinal digestion experiments showed the higher stability of rMYL1 as compared to rMYL3. Exposure of a monolayer of Caco-2 cells to rMYL1 indicated that the molecule is able to cross intestinal epithelial cells without disturbing the integrity of the tight junctions, suggesting the sensitizing capacity of MYL1. CONCLUSION: MYLs are identified as novel heat-stable bovine meat allergens.

Caenorhabditis elegans N-glycan core beta-galactoside confers sensitivity towards nematotoxic fungal galectin CGL2.

The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galbeta1,4Fucalpha1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galbeta1,4Fucalpha1,6GlcNAc trisaccharide at 1.5 A resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms.

A Pilot Study on the Urine Proteome of Cats Fed a High-Protein Complete Diet, Supplemented with or without Arginine, Ornithine or Zeolite.

Proteome analyses can be used to detect biomarkers for the healthy and diseased organism. However, data in cats are scarce, and no information is available on the potential impact of nutritional interventions on the feline urine proteome. In the present study, a label-free shotgun proteomics approach was performed to investigate the urinary proteins of four healthy adult cats. Each animal received a high-protein complete diet without (w/o) or with supplements that could affect the protein metabolism: arginine (+100% compared to the arginine concentration in the w/o diet), ornithine (+200% compared to the arginine concentration in the w/o diet) or zeolite (0.375 g/kg body weight/day). Our results demonstrate a huge number of proteins in the urine of cats (516 ± 49, 512 ± 39, 399 ± 149 and 455 ± 134 in the w/o, arginine, ornithine and zeolite group, respectively), which are associated with several biological processes. In addition, up- and downregulated urinary proteins could be detected in the dietary supplementation periods. Overall, the present pilot study provides basic data on the urine proteome of healthy adult cats. With increasing information, the numerousness of urinary proteins implies the potential to identify biomarkers and metabolic pathways in the feline organism.

Distantly related plant and nematode core α1,3-fucosyltransferases display similar trends in structure-function relationships.

Here, we present a comparative structure-function study of a nematode and a plant core α1,3-fucosyltransferase based on deletion and point mutations of the coding regions of Caenorhabditis elegans FUT-1 and Arabidopsis thaliana FucTA (FUT11). In particular, our results reveal a novel "first cluster motif" shared by both core and Lewis-type α1,3-fucosyltransferases of the GT10 family. To evaluate the role of the conserved serine within this motif, this residue was replaced with alanine in FucTA (S218) and FUT-1 (S243). The S218A replacement completely abolished the enzyme activity of FucTA, while the S243A mutant of FUT-1 retained 20% of the "wild-type" activity. Based on the results of homology modeling of FucTA, other residues potentially involved in the donor substrate binding were examined, and mutations of N219 and R226 dramatically affected enzymatic activity. Finally, as both FucTA and FUT-1 were shown to be N-glycosylated, we examined the putative N-glycosylation sites. While alanine replacements at single potential N-glycosylation sites of FucTA resulted in a loss of up to 80% of the activity, a triple glycosylation site mutant still retained 5%, as compared to the control. In summary, our data indicate similar trends in structure-function relationships of distantly related enzymes which perform similar biochemical reactions and form the basis for future work aimed at understanding the structure of α1,3-fucosyltransferases in general.

Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation.

The pUL97 protein kinase encoded by human cytomegalovirus is a multifunctional determinant of the efficiency of viral replication and phosphorylates viral as well as cellular substrate proteins. Here, we report that pUL97 is expressed in two isoforms with molecular masses of approximately 90 and 100 kDa. ORF UL97 comprises an unusual coding strategy in that five in-frame ATG start codons are contained within the N-terminal 157 aa. Site-directed mutagenesis, transient expression of point and deletion mutants and proteomic analyses accumulated evidence that the formation of the large and small isoforms result from alternative initiation of translation, with the start points being at amino acids 1 and 74, respectively. In vitro kinase assays demonstrated that catalytic activity, in terms of autophosphorylation and histone substrate phosphorylation, was indistinguishable for the two isoforms. An analysis of the intracellular distribution of pUL97 by confocal laser-scanning microscopy demonstrated that both isoforms have a pronounced nuclear localization. Surprisingly, mapping experiments performed to identify the nuclear localization signal (NLS) of pUL97 strongly suggest that the mechanism of nuclear transport is distinct for the two isoforms. While the extreme N terminus (large isoform) comprises a highly efficient, bipartite NLS (amino acids 6-35), a second sequence apparently conferring a less efficient mode of nuclear translocation was identified downstream of amino acid 74 (small and large isoforms). Taken together, the findings argue for a complex mechanism of nuclear translocation for pUL97 which might be linked with fine-regulatory differences between the two isoforms.

Effects of Yersinia ruckeri invasion on the proteome of the Chinook salmon cell line CHSE-214.

Yersinia ruckeri is an important bacterial pathogen of fish, in particular salmonids, it has been associated with systemic infections worldwide and, like many enteric bacteria, it is a facultative intracellular pathogen. However, the effect of Y. ruckeri's interactions with the host at the cellular level have received little investigation. In the present study, a culture of Chinook Salmon Embryo (CHSE) cell line was exposed to Y. ruckeri. Afterwards, the proteins were investigated and identified by mass spectrometry and compared to the content of unexposed cultures. The results of this comparison showed that 4.7% of the identified proteins were found at significantly altered concentrations following infection. Interestingly, infection with Y. ruckeri was associated with significant changes in the concentration of surface adhesion proteins, including a significantly decreased presence of ß-integrins. These surface adhesion molecules are known to be the target for several adhesion molecules of Yersiniaceae. The concentration of several anti-apoptotic regulators (HSP90 and two DNAj molecules) appeared similarly downregulated. Taken together, these findings suggest that Y. ruckeri affects the proteome of infected cells in a notable manner and our results shed some light on the interaction between this important bacterial pathogen and its host.

Author Correction: Shotgun proteomics reveals putative polyesterases in the secretome of the rock-inhabiting fungus Knufia chersonesos.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Shotgun proteomics reveals putative polyesterases in the secretome of the rock-inhabiting fungus Knufia chersonesos.

Knufia chersonesos is an ascomycotal representative of black fungi, a morphological group of polyextremotolerant melanotic fungi, whose ability to resort to recalcitrant carbon sources makes it an interesting candidate for degradation purposes. A secretome screening towards polyesterases was carried out for the fungus and its non-melanized mutant, grown in presence of the synthetic copolyester Polybutylene adipate terephthalate (PBAT) as additional or sole carbon source, and resulted in the identification of 37 esterolytic and lipolytic enzymes across the established cultivation conditions. Quantitative proteomics allowed to unveil 9 proteins being constitutively expressed at all conditions and 7 which were instead detected as up-regulated by PBAT exposure. Protein functional analysis and structure prediction indicated similarity of these enzymes to microbial polyesterases of known biotechnological use such as MHETase from Ideonella sakaiensis and CalA from Candida albicans. For both strains, PBAT hydrolysis was recorded at all cultivation conditions and primarily the corresponding monomers were released, which suggests degradation to the polymer's smallest building block. The work presented here aims to demonstrate how investigations of the secretome can provide new insights into the eco-physiology of polymer degrading fungi and ultimately aid the identification of novel enzymes with potential application in polymer processing, recycling and degradation.