A screen for peptide agonists of the G-CSF receptor

<p>Abstract</p> <p>Background</p> <p>Granulocyte-colony stimulating factor (G-CSF) is one of the most important pharmacologically used proteins. Potential uses beyond the stimulation of neutrophilic granulocytes are the treatment of CNS disorders. Disadvantages of the G-CSF protein as a drug are its moderate plasma half-life time and considerable production costs. We therefore conducted a screen for peptide agonists derived from the sequence of human G-CSF.</p> <p>Findings</p> <p>Despite of the high sensitivity of our screening system we could not detect any positive hits in a single peptide approach. In a multiplex approach using a permutation of any combination of 10 different peptides we could also not detect a positive block.</p> <p>Conclusions</p> <p>We conclude that larger coherent parts of the protein or dimerising peptides may be needed to achieve activation of the receptor.</p

Entwicklung von Nachweismethoden für Lysozym und Nisin in Milchprodukten

Both antibacterial substances lysozyme and nisin can inhibit the growth of grampositive bacteria. Therefore they can be applied as preservatives in cheese to prevent late gas blowing of cheese caused by the bacteria Clostridium tyrobutyricum. These preservatives also take effect on other gram-positive food-borne pathogens and spoilage organisms such as Listeria monocytogenes. The aim of this study was to develop detection and quantification methods for both lysozyme and nisin and to optimize these methods for detection in the cheese matrix. They were to be developed in a way that made them suitable for routine use in public food control. An indirect, competitive ELISA was developed for the quantification of lysozyme in cheese using a commercially available monoclonal anti-lysozyme antibody. The method yielded excellent specifity and sensitivity. The anti-lysozyme antibody neither showed unspecific interaction with the cheese matrix, nor cross-reactivity with proteins sharing a high sequence homology with lysozyme such as the whey protein α-lactalbumin. To confirm the reliability and accuracy of the ELISA, cheese samples containing lysozyme were remeasured using HPLC with fluorescence detection as a reference method. The results were in good agreement for both spiked cheese samples and commercially available cheese samples containing lysozyme. In addition, a method based on immunoaffinity purification combined with mass spectrometric detection was developed. For immunoaffinity chromatography, magnetic beads with protein G on their surface were used. Anti-lysozyme antibodies were immobilized on the magnetic beads. Lysozyme from the cheese extracts was selectively enriched with the help of the magnetic beads. Afterwards, lysozyme was detected highly specifically by its mass of 14313 Da by MALDI-TOF-MS. A reliable distinction between lysozyme-free and lysozyme-containing cheese samples was achieved. The principle of the method can be easily adapted to other target proteins in complex food matrices and therefore offers a broad application potential, for example in the analysis of allergens. Furthermore, an HPLC method with fluorescence detection for the quantification of lysozyme in cheese was optimized and validated. The method yielded excellent precision and recovery rates. The limit of detection was 7.3 mg lysozyme/kg cheese. Lysozyme concentrations of commercially available cheese samples were determined with this method. Lysozyme was detected in 30 out of 46 cheese samples. On seven of the food packages lysozyme was not listed as an ingredient. The content of lysozyme was ranging from 30.8 to 386.2 mg lysozyme/kg cheese. Lysozyme-containing cheese was stored to analyze the stability of lysozyme in cheese. It was found that lysozyme remains quite stable during the ripening of the cheese wheels. An LC-MS/MS method was developed for the detection and quantification of the preservative nisin. An existing ISO standard method for the quantification of nisin A with LC-MS/MS was optimized and validated. With regards to the nisin variant nisin Z, an SRM method was newly developed and integrated in the existing quantification method for nisin A. Furthermore, the stability of nisin during processed cheese manufacturing was examined. For this purpose, nisin-containing processed cheese was produced. By means of heating up, recovery rates of nisin decreased significantly. An method for the three predominant degradation products of nisin, for instance nisin + H2O, was developed to analyze which degradation products of nisin were produced during heating. No significant increase of one of the degradation products was measured. However, the high amount of nisin + H2O in the nisin standard was remarkable. About one third of the nisin was present as nisin + H2O. The majority of this modification has already been present in the nisin standard. A minor part was formed during the extraction of nisin from the cheese sample. In addition, the peptides nisin A, nisin A + H2O, nisin Z and nisin Z + H2O were quantified in commercially available processed cheese samples with the help of a multimethod. In two of the cheese samples, the nisin Z was detected. This shows that the ISO standard method for the detection of nisin A is not sufficient. The detection of nisin Z should be included in the standard method.Die beiden antibakteriell wirksamen Substanzen Lysozym und Nisin wirken gegen grampositive Bakterien und können als Konservierungsstoffe für Käse eingesetzt werden. Dadurch kann die Spätblähung des Käses, verursacht durch das Bakterium Clostridium tyrobutyricum verhindert werden. Daneben wirken diese Konservierungsstoffe aber auch gegen andere grampositive Verderbnis- und/oder Krankheitserreger, wie z. B. den pathogenen Keim Listeria monocytogenes. Ziel dieser Arbeit war die Entwicklung von Nachweis- und Quantifizierungsmethoden für die beiden Konservierungsstoffe. Dabei sollten die Methoden für die Detektion in der Käsematrix optimiert werden. Weiterhin sollten die Nachweismethoden als Routinemethoden in der staatlichen Lebensmittelüberwachung geeignet sein. Unter Verwendung eines monoklonalen, kommerziell erhältlichen anti-Lysozym Antikörpers wurde ein indirekter, kompetitiver ELISA zum Nachweis von Lysozym in Käse entwickelt. Die Methode zeigte eine sehr gute Spezifität und Empfindlichkeit. Der verwendete anti-Lysozym-Antikörper zeigte weder unspezifische Wechselwirkungen mit der Käsematrix, noch Kreuzreaktionen zu Proteinen mit einer hohen Sequenzhomologie zu Lysozym, wie z. B. dem Molkenprotein α-Lactalbumin. Um die Zuverlässigkeit und Richtigkeit der ELISA Methode zu bestätigen, wurden lysozymhaltige Käseproben neben der Quantifizierung mit ELISA mit einer HPLC-FLD Methode als Referenzmethode gemessen. Sowohl für dotierte Käseproben als auch für lysozymhaltige Handelsproben wurde eine sehr gute Korrelation der Lysozymgehalte erzielt. Des Weiteren wurde eine Nachweismethode entwickelt, deren Prinzip auf einer massenspektrometrischen Detektion des Lysozyms nach immunochemischer Aufreinigung beruht. Für die immunochemische Aufreinigung wurden magnetische Partikel verwendet, auf deren Oberfläche Protein G gebunden war. An diesen Partikeln wurden dann die anti-Lysozym IgG Antikörper immobilisiert. Mit Hilfe dieser Partikel konnte das Lysozym aus den Käseextrakten selektiv angereichert werden. Im Anschluss wurde das Lysozym hochspezifisch über seine Masse von 14313 Da mittels MALDI-TOF-MS nachgewiesen. Mit dieser Methode konnte zuverlässig zwischen lysozymhaltigen und lysozymfreien Käseproben unterschieden werden. Das Prinzip der Nachweismethode kann leicht auf andere Zielproteine in komplexen Lebensmittelmatrices übertragen werden und bietet daher ein breites Anwendungspotential, z. B. in der Allergenanalytik. Weiterhin wurde eine HPLC Methode mit Fluoreszenzdetektion zur Quantifizierung von Lysozym in Käse optimiert und validiert. Die Methode zeigte sehr gute Präzision und Wiederfindungsraten. Die Nachweisgrenze dieser Methode lag bei 7,3 mg Lysozym/kg Käse. Mit dieser Methode wurden die Lysozymgehalte kommerziell erhältlicher Käseproben bestimmt. Lysozym war in 30 von 46 Käseproben nachweisbar. In sieben Fällen fehlte die Deklaration des Lysozyms auf der Lebensmittelverpackung. Die Lysozymgehalte lagen zwischen 30,8 und 386,2 mg Lysozym/kg Käse. Mit einem Lagerversuch von lysozymhaltigen Käse wurde die Stabilität von Lysozym während der Käsereifung untersucht. Es zeigte sich, dass Lysozym während der Reifung weitgehend stabil ist. Für den Nachweis und die Quantifizierung des Konservierungsstoffs Nisin wurde eine LC-MS/MS Methode entwickelt. Eine bestehende ISO Standard Methode zur Quantifizierung von Nisin A mittels LC-MS/MS wurde optimiert und validiert. Für die Nisinvariante Nisin Z wurden eine SRM-Methode neu entwickelt und in die Methode zur Quantifizierung von Nisin A integriert. Weiterhin wurde die Stabilität des Nisins bei der Schmelzkäseherstellung untersucht. Dafür wurde nisinhaltiger Schmelzkäse selbst hergestellt. Durch die Erhitzung sanken die Wiederfindungsraten von Nisin signifikant. Um zu klären, zu welchen Produkten Nisin abgebaut wird, wurde eine Methode zum Nachweis der drei bekannten Hauptabbauprodukte von Nisin, unter anderem Nisin + H2O, entwickelt. Es konnte kein signifikanter Anstieg von einem dieser Nisinabbauprodukte festgestellt werden. Bemerkenswert war jedoch der hohe Anteil von Nisin + H2O in den Nisinstandards. Ungefähr ein Drittel des gesamten Nisins lag als Nisin + H2O vor. Der Großteil dieser Modifikation war schon im Nisinstandard vorhanden. Ein Teil davon bildete sich aber auch während der Extraktion des Nisins aus dem Käse. Schließlich wurden mit einer Multimethode die Peptide Nisin A, Nisin A + H2O, Nisin Z und Nisin Z + H2O in kommerziell erhältlichen Schmelzkäseproben quantifiziert. In zwei Proben wurde Nisin Z nachgewiesen. Damit wurde gezeigt, dass die ISO Standard Methode zum Nachweis von Nisin A nicht ausreicht und der Nachweis von Nisin Z in die Standardmethode aufgenommen werden sollte

Satellite-based analysis of clouds and radiation properties of different vegetation types in the Brazilian Amazon region

Land-use changes impact the energy balance of the Earth system, and feedbacks in the Earth system can dampen or amplify this perturbation. We analyze here from satellite data the response of clouds and subsequently radiation to a change of land use for the example of deforestation in the Amazon Basin. In this region, the characteristics of different cloud types over two vegetation types (forest and crop-/grasslands) were calculated for a time period of five years by using satellite data from the instruments MODIS and CERES. The cloud types are defined according to height, optical thickness, and fraction of cloud cover. For calculating the radiative forcing caused by deforestation, the dependency of spatial and temporal averages for the reflected shortwave and outgoing longwave radiation of the top of the atmosphere on vegetation types were determined as well. The results show distinct differences in cloud cover and radiative forcing over crop-/grasslands and forests for the two vegetation regimes, implying a potentially significant positive cloud feedback to deforestation

The AQP2 mutation V71M causes nephrogenic diabetes insipidus in humans but does not impair the function of a bacterial homolog

Several point mutations have been identified in human aquaporins, but their effects on the function of the respective aquaporins are mostly enigmatic. We analyzed the impact of the aquaporin 2 mutation V71M, which causes nephrogenic diabetes insipidus in humans, on aquaporin structure and activity, using the bacterial aquaglyceroporin GlpF as a model. Importantly, the sequence and structure around the V71M mutation is highly conserved between aquaporin 2 and GlpF. The V71M mutation neither impairs substrate flux nor oligomerization of the aquaglyceroporin. Therefore, the human aquaporin 2 mutant V71M is most likely active, but cellular trafficking is probably impaired

Formulation of Cannabidiol in Colloidal Lipid Carriers

In this study, the general processability of cannabidiol (CBD) in colloidal lipid carriers was investigated. Due to its many pharmacological effects, the pharmaceutical use of this poorly water-soluble drug is currently under intensive research and colloidal lipid emulsions are a well-established formulation option for such lipophilic substances. To obtain a better understanding of the formulability of CBD in lipid emulsions, different aspects of CBD loading and its interaction with the emulsion droplets were investigated. Very high drug loads (>40% related to lipid content) could be achieved in emulsions of medium chain triglycerides, rapeseed oil, soybean oil and trimyristin. The maximum CBD load depended on the type of lipid matrix. CBD loading increased the particle size and the density of the lipid matrix. The loading capacity of a trimyristin emulsion for CBD was superior to that of a suspension of solid lipid nanoparticles based on trimyristin (69% vs. 30% related to the lipid matrix). In addition to its localization within the lipid core of the emulsion droplets, cannabidiol was associated with the droplet interface to a remarkable extent. According to a stress test, CBD destabilized the emulsions, with phospholipid-stabilized emulsions being more stable than poloxamer-stabilized ones. Furthermore, it was possible to produce emulsions with pure CBD as the dispersed phase, since CBD demonstrated such a pronounced supercooling tendency that it did not recrystallize, even if cooled to −60 °C

Assessing the "Good Life” in a Military Context: How Does Life and Work-Satisfaction Relate to Orientations to Happiness and Career-Success Among Swiss Professional Officers?

The study examines work- and life satisfaction along with orientation to happiness, and their relation to subjective but also objective career success, among Swiss military professional officers. They frequently report worsening of their working conditions due to two reforms that have recently been conducted. A total of N=221 Swiss career officers (mainly Land Forces; from Colonel to First Lieutenant) completed several questionnaires in an online survey. As expected, pleasure, engagement and meaning were positively related to satisfaction with life and the meaningful life also correlated with the (overall) work satisfaction. Higher subjective career success was related to higher satisfaction with life, content-related aspects of work satisfaction, and higher endorsements to the engaged and the meaningful life. Belonging to the general staff was considered as an objective criterion of career success and those who were in the general staff, were higher in their overall work satisfaction, content-related aspects of their work and, again, higher inclination to the life of engagement and the life of meaning. The study suggests that variables of positive psychological functioning are useful additions in the field of military psychology and that they might hold a key for the development of strategies for improving both, work- and life satisfaction among military personne