Immunoaffinity-Based Mass Spectrometry for the Species Identification and Quantification of Processed Animal Proteins in Feed

The present work introduced immunoaffinity-based mass spectrometry to feed analysis and improved the detection of banned processed animal proteins (PAPs) in animal feed. Current analytical methods show deficiencies in either sensitivity, species and tissue specificity or quantification ability. To address this issue, a peptide-centric workflow that comprises a more efficient sample preparation, an immunoaffinity enrichment of species- and tissue-specific peptides, and a LC-MS/MS analysis for identification and quantification using stable isotope labeled standard peptides, was established. The release of peptides from poorly soluble PAPs and blood products was improved by a direct digestion in suspension. Further time-consuming clean-up steps are not necessary since reagents and salts are removed during the immunoenrichment. The enrichment also allows a fast peptide separation using short gradients with a 10 min cycle time and therefore an increased sample throughput. The species differentiation of the 8 livestock species cattle, sheep/goat, pig, horse, turkey chicken, duck and goose, was addressed in a multispecies approach. Therefore, a cross-species polyclonal antibody was generated, which is able to enrich 8 homologous peptides from processed meat and bone meal, blood meal and spray-dried plasma, hence allowing a comprehensive analysis of common feed additives. A second multiplex assay was developed to differentiate ruminant tissues by targeting 7 peptides of meat, bone, cartilage, blood and milk proteins. This allows a differentiation of legal and illegal ruminant protein additives. The assays’ basic analytical parameters were validated. Both assays showed a detection limit in the picomolar concentration range allowing a qualitative detection over 4 to 5 orders of magnitude and a quantification over 3 to 4 orders of magnitude. Depending on the tissue type, 0.05%-0.75% PAP was specifically and quantitatively determined in an animal feed background. The multiplex assays were finally applied to official proficiency test samples from the European Reference Laboratory for Animal Proteins (EURL-AP, Gembloux, Belgium). The developed assays showed an unambiguous differentiation and quantification of species and tissues on a contamination level of 0.1% PAP in feed. As a final conclusion, immunoaffinity-based mass spectrometry was shown to overcome the current limitations in PAP detection and meets the requirements for future feed authentication methods

5-Lipoxygenase contributes to PPAR [gamma] activation in macrophages in response to apoptotic cells

Background: One hallmark contributing to immune suppression during the late phase of sepsis is macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells (AC). Taking the important role of the nuclear receptor PPARγ for this phenotype switch into consideration, it remains elusive how AC activate PPARγ in macrophages. Therefore, we were interested to characterize the underlying principle. Methods: Apoptosis was induced by treatment of Jurkat T cells for 3 hours with 0.5 μg/ml staurosporine. Necrotic cells (NC) were prepared by heating cells for 20 minutes to 65°C. PPARγ activation was followed by stably transducing RAW264.7 macrophages with a vector encoding the red fluorescent protein mRuby after PPARγ binding to 4 × PPRE sites downstream of the reporter gene sequence. This readout was established by treatment with the PPARγ agonist rosiglitazone (1 μM) and AC (5:1). Twenty-four hours after stimulation, mRuby expression was analysed by fluorescence microscopy. Lipid rafts of AC, NC, as well as living cells (LC) were enriched by sucrose gradient centrifugation. Fractions were analysed for lipid raft-associated marker proteins. Lipid rafts were incubated with transduced RAW264.7 macrophages as described above. 5-Lipoxygenase (5-LO) involvement was verified by pharmacological inhibition (MK-866, 1 μM) and overexpression. Results: Assuming that the molecule responsible for PPARγ activation in macrophages is localized in the cell membrane of AC, most probably associated to lipid rafts, we isolated lipid rafts from AC, NC and LC. Mass spectrometric analysis of lipid rafts of AC showed the expression of 5-LO, whereas lipid rafts of LC did not. Moreover, incubating macrophages with lipid rafts of AC induced mRuby expression. In contrast, lipid rafts of NC and LC did not. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated for 30 minutes with the 5-LO inhibitor MK-866 (1 μM) before apoptosis induction. In line with our hypothesis, these AC did not induce mRuby expression. Finally, although living Jurkat T cells overexpressing 5-LO did not activate PPARγ in macrophages, mRuby expression was significantly increased when AC were generated from 5-LO overexpressing compared with wild-type Jurkat cells. Conclusion: Our results suggest that induction of apoptosis activates 5-LO, localizing to lipid rafts, necessary for PPARγ activation in macrophages. Therefore, it will be challenging to determine whether 5-LO activity in AC, generated from other cell types, correlates with PPARγ activation, contributing to an immune-suppressed phenotype in macrophages

CD69 is a TGF-β/1α,25-dihydroxyvitamin D3 target gene in monocytes

CD69 is a transmembrane lectin that can be expressed on most hematopoietic cells. In monocytes, it has been functionally linked to the 5-lipoxygenase pathway in which the leukotrienes, a class of highly potent inflammatory mediators, are produced. However, regarding CD69 gene expression and its regulatory mechanisms in monocytes, only scarce data are available. Here, we report that CD69 mRNA expression, analogous to that of 5-lipoxygenase, is induced by the physiologic stimuli transforming growth factor-β (TGF-β) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in monocytic cells. Comparison with T- and B-cell lines showed that the effect was specific for monocytes. CD69 expression levels were increased in a concentration-dependent manner, and kinetic analysis revealed a rapid onset of mRNA expression, indicating that CD69 is a primary TGF-β/1α,25(OH)2D3 target gene. PCR analysis of different regions of the CD69 mRNA revealed that de novo transcription was initiated and proximal and distal parts were induced concomitantly. In common with 5-lipoxygenase, no activation of 0.7 kb or ~2.3 kb promoter fragments by TGF-β and 1α,25(OH)2D3 could be observed in transient reporter assays for CD69. Analysis of mRNA stability using a transcription inhibitor and a 3′UTR reporter construct showed that TGF-β and 1α,25(OH)2D3 do not influence CD69 mRNA stability. Functional knockdown of Smad3 clearly demonstrated that upregulation of CD69 mRNA, in contrast to 5-LO, depends on Smad3. Comparative studies with different inhibitors for mitogen activated protein kinases (MAPKs) revealed that MAPK signalling is involved in CD69 gene regulation, whereas 5-lipoxygenase gene expression was only partly affected. Mechanistically, we found evidence that CD69 gene upregulation depends on TAK1-mediated p38 activation. In summary, our data indicate that CD69 gene expression, conforming with 5-lipoxygenase, is regulated monocyte-specifically by the physiologic stimuli TGF-β and 1α,25(OH)2D3 on mRNA level, although different mechanisms account for the upregulation of each gene

Elektrochemische Untersuchung und physikalische Charakterisierung von Katalysatoren für die Sauerstoffreduktion in der DMFC

In allen Niedertemperatur-Brennstoffzellen hat die Sauerstoffreduktion einen wesentlichen Anteil an den Wandlungsverlusten von chemischer in elektrische Energie. Für die Reaktionskinetik und die mit der Sauerstoffreduktionsreaktion korrelierten Verluste sind die Katalysatoren sowie die Elektrodenstruktur von entscheidender Bedeutung. Bei der Direktmethanol-Brennstoffzelle (DMFC) werden an der Kathode weitere Verluste durch den Durchtritt von Methanol von der Anode zur Kathode und die dort ablaufende Methanoloxidation verursacht. Da zum einen die Brennstoffausnutzung reduziert wird und zum anderen sich an der Kathode durch die gleichzeitig ablaufende Sauerstoffreduktionsreaktion und die Methanoloxidationsreaktion ein Mischpotential ausbildet, kommt es zur Absenkung der nutzbaren Zellspannung. Um die Wandlungsverluste in der Brennstoffzelle zu senken, wurden alternative Katalysatoren für die Sauerstoffreduktion in Niedertemperatur-Brennstoffzellen getestet. Diese wurden in DMFC-Einzelzellen mit Strom-Spannungs-Kennlinien bezüglich der Leistungsfähigkeit untersucht. Da die Strom-Spannungs-Kennlinien allerdings keine detaillierten Informationen über die Reaktion liefern, wurden die DMFC-Zellen zusätzlich mit Hilfe der Elektrochemischen Impedanzspektroskopie (EIS) untersucht. Unterschiede zwischen verschiedenen Katalysatoren lassen sich mit Hilfe der EIS klarer bestimmen. Außerdem wurden CO2-Konzentrationen an der Kathode gemessen, die Informationen über die Methanoltoleranz der getesteten Katalysatoren liefern. Neben der chemischen Zusammensetzung der Katalysatoren spielen eine Reihe von physikalischen Eigenschaften für den Einsatz in Brennstoffzellenelektroden eine Rolle, insbesondere die Größe und die spezifische Oberfläche der Katalysatoren. Diese wurden mit Porosimetrie und temperaturprogrammierter Reduktion und Desorption ermittelt. Eine weitere wichtige Eigenschaft für den Einsatz alternativer Katalysatoren ist neben der Verbesserung der Sauerstoffreduktionsreaktion in der Brennstoffzelle auch die Stabilität der Katalysatoren. Um diese zu untersuchen, wurden die Katalysatoren vor und nach dem Einsatz in der Brennstoffzelle mit oberflächenphysikalischen Methoden, insbesondere der Röntgenphotoelektronenspektroskopie (XPS), charakterisiert