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Glycans play key roles in host-pathogen interactions; thus, knowing the N-glycomic repertoire of a pathogen can be helpful in deciphering its methods of establishing and sustaining a disease. Therefore, we sought to elucidate the glycomic potential of the facultative amoebal parasite Acanthamoeba. This is the first study of its asparagine-linked glycans, for which we applied biochemical tools and various approaches of mass spectrometry. An initial glycomic screen of eight strains from five genotypes of this human pathogen suggested, in addition to the common eukaryotic oligomannose structures, the presence of pentose and deoxyhexose residues on their N-glycans. A more detailed analysis was performed on the N-glycans of a genotype T11 strain (4RE); fractionation by HPLC and tandem mass spectrometric analyses indicated the presence of a novel mannosylfucosyl modification of the reducing terminal core as well as phosphorylation of mannose residues, methylation of hexose and various forms of pentosylation. The largest N-glycan in the 4RE strain contained two N-acetylhexosamine, thirteen hexose, one fucose, one methyl, and two pentose residues; however, in this and most other strains analyzed, glycans with compositions of Hex(8-9)HexNAc(2)Pnt(0-1) tended to dominate in terms of abundance. Although no correlation between pathogenicity and N-glycan structure can be proposed, highly unusual structures in this facultative parasite can be found which are potential virulence factors or therapeutic targets.

The modification of α1,6-linked fucose residues attached to the proximal (reducing-terminal) core N-acetylglucosamine residue of N-glycans by β1,4-linked galactose (“GalFuc” epitope) is a feature of a number of invertebrate species including the model nematode Caenorhabditis elegans. A pre-requisite for both core α1,6-fucosylation and β1,4-galactosylation is the presence of a nonreducing terminal N-acetylglucosamine; however, this residue is normally absent from the final glycan structure in invertebrates due to the action of specific hexosaminidases. Previously, we have identified two hexosaminidases (HEX-2 and HEX-3) in C. elegans, which process N-glycans. In the present study, we have prepared a hex-2;hex-3 double mutant, which possesses a radically altered N-glycomic profile. Whereas in the double mutant core α1,3-fucosylation of the proximal N-acetylglucosamine was abolished, the degree of galactosylation of core α1,6-fucose increased, and a novel Galα1,2Fucα1,3 moiety attached to the distal core N-acetylglucosamine residue was detected. Both galactosylated fucose moieties were also found in two parasitic nematodes, Ascaris suum and Oesophagostomum dentatum. As core modifications of N-glycans are known targets for fungal nematotoxic lectins, the sensitivity of the C. elegans double hexosaminidase mutant was assessed. Although this mutant displayed hypersensitivity to the GalFuc-binding lectin CGL2 and the N-acetylglucosamine-binding lectin XCL, the mutant was resistant to CCL2, which binds core α1,3-fucose. Thus, the use of C. elegans mutants aids the identification of novel N-glycan modifications and the definition of in vivo specificities of nematotoxic lectins with potential as anthelmintic agents.

Fifty samples consisted of 24 maize kernels and 26 maize based-food products from retail market in Yogyakarta, Indonesia were analysed for deoxynivalenol (DON) using high performance liquid chromatography (HPLC) combined with ultraviolet detection after immunoaffinity column (IAC) clean-up process. Prior to use, performance of the analytical method was evaluated in term of recovery, repeatability, and detection limit. The recoveries of DON in spiked samples (50–360 μg DON/kg sample) were obtained in a range of 70.9–97.3% with relative standard deviation (RSD) for repeatability within a day less than 16%. Limit of detection based on the response ratio of signal to noise (3/1) was noted being 20 μg DON/kg sample. All analysed samples contained DON ranging between 47 and 348 μg/kg with median and means of 111.0 and 124.6 μg/kg, respectively. The contamination of DON in the studied samples may not be taken into account as hazard for human and animal health since the concentrations were lower than maximum limit of DON contamination in foodstuffs (500 μg/kg) established by European Commission (2006).

Concentration profile of zearalenone (ZON) and its metabolites in plasma, urine and faeces samples of horses fed with Fusarium toxin-contaminated oats is described. In plasma, β-zearalenol (β-ZOL) was detected at high levels on day 10 of the study (3.21-6.24 μg/l). β-Zearalenol and α-zearalenol were the major metabolites in urine. Zearalenone, α-ZOL and β-ZOL were predominantly found in faeces. Zearalanone could also be detected in urine (1.34-5.79 μg/l) and faeces (1 μg/kg). The degree of glucuronidation was established in all sample types, approximately 100% in urine and plasma. Low per cent of glucuronidation (4-15%) was found in faeces samples. The results indicate the main conversion of ZON into β-ZOL in horse. This finding could explain why horse is not susceptible to ZON in comparison with swine which produce α-ZOL as a predominant metabolite.

Trichomonad species are widespread unicellular flagellated parasites of vertebrates which interact with their hosts through carbohydrate-lectin interactions. In the past, some data have been accumulated regarding their lipo(phospho)glycans, a major glycoconjugate on their cell surfaces; on the other hand, other than biosynthetic aspects, few details about their N-linked oligosaccharides are known. In this study, we present both mass spectrometric and high-performance liquid chromatography data about the N-glycans of different strains of Trichomonas vaginalis, a parasite of the human reproductive tract. The major structure in all strains examined is a truncated oligomannose form (Man(5)GlcNAc(2)) with α1,2-mannose residues, compatible with a previous bioinformatic examination of the glycogenomic potential of T. vaginalis. In addition, dependent on the strain, N-glycans modified by pentose residues, phosphate or phosphoethanolamine and terminal N-acetyllactosamine (Galβ1,4GlcNAc) units were found. The modification of N-glycans by N-acetyllactosamine in at least some strains is shared with the lipo(phospho)glycan and may represent a further interaction partner for host galectins, thereby playing a role in binding of the parasite to host epithelia. On the other hand, the variation in glycosylation between strains may be the result of genetic diversity within this species.

Aflatoxin B(1) is a common contaminant of poultry feeds in tropical and subtropical climates. Research during the last five decades has well established the negative effects of the mycotoxin on health of poultry. However, the last ten years of relevant data have accentuated the potential of low levels of aflatoxin B(1) to deteriorate broiler performance. In this regard, any attempt to establish a dose-effect relationship between aflatoxin B(1) level and broiler performance is also complicated due to differences in types of broilers and length of exposure to the mycotoxin in different studies. Contrary to the prevalent notion regarding literature saturation with respect to aflatoxicosis of chicken, many areas of aflatoxicosis still need to be explored. Literature regarding effects of the mycotoxin on the gastrointestinal tract in this regard is particular scanty and non-conclusive. In addition to these issues, the metabolism of aflatoxin B(1) and recently proposed hypotheses regarding biphasic effects of the mycotoxin in broilers are briefly discussed.

Planarial species are of especial interest to biologists due to the phenomenon of pluripotency and, in comparison to other developmental processes, it can be hypothesised that glycan–lectin interactions may play a role. In order to examine the N-glycans of one of these organisms, Dugesia japonica, peptide:N-glycosidase A was employed and the released glycans were subject to pyridylamination, HPLC and mass spectrometric analysis. A range of oligomannosidic glycans was observed with a trimethylated Man5GlcNAc2 structure being the dominant species. Three glycans were also observed to contain deoxyhexose; in particular, a glycan with the composition Hex4HexNAc2Fuc1Me2 was revealed by exoglycosidase digestion, in combination with MS/MS, to contain a galactosylated core α1,6-fucose residue, whereas this core modification was found to be capped with a methylhexose residue in the case of a Hex5HexNAc2Fuc1Me3 structure. This is the first report of these types of structures in a platyhelminth and indicates that the ‘GalFuc’ modification of N-glycans is not just restricted to molluscs and nematodes. Copyright © 2011 John Wiley & Sons, Ltd.

The paper presents a new sample clean-up method based on immuno-ultrafiltration for the analysis of ochratoxin A in cereals. In contrast to immunoaffinity chromatography, in immuno-ultrafiltration, the antibodies are used in non-immobilised form. Ochratoxin A was extracted with ACN/water (60/40, v/v), and the extract was loaded onto the ultrafiltration device. After a washing step with phosphate-buffered saline, containing 0.05% Tween 20, ochratoxin A was eluted with MeOH/acetic acid (99/1, v/v). The detection of ochratoxin A was carried out with high-performance liquid chromatography and a fluorescence detector coupled to an electrochemical cell (Coring cell). The electrochemical cell was used to eliminate matrix interferences by oxidising matrix compounds. The method was validated by repeatedly analysing spiked barley and rye samples as well as a certified wheat reference material. Recoveries and standard deviations (1 SD) were found to be 71 ± 9%, 77 ± 12% and 77 ± 8% in wheat, barley and rye, respectively. The limit of detection (S/N = 3) and limit of quantitation (S/N = 10) were determined to be 0.4 μg kg(-1) and 1 μg kg(-1). The analysis of the certified reference material resulted in ochratoxin A concentrations which were in the range assigned by the producer. Additionally, the effect of the electrochemical cell on other widely used clean-up techniques, namely the immunoaffinity clean-up and multifunctional columns (Mycosep #229), was evaluated. In all clean-up methods, an improvement of the chromatogram quality was registered.

Saliva contains a number of proteins that may be useful as biomarkers of health and disease and can be easily obtained from large numbers of animals in a non-invasive, stress-free way. The objective of this study was to explore the protein composition of porcine saliva from 10 specific pathogen free pigs using first one-dimensional SDS–PAGE and then two-dimensional electrophoresis and mass spectrometry. A reference proteome pattern for porcine saliva was established with the identification of 13 different, mainly saliva-specific, proteins. These reference data will facilitate the investigation of salivary proteins potentially altered in disease and could serve as novel diagnostic biomarkers.

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Art v 1, the major pollen allergen of the composite plant mugwort (Artemisia vulgaris) has been identified recently as a thionin-like protein with a bulky arabinogalactan-protein moiety. A close relative of mugwort, ragweed (Ambrosia artemisiifolia) is an important allergen source in North America, and, since 1990, ragweed has become a growing health concern in Europe as well. Weed pollen-sensitized patients demonstrated IgE reactivity to a ragweed pollen protein of apparently 29-31 kDa. This reaction could be inhibited by the mugwort allergen Art v 1. The purified ragweed pollen protein consisted of a 57-amino acid-long defensin-like domain with high homology to Art v 1 and a C-terminal proline-rich domain. This part contained hydroxyproline-linked arabinogalactan chains with one galactose and 5 to 20 and more alpha-arabinofuranosyl residues with some beta-arabinoses in terminal positions as revealed by high field NMR. The ragweed protein contained only small amounts of the single hydroxyproline-linked beta-arabinosyl residues, which form an important IgE binding determinant in Art v 1. cDNA clones for this protein were obtained from ragweed flowers. Immunological characterization revealed that the recombinant ragweed protein reacted with30% of the weed pollen allergic patients. Therefore, this protein from ragweed pollen constitutes a novel important ragweed allergen and has been designated Amb a 4.

Eighty-one rice samples were purchased from different markets in Vienna and were analysed for their aflatoxin content. The samples were extracted using methanol in water (80/20 v/v) followed by immunoaffinity clean up. The determination was carried out by HPLC–FLD coupled to a Kobracell. Different samples including basmati rice, whole grain rice, long grain rice, short grain rice as well as puffed rice were investigated. Moreover, conventionally and organically produced rice were compared. The results revealed that 24 out of 81 samples contained detectable amounts of aflatoxins. Aflatoxin B1 could be quantified in 15 samples and aflatoxin B2 in one sample. The contamination range was noted to be between 0.45 μg kg−1 and 9.86 μg kg−1 for aflatoxin B1 and 1.5 μg kg−1 for aflatoxin B2. Aflatoxins G1 and G2 were not detected in any sample. Three samples exceeded the maximum levels set in the European Union; having AFB1 concentrations of 2.16, 2.85 and 9.86 μg kg−1. In the three organic produced rice samples only traces of aflatoxins were found.

Galectin CGL2 from the ink cap mushroom Coprinopsis cinerea displays toxicity toward the model nematode Caenorhabditis elegans. A mutation in a putative glycosyltransferase-encoding gene resulted in a CGL2-resistant C. elegans strain characterized by N-glycans lacking the β1,4-galactoside linked to the α1,6-linked core fucose. Expression of the corresponding GALT-1 protein in insect cells was used to demonstrate a manganese-dependent galactosyltransferase activity. In vitro, the GALT-1 enzyme showed strong selectivity for acceptors with α1,6-linked N-glycan core fucosides and required Golgi- dependent modifications on the oligosaccharide antennae for optimal synthesis of the Gal-β1,4-fucose structure. Phylogenetic analysis of the GALT-1 protein sequence identified a novel glycosyltransferase family (GT92) with members widespread among eukarya but absent in mammals.

This paper presents a limited survey of aflatoxin M1 (AFM1) in Indonesian milk. AFM1 concentrations of 113 fresh milk samples, collected in 2006 from farms in five different areas of the Yogyakarta Province were analysed. The fresh milk samples were taken directly from dairy farms before pasteurisation. Enzyme-linked immunosorbent assay (ELISA) was used for the analysis of milk samples. Results show that in 48 samples (42.5%) the AFM1 concentrations were less than 5 ng/L and in 31 samples (27.4%) AFM1 was found between 5 and 10 ng/L. In 34 samples (30.1%) the concentrations were above 10 ng/L. None of contaminated samples exceeded the European Union regulation limits of 25 and 50 ng AFM1/L for infant and adult consumption, respectively. Since AFM1 is derived from aflatoxin B1 (AFB1) contained in cow feedstuffs, based on the contamination levels of AFM1 found in this study, the exposure of animals to AFB1 seems to be low.

Overexpression of matrix metalloproteinases (MMPs) has been associated with increased tumor aggressiveness and metastasis dissemination. We investigated whether the contrasting metastatic behavior of feline and canine osteosarcoma is related to levels and activities of MMP2 and MMP9. Zymography and immunohistochemistry were used to determine expression levels of MMP2 and MMP9 in canine and feline osteosarcoma. Using immunohistochemistry, increased MMP9 levels were identified in most canine osteosarcomas, whereas cat samples more often displayed moderate levels. High levels of pro-MMP9, pro-MMP2, and active MMP2 were detected by gelatin zymography in both species, with significantly higher values for active MMP2 in canine osteosarcoma. These findings indicate that MMP2 is probably involved in canine and feline osteosarcoma and their expression and activity could be associated with the different metastatic behavior of canine and feline osteosarcoma.La surexpression de métalloprotéases de matrice (MPMs) a été associée avec une augmentation de l’agressivité des tumeurs et de la dissémination métastasique. Nous avons cherché à savoir si le comportement métastasique contrastant d’ostéosarcomes félin et canin est relié aux quantités et à l’activité de MPM2 et MPM9. La zymographie et l’immunohistochimie ont été utilisées afin de déterminer les niveaux d’expression de MPM2 et de MPM9 dans des ostéosarcomes canins et félins. En utilisant l’immunohistochimie, des quantités augmentées de MPM9 ont été identifiées dans la plupart des ostéosarcomes canins, alors que les échantillons félins montraient plus souvent des quantités modérées. Des niveaux élevés de pro-MPM9, pro-MPM2, et de la MPM2 active ont été détectés par zymographie sur gélatine chez les deux espèces, avec des valeurs significativement plus élevées pour de la MPM2 active dans les ostéosarcomes canins. Ces données indiquent que MPM2 est probablement impliquée dans les ostéosarcomes canins et félins et que leur expression et activité pourraient être associé avec le comportement métastasique différent des ostéosarcomes canins et félins.(Traduit par Docteur Serge Messier).

Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100⿿200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1ÿ109 particles, could be processed with a 1mL monolith within 47min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity.

The foodborne pathogen Listeria monocytogenes, responsible for listeriosis a rare but severe infection disease, can survive in the food processing environment for month or even years. So-called persistent L. monocytogenes strains greatly increase the risk of (re)contamination of food products, and are therefore a great challenge for food safety. However, our understanding of the mechanism underlying persistence is still fragmented. In this study we compared the exoproteome of three persistent strains with the reference strain EGDe under mild stress conditions using 2D differential gel electrophoresis. Principal component analysis including all differentially abundant protein spots showed that the exoproteome of strain EGDe (sequence type (ST) 35) is distinct from that of the persistent strain R479a (ST8) and the two closely related ST121 strains 4423 and 6179. Phylogenetic analyses based on multilocus ST genes showed similar grouping of the strains. Comparing the exoproteome of strain EGDe and the three persistent strains resulted in identification of 22 differentially expressed protein spots corresponding to 16 proteins. Six proteins were significantly increased in the persistent L. monocytogenes exoproteomes, among them proteins involved in alkaline stress response (e.g. the membrane anchored lipoprotein Lmo2637 and the NADPH dehydrogenase NamA). In parallel the persistent strains showed increased survival under alkaline stress, which is often provided during cleaning and disinfection in the food processing environments. In addition, gene expression of the proteins linked to stress response (Lmo2637, NamA, Fhs and QoxA) was higher in the persistent strain not only at 37 °C but also at 10 °C. Invasion efficiency of EGDe was higher in intestinal epithelial Caco2 and macrophage-like THP1 cells compared to the persistent strains. Concurrently we found higher expression of proteins involved in virulence in EGDe e.g. the actin-assembly-inducing protein ActA and the surface virulence associated protein SvpA. Furthermore proteins involved in cell wall modification, such as the lipoteichonic acid primase LtaP and the N-acetylmuramoyl-l-alanine amidase (Lmo2591) are more abundant in EGDe than in the persistent strains and could indirectly contribute to virulence. In conclusion this study provides information about a set of proteins that could potentially support survival of L. monocytogenes in abiotic niches in food processing environments. Based on these data, a more detailed analysis of the role of the identified proteins under stresses mimicking conditions in food producing environment is essential for further elucidate the mechanism of the phenomenon of persistence of L. monocytogenes.

The paper describes the development of a simple and highly selective method for the determination of deoxynivalenol (DON) in food and feed samples. It combines sample cleanup with sol-gel immunoaffinity columns containing monoclonal anti-DON antibodies and quantification of DON by high-performance liquid chromatography with ultraviolet detection. The sol-gel immunoaffinity columns are as selective as commercial DON immunoaffinity columns but superior with regard to production costs, storage stability, and reusability. In applying the method for the analysis of maize, wheat, and spaghetti samples, it offers detection limits (LOD, S/N = 3) of 240, 200, and 207 ng/g, and recoveries of 83, 99, and 97%, respectively.

The paper describes a method for the sensitive and selective determination of zearalenone and its metabolites in urine, plasma and faeces of horses by high performance liquid chromatography and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS). While only one step sample clean-up by an immunoaffinity column (IAC) was sufficient for plasma samples, urine and faeces samples had to be prepared by a combination of a solid-phase extraction (SPE) and an immunoaffinity column. The method allows the simultaneous determination of zearalenone and all of its metabolites; alpha-zearalenol, beta-zearalenol, alpha-zearalanol, beta-zearalanol and zearalanone. Dideuterated zearalanone was used as internal standard for quantification and the study of the matrix effect. Recovery rates between 56 and slightly above 100% were achieved in urine samples, and more than 80% in plasma and faeces samples. The limits of detection ranged from 0.1-0.5 microg/l or microg/kg, the limits of quantification from 0.5-1.0 microg/l or microg/kg. The practical use of the method is demonstrated by the analysis of spiked and naturally contaminated urine, plasma and faeces of horses.

The effect of pan-frying on the formation of cholesterol oxidation products (COPs) in different processed meat samples (beef patties, braised meat, and fillets of pork) was studied. Samples were pan-fried with or without addition of oil. Different unsaturated oils (olive oil, corn oil or partly hydrogenated plant oil) were used throughout the study. After extraction, seven toxicologically relevant COPs were analyzed using LC–MS. Prior to heat processing up to 6.7 mg COPs/kg extracted fat could be detected in the raw material. Neither the cholestanetriol nor 25-hydroxycholesterol, which are the most cytotoxic COPs in vitro, were detectable in any sample. Differences in the COPs contents were observed between beef (up to 16.5 mg/kg extracted fat) and pork (up to 22.2 mg/kg extracted fat) samples. In prepared samples higher COPs content was noted compared with raw samples. Generally, a certain order of COPs increase dependent on the plant oil used could be recognized: corn oil < partially hydrogenated plant oil < olive oil. It appears that short heating time, mild heating conditions, and the use of fresh and shortly stored raw materials keep COPs levels low.

An experiment was conducted to study the effects of deoxynivalenol (DON) on the performance of broilers, organ weights, and intestinal histology and to evaluate the efficacy of a probiotic feed additive (PB, Eubacterium sp.) with the ability to deepoxidize DON. Two hundred seventy-seven 1-d-old broiler chicks were randomly assigned to 1 of the 3 dietary treatments for 6 wk. The dietary treatments were 1) control; 2) artificially contaminated diets with 10 mg of DON/kg of diet; 3) DON-contaminated diets plus probiotic feed additive (DON-PB). The BW and the efficiency of feed utilization were not adversely affected (P > 0.05) by the inclusion of DON in the diets. A slight improvement in feed intake and BW gain over the course of the experiment was observed in broilers fed DON-PB with no change in feed efficiency. The absolute or relative organ weights were not altered (P > 0.05) in broilers fed the diet containing DON compared with controls and the DON-PB group. The absolute liver weights were numerically increased (P < 0.1) for broilers receiving the diet containing DON-PB. There were no significant differences in the absolute and relative weights of the gizzard, duodenum, pancreas, heart, and spleen. However, the absolute and relative weights of the jejunum and cecum were increased for DON-PB-fed broilers compared with the controls and DON group. No pathological lesions were found in the gut of birds fed DON-contaminated diets during the feeding trial, but mild intestinal changes were observed. The DON altered small intestinal morphology, especially in the duodenum and jejunum, where villi were shorter and thinner (P < 0.05). The addition of the eubacteria to the DON-contaminated feed of the broilers effectively alleviated the histological alterations caused by DON and led to comparable villus length as in the control group. In conclusion, diets with DON contamination below levels that induce a negative impact on health and performance could affect small intestinal morphology in broilers. The histological alterations caused by DON were reduced by supplementing the DON-containing diets with PB. This indicates that in case of DON contamination of feedstuffs, the addition of PB would be a proper way to counteract the possible effects caused by this mycotoxin.

A feeding trial was conducted to evaluate the effects of moderate dietary concentrations of deoxynivalenol (DON) during a 21-day feeding experiment on the performance of broilers. Fifteen 1-day-old broiler chicks were randomly divided into two groups. The control group was fed non-contaminated diet. Another group of broilers was fed a diet naturally contaminated with 5 mg DON/kg diet. Deoxynivalenol had no effect (p0.05) on feed consumption, feed conversion, body-weight gain, live body weight or mortality. The absolute and relative weight of the organs (gizzard, pancreas, heart, spleen, colon and caecum) were not altered by the dietary inclusion of DON contaminated grain. However, both the absolute and relative weight of small intestine was decreased (p0.01) in DON fed broilers compared to the controls. No gross lesions were detected in any of the organs of birds fed contaminated wheat during the feeding trial. The microscopic examination revealed that, the height and the width of villi in duodenum decreased (p0.05) in birds fed DON contaminated wheat compared to controls. On the other hand the height and the width of jejunum villi were not affected (p0.05). This study indicates that feeding DON for 21 days to broiler chickens at a concentration of up to 5 mg/kg of diet influenced the weight of the small intestine as well as intestinal histology, especially the duodenum, as evidenced by shorter and thinner villi. In conclusion, diets with DON contamination below levels that induce negative impact on health and performance could affect small intestinal morphology in broilers.

Deoxynivalenol (DON) is common in European cereal grains, and of all the trichothecenes, poses the greatest problems to animal health. The present study investigated the effects of DON on electrophysiological parameters in laying hens' jejunum mounted in Ussing chambers. In vitro studies were performed to measure the effects of different luminal concentrations of DON (0.5, 1, 5, and 10 microg/mL) on the transmural potential difference, electrical tissue resistance, and electrogenic ion flux rates (short-circuit current, Isc) across the isolated gut mucosa. Deoxynivalenol did not alter (P0.05) the transmural potential difference. Resistance was higher (P0.05) in the tissues exposed to DON compared with basal values. Deoxynivalenol caused a dose-dependent decrease in Isc (P0.05). To investigate the mechanism of action of DON, amiloride (a specific inhibitor for Na+ transport) was added after incubation of the tissue with DON. Amiloride did not decrease (P0.05) Isc under these conditions. This may indicate that DON inhibited the Na+ transport before addition of amiloride, which did not then show further inhibitory effects. The addition of D-glucose (5 mmol/L) on the luminal side of the isolated mucosa increased (P0.05) Isc, and this effect was reversed by phlorizin (a specific inhibitor of sodium/glucose transporter 1), indicating that the glucose-induced Isc increase may be due to Na+-D-glucose cotransport. In our study, DON decreased (P0.05) the glucose-induced Isc in a similar way to phlorizin. The remarkable similarity between the effects of phlorizin and DON on electrical properties seemed to be consistent with their common ability to inhibit Na+-D-glucose cotransport. In conclusion, DON decreased the Isc via inhibition of Na+ transport. The effect on intestinal electrical properties was similar to that of phlorizin after addition of glucose, suggesting that DON may inhibit Na+-D-glucose cotransport. The inhibition of Na+ transport and Na+-D-glucose cotransport are important mechanisms of DON toxicity in the intestine of laying hens.

Most amino acids are cotransported with sodium. Deoxynivalenol (DON) decreases glucose absorption in the chicken small intestine in vivo and in vitro, and this effect is apparently mediated by the inhibition of the sodium D-glucose cotransporter. DON could selectively modulate the activities of other intestinal transporters. In order to assess this hypothesis, a study was conducted to characterize the in vitro effects of DON in the presence of mucosal amino acids, using L-proline as a model, on the electrophysiological parameters in the jejunums of laying hens. L-Proline (mucosal concentration of 1 mmol/L) was added to a stripped proximal part of jejunum sheets mounted in Ussing chambers in Ringer buffer, and the electrical properties were measured. The transmural potential difference (PD) was nearly constant between the treatments. The tissue resistance (Rt) was higher (P0.05) in the tissues exposed to DON compared with basal values and the values after addition of L-proline. Addition of L-proline on the luminal side of the isolated mucosa increased (P0.05) the short circuit-current (Isc), and it decreased (P0.05) after addition of DON, indicating that the proline-induced Isc was altered by DON. The addition of proline after incubation of the tissues with DON had no effect (P0.05) on PD or Rt. Proline did not increase the Isc under these conditions. DON decreased (P0.1) the Isc after addition of proline, indicating that DON inhibited the Na+-amino acid co-transport. We concluded from the present study that the amino acid cotransporter activity appears to be highly sensitive to DON suppression.

A survey of zearalenone (ZON) in Indonesian maize-based food and feed was performed in 89 samples using an enzyme-linked immunosorbent assay (ELISA). Results indicated that 32 samples (36.0%) were contaminated in a range from 5.5 to 526 μg/kg. Only one highly contaminated sample was observed in a category of home-made food samples (>500 μg/kg) and the highest percentage of contaminated samples (>85.7%) was noted in a category of poultry feed. The positive samples based on the ELISA analyses were then confirmed using a high performance liquid chromatography (HPLC) method in combination with an immuno-affinity column for clean-up. Prior to sample measurement, the HPLC method was validated using a blank maize matrix. The overall average recovery of blank maize spiked with ZON at levels of 200–750 μg/kg were 100.2%. Detection limit based on a signal-to-noise ratio 3:1 was 3.0 μg/kg for ZON in maize samples and the linear range applicable for analyses was 5–900 μg/kg of ZON. Of 32 samples confirmed by HPLC, 25 samples were observed to be contaminated in a comparable range from 6.9 to 589 μg/kg. Comparative analysis of 25 positive samples using the ELISA and HPLC methods showed a reasonable correlation (R2=0.9258).

A feeding trial was conducted to evaluate the effects of diets contaminated with deoxynivalenol (DON on the performance of broilers and on the electro-physiological parameters of the gut. The control group was fed the starter and finisher diets without addition of DON. Another group of broilers was fed the starter and finisher diets with 10 mg/kg DON, whereas another group was fed the DON-contaminated diets supplemented with a microbial feed additive (Eubacterium sp.). The diets were provided ad libitum for 6 wk. DON had no effect (P > 0.05) on feed consumption, feed conversion, or body weight. The effect of DON on the electrophysiological parameters of the jejunum was studied in vitro using isolated gut mucosa in Ussing chambers. At the end of the feeding period, 7 birds from each group were killed, and the basal and glucose stimulated transmural potential difference (PD), short-circuit current (Isc), and electrical resistance (R) were measured in the isolated gut mucosa to characterize the electrical properties of the gut. The transmural PD did not differ (P > 0.05) among groups. The tissue resistance was greater (P < 0.05) in birds receiving DON and the microbial feed additive than in the controls and DON group. Addition of D-glucose on the luminal side of the isolated mucosa increased (P < 0.05) Isc in the control and DON-probiotic (Eubacterium sp.; PB) groups, whereas it decreased (P < 0.05) in the DON group indicating that the glucose-induced Isc was altered by DON. Addition of the eubacteria to the DON contaminated feed of the broilers led to electrophysiological properties in the gut that were comparable with those of the control group. It could be concluded that 10 mg/kg DON in the diet impaired the Na(+)-D-glucose cotransport in the jejunum of broilers. In the absence of clinical signs, and without impaired performance, DON appeared to alter the gut function of broilers. The addition of Eubacterium sp. may be useful in counteracting the toxic effects of DON on intestinal glucose transport.

One hundred and sixty-eight 1-day-old Cobb broiler chickens were randomly assigned to 12 broiler cages to determine the dietary effect of different sources of oils rich in long-chain omega-3 polyunsaturated fatty acids (LCω3PUFA). The following oils were added to the feed at a concentration of 12.5 g kg−1: a highly refined seal blubber oil (SBO) containing 225 g kg−1 LCω3PUFA; a fish oil (FO) concentrate containing >800 g kg−1 LCω3PUFA in the form of triglycerides; and an FO concentrate containing >600 g kg−1 LCω3PUFA in the form of ethyl esters. Fatty acid profiles of the breast and thigh meat were measured in order to determine the deposition of LCω3PUFA into the body tissues, whilst fatty acid profiles of the liver and the portal blood were taken in order to deduce consequences of the different ester forms on the absorption process. A triangular sensory evaluation was performed on the breast meat. The levels of LCω3PUFA in all tissues and the portal blood reflected the concentrations of LCω3PUFA in the diets. The sensory properties of the breast meat were negatively influenced by ethyl ester supplementation only. Copyright © 2004 Society of Chemical Industry

Seventy-two 26-wk-old Single Comb White Leghorn laying hens were randomly assigned to 36 cages (2 per cage) in a 3-orthogonal 4 x 4 latin square, with the fourth row suppressed, to assess the effect of feeding refined seal blubber oil (SBO, containing 22.2% omega-3 fatty acids) on the fatty acid composition and position in the egg yolk lipids. The experiment was conducted over a period of 9 wk. Eggs were collected and numbered, and the weights were recorded for each week and cage. Eggs collected at wk 5 and 9 were used for total lipid, lipid class, fatty acid, and positional analyses. Sensory evaluation was carried out on eggs collected at wk 6 and 7. Feeding SBO at 1.25% led to an increase (P0.0001) in the long-chain omega-3 polyunsaturated fatty acids (LCn3PUFA) and a concomitant decrease (P0.0001) in arachidonic acid (ARA) in the egg yolk lipids. Yet this amount of SBO in the diet had no effect (P0.1) on the sensory attributes of the egg and on production parameters such as egg weight, number of eggs laid, and feed intake (P0.05). When feeding SBO in amounts higher than 1.25% proportionately, a plateau effect of the LCn3PUFA content of the eggs was observed. This appears to be because the PUFA content in the sn-2 position of the phospholipids cannot exceed a certain amount. When this amount is reached, the LCn3PUFA will be increasingly stored in triglycerides. The results presented here clearly indicate how eggs can be produced with optimized composition of LCn3PUFA without affecting (P0.1) the sensory properties of the eggs. The procedures elaborated herein provide directly applicable consequences for the food industry.

An approach for simultaneous determination of the main type A-trichothecenes by liquid chromatography and atmospheric pressure chemical ionization mass spectrometry is described. Parameters for coupling of LC-MS such as cone voltage, nebulizing temperature and the LC flow-rate, were optimized to provide detection of mycotoxins with maximum sensitivity. Furthermore, the effects of cone voltage and temperature on the fragmentation pattern of the tested toxins were studied. Main type A-trichothecenes such as T-2 Toxin, HT-2 Toxin, acetyl T-2 Toxin, diacetoxyscirpenol, monoacetoxyscirpenol (15-acetoxyscirpenol) and neosolaniol were separated on a reversed-phase narrow bore C18 column, using a linear gradient and a flow-rate of 0.3 ml/min. Mass spectra were obtained in positive ion mode for confirmation and quantitation. The method involves extraction and purification of toxins by using multifunctional Mycosep columns. Deuterated T-2 Toxin was used as an internal standard. A linear working range between 80 and 500 microg/kg in matrix with an acceptable correlation coefficient was observed. The developed method was validated by using a blank oats sample. The detection limit in the matrix was found to be between 50 and 85 microg/kg in selected ion mode for all tested A-trichothecenes. Recovery data were found to be between 77 and 101%. Within run and day-to-day precision were determined as having comparable levels to those found using GC methods. Furthermore, the matrix effect was investigated by comparing the internal standard versus the external standard method in quantification studies. In addition, the developed method was applied for the analysis of naturally contaminated oats, maize, barley and wheat samples.

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).

Fused lobes (FDL) hexosaminidases are the most recently genetically defined glycosidases involved in the biosynthesis of N-glycans in invertebrates, and their narrow specificity is essential for the generation of paucimannosidic N-glycans in insects. In this study, we explored the potential of FDL hexosaminidases in the utilization of different artificial and natural substrates, both as purified, native compounds or generated in vitro using various relevant glycosyltransferases. In addition to the already-known FDL enzyme from Drosophila melanogaster, we now have identified and characterized the Apis mellifera FDL homolog. The enzymatic properties of the soluble forms of the affinity-purified insect FDL enzymes, expressed in both yeast and insect cells, were compared with those of the phylogenetically distinct recombinant Caenorhabditis elegans FDL-like enzymes and the N-acetylgalactosamine (GalNAc)-specific Caenorhabditis hexosaminidase HEX-4. In tests with a range of substrates, including natural N-glycans, we show that the invertebrate FDL(-like) enzymes are highly specific for N-acetylglucosamine attached to the α1,3-mannose, but under extreme conditions also remove other terminal GalNAc and N-acetylglucosamine residues. Recombinant FDL also proved useful in the analysis of complex mixtures of N-glycans originating from wild-type and mutant Caenorhabditis strains, thereby aiding isomeric definition of paucimannosidic and hybrid N-glycans in this organism. Furthermore, differences in activity and specificity were shown for two site-directed mutants of Drosophila FDL, compatible with the high structural similarity of chitinolytic and N-glycan degrading exohexosaminidases in insects. Our studies are another indication for the variety of structural and function aspects in the GH20 hexosaminidase family important for both catabolism and biosynthesis of glycoconjugates in eukaryotes.

Apolipoprotein A-I (Apo A-I) is a major protein in lipid/lipoprotein metabolism and decreased serum levels have been observed in many species in response to inflammatory and infectious challenges. Little is known about the porcine homologue, therefore in this work we have characterized it through biochemical and proteomic techniques. In 2DE, porcine serum Apo A-I is found as three spots, the two more acidic ones corresponding to the mature protein, the more basic spot to the protein precursor. Despite high sequence coverage in LC-MS/MS, we did not find a sequence or PTM difference between the two mature protein species. Besides this biochemical characterization, we measured overall levels and relative species abundance of serum Apo A-I in four different viral and bacterial porcine infectious diseases. Lower overall amounts of Apo A-I were observed in Salmonella typhimurium and Escherichia coli infections. In the 2DE protein pattern, an increase of the protein precursor together with a lower level of mature protein species were detected in the porcine circovirus type 2-systemic disease and S. typhimurium infection. These results reveal that both the porcine serum Apo A-I concentration and the species pattern are influenced by the nature of the infectious disease.

The present work describes the development and application of an on-line atmospheric pressure ionisation (APCI) LC-MS interface for the simultaneous determination of seven toxicologically relevant cholesterol oxides (7alpha-hydroxycholesterol, 7beta-hydroxycholesterol, 25-hydroxycholesterol, 7-ketocholesterol, 5,6alpha-, 5,6beta-epoxycholesterol and cholestan-3beta,5alpha,6beta-triol). The HPLC method has been optimised to reach better separation of all tested compounds. The influences of APCI parameters (nebulising temperature, cone voltage, source temperature) on signal intensity and fragmentation pattern were investigated for all tested cholesterol oxides compounds. This is the first report on optimisation and determination of two compounds 7alpha-hydroxycholesterol and 5,6beta-epoxycholesterol in processed food using LC-MS. After extraction with hexane, clean-up was carried out using solid-phase extraction on a silica column. For the chromatographic separation of cholesterol oxides an Aquasil C18 column was used with acetonitrile-methanol (60:40) as mobile phase. For the first time we report the use of such a C18 column with a relatively hydrophilic nature for the separation of cholesterol oxides. APCI-MS detection was then applied in selected ion monitoring and positive ion modes by using the molecular ions and the main fragments. The developed method shows good linearity, high repeatability and good recovery for all tested cholesterol oxides. The method was applied for determination of seven selected cholesterol oxidation products in different foodstuffs such as butter, butteroil, lard and egg powder.

The present study shows how specific and selective particle-beam liquid chromatography mass spectrometry (PB-LC/MS) can be for certain biopharmaceutical applications and by which strategy maximum sensitivity can be achieved. The utility of PB-LC/MS in the analysis of different pharmaceutical drugs and endogenic substances with different molecular structures has been investigated. Using the PB interface in combination with a triple quadrupole mass spectrometer, the effects of mobile phase composition and flow rate, interface parameters---such as desolvation temperature and pressure of the nebulization gas---and the temperature of the ion source, on detection limits and sensitivity, are demonstrated. A particular substance specificity of this interface type for creatinine, naloxone and methadone could be observed after optimizing the transmission of the interface. The best sensitivity is achieved only in a narrow working range, indicating the necessity of an individual optimization of interface conditions for each substance. Linearity, sensitivity and precision of transfer rate using the EI and selected-ion monitoring modes were demonstrated. Investigation of the so-called “carrier effect” on the transfer of the selected compounds indicated that the addition of ammonium acetate as “carrier” changed the detection sensitivity in a completely different order. Copyright © 1998 John Wiley & Sons, Ltd.

Haptoglobin (Hp) and immunoglobulins are plasma glycoproteins involved in the immune reaction of the organism after infection and/or inflammation. Porcine circovirus type 2-systemic disease (PCV2-SD), formerly known as postweaning multisystemic wasting syndrome (PMWS), is a globally spread pig disease of great economic impact. PCV2-SD affects the immunological system of pigs causing immunosuppression. The aim of this work was to characterize the Hp protein species of healthy and PCV2-SD affected pigs, as well as the protein backbone and the glycan chain composition of porcine Hp. PCV2-SD affected pigs had an increased overall Hp level, but it did not affect the ratio between Hp species. Glycoproteomic analysis of the Hp β subunits confirmed that porcine Hp is N-glycosylated and, unexpectedly, O-glycosylated, a PTM that is not found on Hp from healthy humans. The glyco-profile of porcine IgG and IgA heavy chains was also characterized; decreased levels of both proteins were found in the investigated group of PCV2-SD affected pigs. Obtained results indicate that no significant changes in the N- and O-glycosylation patterns of these major porcine plasma glycoproteins were detectable between healthy and PCV2-SD affected animals. Biological significance PCV2-SD is a disease of great economic importance for pig production, characterized by a complex response of the immune system. In the search of a better diagnostic/prognostic marker for porcine PCV2-SD, extensive analyses of the Hp protein backbone and the glycan chains were thoroughly analyzed by various techniques. This resulted in detection and confirmation of Hp O-glycosylation and the glyco-profiling of porcine IgG and IgA. The N- and O-glycosylation of these major porcine plasma glycoproteins appears to be not affected by PCV2-SD infection. Interestingly, these data suggest that this viral infection, which significantly affects the immune responses of the host, leaves the biosynthetic glycosylation processes in the liver and immune cells unaffected. Lack of PTM changes is in contrast to findings in humans where for both proteins pattern changes have been reported in several chronic and inflammatory diseases. This underlines the importance of studying species in detail and not reaching to conclusions by analogy. Furthermore, since Hp is usually quantified by immunoassays in clinical routine analyses, our findings indicate that no bias in Hp determination capabilities due to an altered carbohydrate pattern is to be expected.

Polyclonal immunoglobulin (Ig) concentrates are important biological medicinal products and the assurance of their quality and safety is crucial. In our present approach we used proteomic methods to check the purity of commercial Ig products of different origin. The experimental setup included nonreducing 2DE or DIGE combined with MALDI-TOF and the thrombin generation assay, a routine safety test for pharmaceutical Ig preparations, and was complemented by a specific immunoassay. 2DE patterns displayed contaminations with trace amounts of human apolipoprotein H (Apo-H), transferrin, albumin, and its fragments. In contrast to the latter, Apo-H is a protein that is active in the coagulation cascade, and thus a potential involvement in thromboembolic events in vivo cannot be excluded. It was found by 2DE and MALDI-TOF to be a contaminant of several Ig preparations. Spiking experiments of Ig preparations with pure Apo-H demonstrated an Apo-H concentration dependent increase in thrombin generation assay values. Traces of Apo-H are possibly also contributing to unwanted side effects, as already known for factor XIa. The significance of Apo-H contaminations for these side effects might be verified by detailed analyses of pharmacovigilance data.

Energy-rich diets can challenge metabolic and protective functions of the rumen epithelial cells, but the underlying factors are unclear. This study sought to evaluate proteomic changes of the rumen epithelium in goats fed a low, medium, or high energy diet. Expression of protein changes were compared by two-dimensional differential gel electrophoresis followed by protein identification with matrix assisted laser desorption ionisation tandem time-of-flight mass spectrometry. Of about 2,000 spots commonly detected in all gels, 64 spots were significantly regulated, which were traced back to 24 unique proteins. Interestingly, the expression profiles of several chaperone proteins with important cellular protective functions such as heat shock cognate 71 kDa protein, peroxiredoxin-6, serpin H1, protein disulfide-isomerase, and selenium-binding protein were collectively downregulated in response to high dietary energy supply. Similar regulation patterns were obtained for some other proteins involved in transport or metabolic functions. In contrast, metabolic enzymes like retinal dehydrogenase 1 and ATP synthase subunit beta, mitochondrial precursor were upregulated in response to high energy diet. Lower expressions of chaperone proteins in the rumen epithelial cells in response to high energy supply may suggest that these cells were less protected against the potentially harmful rumen toxic compounds, which might have consequences for rumen and systemic health. Our findings also suggest that energy-rich diets and the resulting acidotic insult may render rumen epithelial cells more vulnerable to cellular damage by attenuating their cell defense system, hence facilitating the impairment of rumen barrier function, typically observed in energy-rich fed ruminants.

A chromatographic process based on monoliths for purification of infective baculovirus without prior concentration step has been established. Baculovirus produced in Spodoptera frugiperda cells (Sf-9) were harvested by centrifugation, filtered through 0.8 μm filters and directly loaded onto radial 1 mL anion exchange monoliths with a channel size of 1.5–2.0 μm operated at a volumetric flow rate of one bed volume per minute. Optional an epoxy monolith was used as pre-column to reduce interfering compounds and substances influencing the capacity of anion exchange monoliths for baculovirus infectious virus could be eluted with a step gradient at salt concentrations of 440 mM NaCl. Recovery of infectious virus was highly influenced by composition and age of supernatant and ranged from 20 to >99% active baculovirus. Total protein content could be reduced to 1–8% and DNA content to 38–48% in main virus fraction. Infective virus could be 52-fold concentrated within 20.5 h and simultaneously an 82-fold volume reduction was possible when loading 1150 mL (2.1 × 10 8 pfu/mL) onto 1 mL scale support.

Scope Patients with persistent egg allergy have more immunoglobulin E (IgE) against sequential than conformational epitopes of ovomucoid (OVO). Here, we aimed to identify compounds capable to render sequential epitopes in egg. Methods and results Glutathione was used for in vitro reduction of OVO and circular dichroism analyses were performed. Glutathione reduced OVO in a concentration-dependent manner. Egg white was analyzed for reduced proteins with a thiol probe and by MALDI-TOF/TOF. In unprocessed total egg white, several reduced proteins were detected by the thiol probe, among them reduced ovalbumin could be confirmed with MS analyses. Egg-allergics or sensitized controls were tested serologically (n = 19) for IgE against native and reduced OVO and in skin prick tests (n = 9). More patients had IgE against reduced than native OVO in Western blots. In skin prick test, five out of seven persistent egg-allergics and none of the controls reacted with reduced OVO. Conclusion Reduced egg proteins are present in natural egg white. Glutathione, which is present in egg and furthermore is used as texture-improving additive in processed food, is capable of reducing OVO. Patients with persistent egg allergy reacted rather to reduce the native OVO. Hence, our data indicate that reduction is a novel natural and processing-associated principle, which contributes to the allergenicity of food.

The aim of the present study was to investigate the impact of deoxynivalenol (DON) on cellular and humoral immune parameters in horses. A feeding trial using naturally contaminated oats with high (20.2 mg/kg) and low (0.49 mg/kg) levels of DON was conducted. Two groups of five mares were fed 2 kg oats daily with high or low DON levels for two weeks, using a crossover design with a three-week wash-out period. No adverse effects on general health were observed. Only minor diet-related changes in differential blood counts and serum biochemistry were noted. Serum haptoglobin concentration was significantly elevated after feeding DON (p = 0.04). Lymphocyte subsets (CD4+ CD8+, CD2+, CD21+, MHCII+) and lymphocyte proliferation data (concanavalin A, phytohemagglutinin, pokeweed mitogen) were not different between feeding-groups. It can be concluded that daily DON intakes as high as 6.9 to 9.5 mg/100 kg BW appear to have no major impact on the measured immune response of horses, indicating that this species has a high tolerance for DON.

The objective of this study was to investigate how weight-loss program would alter the proteome of the serum of Beagle dogs. For this purpose, serum samples from 5 Beagle dogs, before and after weight loss, were analyzed using 2-dimensional electrophoresis. Protein profiles of all samples were obtained, divided into 2 classes (obese and lean), and compared using specific 2-dimensional software, giving a total of 144 spot matches. Statistical analysis revealed 3 spot matches whose expressions were modulated in response to weight loss: 2 protein spots were upregulated and 1 protein spot was downregulated in the obese state in comparison with the lean state of the dogs. Mass spectrometric identification of differentially regulated spots revealed that these protein spots corresponded to retinol-binding protein 4, clusterin precursor, and α-1 antitrypsin, respectively, which could be considered potential markers of obesity and obesity-related disease processes in dogs.

The field of system biology among the disciplines, genomics, transcriptomics, proteomics and other “omics” technologies, has become one of the important scientific fields nowadays and will play a major role in investigating biological processes on a global level. Huge financial, technological and organisation efforts are needed to perform system biology based approaches. Progresses in molecular biology have led to the sequencing of the whole genome of several organisms, ranging from bacteria to various mammals including human. While the term “genomics” has already been used for a long time, new technologies such as transcriptomics, proteomics and metabolomics have been introduced during the last two decades and are beginning to expand rapidly. In general the “omics technologies” deal with hundreds or thousands of genes and/or their products such as mRNA, proteins or metabolites. Genomics is the study of genes, their variation and function by sequencing and mapping them. Similarly, transcriptomics deals with information at the messenger RNA (mRNA) level in an organism, tissue or cells at a given time by providing quantitative or semi-quantitative data. The transcriptome is however, not a straight copy of the genome, since the sequence of RNA molecules can be altered due to differential splicing and RNA editing. In contrast to the genome, which is static, the transcriptome is changing depending on environmental signals. Although, genomics and transcriptomics provide huge amount of information for understanding biological processes, the knowledge regarding the products of genes and transcripts remained underutilized. While, in last three decades predominantly genes and gene expression profiles were investigated and genomics has become the major side in biosciences, the analysis of proteins in a global approach was neglected mostly. However, since the 1990s also proteomics has gained increasing interest in the field of biosciences. The term proteomics was mentioned for the first time in Siena/Italy by Marc Wilkins in 1994 on occasion of the symposium on “2D Electrophoresis: from protein maps to genomes" (Wilkins 1997). Proteomics is defined as the qualitative and quantitative comparison of proteins in a cell or organism under defined conditions at a given time point. In other words proteomics is a term coined to comprise a field that attempts to understand the expression, function and regulation of the entire set of proteins encoded by an organism (Liebler 2002). In addition, since the proteins within a cell are the functioning units, their expression is strongly influenced by the environmental signals such as drugs, toxins, stress, age and other surrounding conditions. It is a complementary technology to genomic as well as transcriptomic research (Wilkins et al., 2007). Proteomics aims to deliver statistically

The duplicate method for estimating uncertainty from measurement including sampling is presented in the Eurachem/CITAC guide. The applicability of this method as a tool for verifying sampling plans for mycotoxins was assessed in three case studies with aflatoxin B(1) in animal feedingstuffs. Aspects considered included strategies for obtaining samples from contaminated lots, assumptions about distributions, approaches for statistical analysis, log(10)-transformation of test data and applicability of uncertainty estimates. The results showed that when duplicate aggregate samples are formed by interpenetrating sampling, repeated measurements from a lot can be assumed to approximately follow a normal or lognormal distribution. Due to the large variation in toxin concentration between sampling targets and sometimes very large uncertainty arising from sampling and sample preparation (U(rel) ≥ 50%), estimation of uncertainty from log(10)-transformed data was found to be a more generally applicable approach than application of robust ANOVA.

In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We aimed to establish an ovine model to study thrombin effects in vivo.Thrombin (0.0004-0.42 IU/kg/min) was continuously infused in Austrian Mountain Sheep over five hours in the dose escalation study (n=5 animals; 15 experiments). In the dose verification study animals received 0.42 IU/kg/min of thrombin vs. saline solution in a cross-over design (n=3 animals; 7 experiments).Thrombin at an infusion rate of 0.42 IU/kg/min decreased fibrinogen levels by 75% (p0.001) and increased degradation products of the fibrinogen beta-chain as shown in a proteomic analysis. Thrombin decreased platelet counts by 36% (p=0.006), prolonged thrombin time by 70% (p=0.012) and activated partial thromboplastin time by 32%. Interestingly, thrombin infusion significantly increased the activity of coagulation factors V and X (p0.05) and decreased the activity of the coagulation factors VIII and XIII (p0.05). Accordingly, thrombin displayed predominantly anti-coagulant and anti-platelet effects: 1) thrombin prolonged clotting time/clot formation time 7-fold (p=0.019) and induced a 65% decrease in maximal clot firmness (p0.001); 2) thrombin reduced collagen- induced platelet aggregation by 85% and prolonged collagen/adenosine diphosphate closure time 3-fold; and 3) thrombin caused lung haemorrhage but not thromboembolism.Protracted intravenous infusion of thrombin over a period of five hours offers a new experimental model to study thrombin effects in a large animal species.

Automatic and manual sampling for ochratoxin A (OTA) in barley grain was compared under industrial conditions considering sampling uncertainty as well as practical and technical aspects. Ten tonnes of barley inoculated with Penicillium verrucosum were incubated until the OTA concentration reached approximately 15 µg kg(-1) and sampled with manual and automatic sampling. A nested experimental design and ANOVA was used to estimate variance components from sampling, sample reduction, sample preparation and analysis. Manual sampling resulted in a high sampling uncertainty and OTA concentrations in aggregate samples ranged from 2 to 80 µg kg(-1). When aggregate samples were formed by automatic sampling the uncertainty arising from nugget effects and spatial distribution was practically eliminated. Results from this study show that an automatic sampler mounted after a mixer or conveyer can provide representative samples of OTA from a moving stream of barley. Automatic sampling might present a practical and economical alternative to manual sampling for feed mill operators when monitoring low levels of mycotoxins in grain or other commodities. Despite careful precautions, sample preparation and analysis resulted in a relative uncertainty of ±40% (p = 0.95), which was attributed to the sub-sampling following the two grinding steps. Size fractionation of the coarsely ground barley showed that 40% of the total amount of OTA was present in a small fraction of fine particles with a strong tendency to aggregate or stick to equipment and containers. Thus, in order to take advantage of the automatic sampling, it is crucial to apply an appropriate sub-sampling to prevent segregation of particles which may affect the OTA measurements.

Gallibacterium anatis has been suggested to have a causal role in the salpingitis/peritonitis complex in chickens, beside its isolation from the respiratory tract. As G. anatis strains from different flocks were compared by MALDI-TOF MS proteomic phenotyping it could be demonstrated that in most flocks one clonal lineage was present. This finding is also reflected by data achieved when isolates from different organs within a bird generally belong to the same clonal lineage. In addition, it was also confirmed by two independent experiments, as well as, two MALDI instruments. Altogether, proteomic phenotyping indicates that the nature of a chicken flock may play a certain role in particular clone type selection of G. anatis.

This paper describes the application of sol–gel immunoaffinity columns for clean up of ochratoxin A contaminated cereal crops. Monoclonal antibodies selective for OTA have been entrapped into the pores of a sol–gel matrix in order to prepare immunoaffinity columns. Different parameters such as amount of entrapped antibodies and loading conditions were optimized to obtain highest possible recoveries of OTA. The method has been found to be a suitable tool in sample preparation prior to HPLC-FLD determination and as selective as conventional commercially available immunoaffinity columns. In the clean up of different cereals mean recoveries of 82 ± 5%, 90 ± 6% and 91 ± 3%, were obtained for wheat, barley and rye, respectively, with sol–gel columns containing 1 mg of anti-OTA antibodies. The detection limit (signal-to-noise ratio, 3) was 0.5 μg/kg and the limit of quantification (signal-to-noise ratio, 10) determined to be 1 μg/kg. Sol–gel columns can be reused 7 times without significant loss of recovery. After 10 applications the recovery decreased to approx. 50%.

Risk assessment and monitoring of mycotoxins in agricultural commodities is associated with an increasing demand for appropriate analytical methods. Owing to the introduction of regulatory limits for various mycotoxins, the determination of mycotoxins in food and feed has become an important research issue in routine and governmental laboratories. The most prevalent methods for qualitative and quantitative determination, with the exception of enzyme linked immunosorbent assay (ELISA), are not able to analyse mycotoxins in complex matrices without sample pretreatment. In the same way as for all other analytes, sample preparation is one of the most crucial and important steps in the whole chain of analysis. There are different strategies used to determine mycotoxins in agricultural commodities as well as in biological samples, for example animal tissues, urine and so on. There are various approaches, such as solid phase extraction and liquid–liquid extraction; however, the most important and popular sample preparation technique used in mycotoxin analysis is immunoaffinity chromatography. In recent years there have been efforts to introduce selective and cost-effective strategies in sample pretreatment of mycotoxins. This chapter describes the significance of sample clean up in the analysis of major and abundant mycotoxins. Various conventional methods that are applied to sample preparation of mycotoxins in food, feed and biological samples will be discussed. Recent developments are included and different strategies for achieving more efficient extraction and separation are compared and presented.

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.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) whole-cell fingerprinting was used for characterization of 66 reference strains of Gallibacterium . The 4 recognised Gallibacterium species and Gallibacterium genomospecies 1 yielded reproducible and unique mass spectrum profiles, which were confirmed with Bruker Biotyper reference database version 3. The reproducibility of MALDI-TOF MS results were evaluated varying the age and storage of the cultures investigated. Reliable species identification was possible for up to 8 days of storage at 4 °C and less reliable if the bacteria were stored at room temperature (20 °C). However, if the strains were grown longer than 48 h at 37 °C under microaerobic atmosphere, poor identification results were obtained, due to changes in protein profile. The MALDI-TOF MS results of all 66 strains demonstrated 87.9% concordance with results based upon biochemical/physiological characterization. In addition, diversities outlined by MALDI-TOF MS were verified by sequencing the rpoB ( n = 43), 16S rRNA ( n = 28), infB ( n = 14), and recN ( n = 14) genes (multilocus sequence analysis, MLSA). In addition, discrepancies were observed between some of the genes sequenced. Results obtained demonstrated that MALDI-TOF MS fingerprinting represents a fast and reliable method for identification and differentiation of the 4 recognised Gallibacterium species and possible a fifth species Gallibacterium genomospecies 1, with applications in clinical diagnostics.