Antimicrobial susceptibility in E. coli and Pasteurellaceae at the beginning and at the end of the fattening process in veal calves: Comparing 'outdoor veal calf' and conventional operations.

Animal husbandry requires practical measures to limit antimicrobial resistance (AMR). Therefore, a novel management and housing concept for veal calf fattening was implemented on 19 intervention farms (IF) and evaluated regarding its effects on AMR in Escherichia (E.) coli, Pasteurella (P.) multocida and Mannheimia (M.) haemolytica in comparison with 19 conventional control farms (CF). Treatment intensity (-80%) and mortality (-50%) were significantly lower in IF than in CF, however, production parameters did not differ significantly between groups. Rectal and nasopharyngeal swabs were taken at the beginning and the end of the fattening period. Susceptibility testing by determination of the minimum inhibitory concentration was performed on 5420 isolates. The presence of AMR was described as prevalence of resistant isolates (%), by calculating the Antimicrobial Resistance Index (ARI: number of resistance of one isolate to single drugs/total number of drugs tested), by the occurrence of pansusceptible isolates (susceptible to all tested drugs, ARI=0), and by calculating the prevalence of multidrug (≥3) resistant isolates (MDR). Before slaughter, odds for carrying pansusceptible E. coli were higher in IF than in CF (+65%, p=0.022), whereas ARI was lower (-16%, p=0.003), and MDR isolates were less prevalent (-65%, p=0.001). For P. multocida, odds for carrying pansusceptible isolates were higher in IF before slaughter compared to CF (+990%, p=0.009). No differences between IF and CF were seen regarding the prevalence of pansuceptible M. haemolytica. These findings indicate that easy-to-implement measures to improve calf management can lead to a limitation of AMR in Swiss veal fattening farms

Cellular and molecular characterisation of novel lipopeptides with anti-myeloma activities.

The pleiotropic cytokine interleukin-6 (IL-6) is one of the major growth factors for multiple myeloma cells. It was previously shown that IL-6 induces the activation of Src family kinases Hck, Lyn and Fyn and that Hck is associated with the IL-6-receptor beta chain (gp130) via an acidic domain in gp130. In the first part of this thesis the acidic domain is narrowed down from 41 to 18 amino acids by a peptide-based functional screening assay. A derivative of the acidic domain, an 18mer lipopeptide, peptide 18AD, is characterised on the cellular and molecular level. IL-6-dependent growth of human and murine myeloma cells is inhibited with an IC50 of 25-30 µM by the addition of peptide 18AD to the growth medium. These cells remain unaffected by treatment with a control peptide with scrambled sequence (18sc). Furthermore, growth of IL-6-independent myeloma cells is not inhibited by peptide 18AD. In IL-6-dependent cells, peptide 18AD causes the same degree of apoptosis induction as IL-6 deprivation. On the molecular level it is shown by peptide competition assays that the association of Hck and gp130 is inhibited by peptide 18AD in a concentration dependent way. Peptide 18AD blocked the IL-6-induced activity of the Src family kinases Hck, Lyn and Fyn. Results from different factor-dependent cell lines demonstrate a common mechanism of the molecular peptide effects on Src family kinase activity, but the involved pathways downstream of the kinases appear to differ among species and cell lines tested. Treatment of human IL-6-dependent myeloma cells with peptide 18AD reduced the activating tyrosine phosphorylation of the signal transducer and activator of transcription 3 (STAT3), while STAT3 activation remains unaffected in murine cells. The Co-immunoprecipitations from different overexpressed receptor-deletionmutants and Hck confirms that the association is mainly due to the interaction between the kinase and a 9 amino acids spanning region within the acidic domain which carries the highest accumulation of acidic residues. Apparently, a sequence of 8-9 amino acids within gp130 with the prevalence of acidic side chains resembles a potential pseudosubstrate domain of tyrosine kinases and is responsible for localisation and activation of Src family kinases in response to receptor stimulation. The identification of sequence motives similar to the acidic domain of gp130 in other cytokine receptors, receptor tyrosine kinases and adapter proteins by an in silicio motive scan might suggest a general role of such motives. This could be the efficient recruitment of cytoplasmic kinases to signalling complexes at the time of ligand stimulation. Here, the importance of the acidic domain-kinase interaction for the IL-6-signaling pathway is shown by the growth-inhibiting effect of peptide 18AD on myeloma cells. In the second part, a novel sequence and celltype-specific anti-myeloma agent, peptide 1A, is characterised. It was initially designed as the negative control for a "reverse alanine scan" to define the role of the acidic residues within the sequence of peptide 18AD. Unexpectedly it turned out to be at least a 25-fold more potent growth inhibitor of myeloma cells than peptide 18AD. Similar molecular bases of the observed effects of peptide 18AD and peptide 1A are very unlikely, because in contrast to peptide 18AD, peptide 1A efficiently kills IL-6-independent myeloma cells as well. Moreover, an excess of IL-6 fails to rescue the cells from peptide 1A-induced cell death. Peptide 1A shows, as tested so far, no effects on cells derived from non-tumor tissue or tumor cells of various origins other than multiple myeloma. Peptide 1A specifically kills myeloma cells by the induction of apoptosis and severely disturbs cell cycle progression. Apoptotic cell death induction by peptide 1A is shown by peptide 1A-induced caspase-3 activation and the cleavage of PARP, a substrate of effector caspases. Peptide-induced cell death can partly be inhibited by co-treatment with the pan-caspase inhibitor ZVAD-fmk. Major survival pathways like the STAT3, PI3K/Akt and the MAPK pathway are inhibited in peptide 1A treated cells. If a biotinylated version of the peptide is added to the growth medium, it is incorporated into human myeloma cells and localised in defined regions of the cytoplasm of myeloma cells. Here some of the molecular mechanisms of peptide 1A-induced cell death are elucidated. However, the direct molecular target(s) are still unknown. Despite the potential difference in the molecular mechanisms, both peptides 18AD and 1A are expected to prove useful for developing derivatives with possible applications for the treatment of multiple myeloma

Human Bone Progenitor Cells for Clinical Application: What Kind of Immune Reaction Does Fetal Xenograft Tissue Trigger in Immunocompetent Rats?

The potential of human fetal bone cells for successful bone regeneration has been shown in vivo. In particular, it has been demonstrated that the seeding of these cells in porous poly-(L-lactic acid)/β-tricalcium phosphate scaffolds improved the bone formation compared to cell-free scaffolds in skulls of rats. However, even if the outcome is an improvement of bone formation, a thorough analysis concerning any immune responses, due to the implantation of a xenograft tissue, is not known. As the immune response and skeletal system relationship may contribute to either the success or failure of an implant, we were interested in evaluating the presence of any immune cells and specific reactions of human fetal cells (also called human bone progenitor cells) once implanted in femoral condyles of rats. For this purpose, (1) cell-free scaffolds, (2) human bone progenitor cells, or (3) osteogenic human bone progenitor cells within scaffolds were implanted over 3, 7, 14 days, and 12 weeks. The key finding is that human bone progenitor cells and osteogenic human bone progenitor cells do not trigger any particular specific immune reactions in immunocompetent rats but are noted to delay some bone formation. </jats:p

Investigation of cutting-induced damage in CMC bend bars

Ceramic matrix composites (“CMC”) with a strong fibre-matrix interface can be made damage-tolerant by introducing a highly porous matrix. Such composites typically have only a low interlaminar shear strength, which can potentially promote damage when preparing specimens or components by cutting. In order to investigate the damage induced by different cutting methods, waterjet cutting with and without abrasives, laser-cutting, wire eroding and cutoff grinding were used to cut plates of two different CMCs with a matrix porosity up to 35 vol.-%. For each combination of cutting method and composite, the flexural and interlaminar shear strength of the resulting specimens was determined. Additionally, the integrity of the regions near the cut surfaces was investigated by high-resolution x-ray computer tomography. It could be shown that the geometrical quality of the cut is strongly affected by the cutting method employed. Laser cut and waterjet cut specimens showed damage and delaminations near the cut surface leading to a reduced interlaminar shear strength of short bend bars in extreme cases

Investigation of cutting-induced damage in CMC bend bars

Ceramic matrix composites (“CMC”) with a strong fibre-matrix interface can be made damage-tolerant by introducing a highly porous matrix. Such composites typically have only a low interlaminar shear strength, which can potentially promote damage when preparing specimens or components by cutting. In order to investigate the damage induced by different cutting methods, waterjet cutting with and without abrasives, laser-cutting, wire eroding and cutoff grinding were used to cut plates of two different CMCs with a matrix porosity up to 35 vol.-%. For each combination of cutting method and composite, the flexural and interlaminar shear strength of the resulting specimens was determined. Additionally, the integrity of the regions near the cut surfaces was investigated by high-resolution x-ray computer tomography. It could be shown that the geometrical quality of the cut is strongly affected by the cutting method employed. Laser cut and waterjet cut specimens showed damage and delaminations near the cut surface leading to a reduced interlaminar shear strength of short bend bars in extreme cases

A minimal growth medium for the basidiomycete Pleurotus sapidus for metabolic flux analysis

BACKGROUND: Pleurotus sapidus secretes a huge enzymatic repertoire including hydrolytic and oxidative enzymes and is an example for higher basidiomycetes being interesting for biotechnology. The complex growth media used for submerged cultivation limit basic physiological analyses of this group of organisms. Using undefined growth media, only little insights into the operation of central carbon metabolism and biomass formation, i.e., the interplay of catabolic and anabolic pathways, can be gained. RESULTS: The development of a chemically defined growth medium allowed rapid growth of P. sapidus in submerged cultures. As P. sapidus grew extremely slow in salt medium, the co-utilization of amino acids using 13C-labelled glucose was investigated by gas chromatography-mass spectrometry (GC-MS) analysis. While some amino acids were synthesized up to 90% in vivo from glucose (e.g., alanine), asparagine and/or aspartate were predominantly taken up from the medium. With this information in hand, a defined yeast free salt medium containing aspartate and ammonium nitrate as a nitrogen source was developed. The observed growth rates of P. sapidus were well comparable with those previously published for complex media. Importantly, fast growth could be observed for 4days at least, up to cell wet weights (CWW) of 400gL-1. The chemically defined medium was used to carry out a 13C-based metabolic flux analysis, and the in vivo reactions rates in the central carbon metabolism of P. sapidus were investigated. The results revealed a highly respiratory metabolism with high fluxes through the pentose phosphate pathway and TCA cycle. CONCLUSIONS: The presented chemically defined growth medium enables researchers to study the metabolism of P. sapidus, significantly enlarging the analytical capabilities. Detailed studies on the production of extracellular enzymes and of secondary metabolites of P. sapidus may be designed based on the reported data

Inhibition of IL-6-dependent growth of myeloma cells by an acidic peptide repressing the gp130-mediated activation of Src family kinases.

An acidic domain (AD) of gp130 was previously found to interact with the Src family kinase (SFK) Hck. Here, the influence of myristoylated peptides derived from this AD was assessed in the mouse myeloma cell line, 7TD1. The IL-6-dependent growth of 7TD1 cells was reduced by approximately 75%, if 100 microM of myristoylated 18mer peptide (18AD) was included in the growth medium, but was unaffected by a control peptide with scrambled sequence (18sc). A similar differential inhibition by peptides 18AD and 18sc was observed for the erythropoietin-dependent growth of BaF-EH cells expressing chimeric erythropoietin receptor-gp130 and human Hck and for the human myeloma cell line INA-6. While the peptide 18AD concentration inhibiting 50% was approximately 30 microM in 7TD1 and BaF-EH cells, peptide 18AD did not significantly inhibit growth of IL-6-independent MM1.S myeloma and OKT1 hybridoma cells or of BaF-EH cells supplied with IL-3. Treatment with 100 microM peptide 18AD caused the same degree or 60% of apoptosis induction as IL-6 deprivation in 7TD1 or INA-6 cells, respectively. Co-immunoprecipitation experiments revealed that peptide 18AD interfered with the association of Hck and gp130 in 7TD1 lysates in a concentration-dependent manner. IL-6-treatment of INA-6 cells induced the kinase activities of Fyn, Lyn and Hck, but not Src, and the IL-6-induced SFK activities were inhibited by peptide 18AD. Expression in 7TD1 cells of a kinase-inactive Hck mutant (K269R) elicited a dominant-negative effect on cell number increases providing further evidence that SFKs are required for gp130 signalling in myeloma cells