Impact of Alternaria toxins on CYP1A1-and GST-expression in human tumour cells

Die Alternaria Toxine Alternariol (AOH) und Alternariolmonomethylether (AME) wurden von der EFSA auf die Liste der Mykotoxine gesetzt, die noch einer weitgehenden toxikologische Untersuchungen bedürfen, um eine bessere Risikobewertung für Mensch und Tier zu ermöglichen. Der Konsum von Alternaria spp. kontaminierten Getreide wurde in Linxian, China in Verbindung mit Ösophaguskarzinom gebracht. In bereits veröffentlichten Daten von Fehr (2008) und Schreck et al. (2011) wurde die Induktion von CYP1A1 durch Alternaria Toxine beschrieben, was zu weiteren Untersuchungen über die Rolle des Arylhydrocarbon Rezeptors (AhR) in der AME- und AOH-vermittelten CYP1A1 Induktion in humanen Ösophagus Karzinomzellen (KYSE510) veranlasste. Die Frage, ob die Induktion von CYP1A1 mRNA durch Mykotoxine Ah-Rezeptor vermittelt abläuft, wurde in zwei verschiedene Ansätze geklärt. Zuerst wurden AhR-supprimierte Ösophagus Karzinomzellen mithilfe der RNAi Technologie generiert, die ein geeignetes Modell für die Untersuchung des Einfluss der Alternaria Toxine auf CYP1A1 Genexpression darstellten. Zweitens wurde der Ah-Rezeptor in KYSE510 Zellen durch eine Vorbehandlung mit 10 µM MNF inhibiert. Die signifikante Induktion von CYP1A1 in AhR-exprimierenden Zellen (nicht-transfizierte und dsRNA-transfizierte (Negativkontrolle) KYSE510) nach 24 h war abhängig von der applizierten AME- und AOH-Konzentration. Keine Änderungen der CYP1A1 Transkripte wurden bei geringen Mykotoxinkonzentrationen (≤ 0.1 µM) beobachtet, wobei höhere Konzentrationen (≥ 1 µM) eine maximale, 2.5-fache Induktion für AME und eine 8-fache Induktion für AOH hervor riefen. Vergleichbare Ergebnisse in beiden Zelltypen lassen auf keinerlei Veränderungen durch die Transfektion der KYSE510, in Hinblick auf ihre Reaktion auf AME und AOH, schließen. In AhR-supprimierten (AhR-siRNA transfizierten) KYSE510 Zellen waren die CYP1A1 mRNA Level nach der Inkubation mit AME und AOH im Vergleich zu den Kontrollzellen signifikant erniedrigt, was für die AhR-Abhängigkeit der CYP1A1 Induktion spricht. Das Absinken der CYP1A1 Transkripte unterhalb das endogene Level bei hohen Konzentrationen (10 µM AME, 50 µM AOH) lässt auf die Interferenz eines bisher unbekannten Signalweges vermuten. Die Ergebnisse die mithilfe MNF-inhibierter KYSE510 Zellen ermittelt wurden, sind generell in Einklang mit denen, die mit transient siRNA-transfizierten Zellen erzielt wurden und sprechen für eine entscheidende Rolle des Ah-Rezeptors in der AME-vermittelten CYP1A1 Induktion. Glutathion-S-Transferasen (GST) gehören zu den Phase II Enzymen und tragen einen beachtlichen Anteil zur Detoxifizierung von Xenobiotika bei. Nach der Etablierung und Optimierung des GST Assays nach Habig et al. (1973), wurde der Einfluss von AME und AOH auf die GST Aktivitäten in humanen Kolonkarzinomzellen (HT29) untersucht. Ein marginaler, aber doch signifikant erhöhter Anstieg der GST Aktivität bei hohen AOH Konzentrationen (10 µM, 50 µM) lässt auf einen minimalen Beitrag dieser Enzyme in der Detoxifizierung beider Alternaria Toxine schließen.The Alternaria toxins alternariol (AOH) and alternariol monomethyl ether (AME) are, according to the EFSA, on the list of mycotoxins with an urgent need for further investigations of toxicity, permitting a better risk assessment for man and animal. The consumption of Alternaria spp. contaminated grain is associated with increased incidences of oesophageal cancer in Linxian, China (Liu et al., 1992). Fehr (2008) and Schreck et al. (2011) reported the induction of CYP1A1 by Alternaria toxins, leading to investigations addressing the role of the aryl hydrocarbon receptor (AhR) in AME- and AOH-mediated CYP1A1 induction in human oesophageal carcinoma cells (KYSE510). The question whether mycotoxin-induced CYP1A1 mRNA levels were AhR-dependent was investigated by two different approaches. Firstly, oesophageal AhR knockdown cells were generated by RNAi technology and were used for studying the impact of Alternaria toxins on CYP1A1. Secondly, the Ah-receptor of KYSE510 cells was inhibited in a pre-treatment with 10 µM MNF prior the incubation with the Alternaria toxins. AOH and AME significantly induced CYP1A1 in a concentration dependent manner in AhR-expressing cells (non-transfected and dsRNA-transfected (negative control) KYSE510) after 24 h of incubation. Any changes in CYP1A1 transcript levels were observed at low mycotoxin concentrations (≤ 0.1 µM), whereas at higher concentrations (≥ 1 µM) CYP1A1 induction peaked by 2.5-fold for AME and 8-fold for AOH. Similar results for both cell types suggested, that the transfection procedure did not influence the response of KYSE510 cells towards AME and AOH. In AhR-suppressed (AhR-siRNA transfected) KYSE510 cells, CYP1A1 levels were significantly reduced after AME- as well as AOH-treatment in comparison to control cells, supporting the AhR-dependence of CYP1A1 induction by both mycotoxins. At high concentrations (10 µM AME, 50 µM AOH), CYP1A1 transcripts plummeted below the endogenous level, giving rise to consider the interference of a putative, yet unknown pathway. The results obtained with MNF-inhibited cells were generally in line with those of transiently siRNA-transfected KYSE510 cells, suggesting a crucial role of the Ah-receptor in AME-mediated CYP1A1 induction. Glutathione-S-transferases (GST) are important phase II drug metabolising enzymes, bearing a considerable role in the detoxification of xenobiotics. After establishment and optimisation of the GST assay according to Habig et al. (1973), the impact of AME and AOH on GST activities in human colon carcinoma cells (HT29) was investigated. Only a marginal, yet significant increase in GST activity at high AOH concentrations (10 µM, 50 µM) was observed, suggesting a minor role of GST in the detoxification of both Alternaria toxins

Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride

Gα subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus

Miniaturized Protein Microarray with Internal Calibration as Point-of-Care Device for Diagnosis of Neonatal Sepsis

Neonatal sepsis is still a leading cause of death among newborns. Therefore a protein-microarray for point-of-care testing that simultaneously quantifies the sepsis associated serum proteins IL-6, IL-8, IL-10, TNF alpha, S-100, PCT, E-Selectin, CRP and Neopterin has been developed. The chip works with only a 4 μL patient serum sample and hence minimizes excessive blood withdrawal from newborns. The 4 μL patient samples are diluted with 36 μL assay buffer and distributed to four slides for repetitive measurements. Streptavidin coated magnetic particles that act as distinct stirring detection components are added, not only to stir the sample, but also to detect antibody antigen binding events. We demonstrate that the test is complete within 2.5 h using a single step assay. S-100 conjugated to BSA is spotted in increasing concentrations to create an internal calibration. The presented low volume protein-chip fulfills the requirements of point-of-care testing for accurate and repeatable (CV < 14%) quantification of serum proteins for the diagnosis of neonatal sepsis

A semi-nonparametric mixture model for selecting functionally consistent proteins

Background High-throughput technologies have led to a new era of proteomics. Although protein microarray experiments are becoming more common place there are a variety of experimental and statistical issues that have yet to be addressed, and that will carry over to new high-throughput technologies unless they are investigated. One of the largest of these challenges is the selection of functionally consistent proteins. Results We present a novel semi-nonparametric mixture model for classifying proteins as consistent or inconsistent while controlling the false discovery rate and the false non-discovery rate. The performance of the proposed approach is compared to current methods via simulation under a variety of experimental conditions. Conclusions We provide a statistical method for selecting functionally consistent proteins in the context of protein microarray experiments, but the proposed semi-nonparametric mixture model method can certainly be generalized to solve other mixture data problems. The main advantage of this approach is that it provides the posterior probability of consistency for each protein

From chip surface to signal amplification : protein microarrays for disease biomarker detection

Zsfassung in dt. SpracheProtein Microarrays sind eine zukunftsträchtige Technologie mit zahlreichen Anwendungen in der klinischen Forschung und Diagnostik. Sie erlauben die gleichzeitige Analyse ganzer Sets von Biomarkern, die Rückschlüsse auf Krankheitsstadium und Verlauf zulassen. Dies ermöglicht einen weiteren Schritt in Richtung personalisierter Medizin, in der auf den Patienten abgestimmte, verträglichere Therapien Anwendung finden.Das Melanom, eine besonders aggressive Form des Hautkrebses, zeichnet sich durch einen auffallend unberechenbaren klinischen Verlauf aus, der durch die derzeitigen Bewertungskriterien nur ungenügend erfasst wird.Um die Prognosestellung zu erleichtern wurde in dieser Arbeit ein Protein Microarray zur simultanen und parallelen Analyse von fünf Melanom-Markern in Patientenblut entwickelt. Der Chip quantifiziert sowohl die spezifischen Prognose- und Staging-Faktoren S100B und Vascular Endothelial Growth Factor A (VEGF-A), als auch die krankheitsrelevanten Entzündungsmediatoren und Immunfaktoren C-reaktives Protein (CRP), Interleukin 6 (IL6) und Interleukin 10 (IL10). Die einzelnen Tests sind als Sandwich Immunoassays implementiert und werden mittels Fluoreszenzmessung ausgelesen. Die Optimierung von Druckpuffer, Antikörperkonzentration, Assaypuffer und Inkubationszeit führte zu einer hohen Reproduzierbarkeit (Variabilitätskoeffizient Zur Sensitivitätssteigerung in Bezug auf niedrig konzentrierte Analyten, die nur schwache Fluoreszenzsignale liefern, wurde ein reflektierender Goldchip entwickelt, der mit Polyelektrolytmultilayern aus Xanthan und Chitosan beschichtet war. Auf diesen Signalverstärkungssubstraten waren die gemessenen Fluoreszenzintensitäten bis zu 50-mal so hoch wie auf Glaschips. In Bezug auf kommerzielle Chips wurde das Signal-Rausch-Verhältnis um bis zu Faktor 11 verbessert. Auch die Sensitivität stieg bis zu 38-fach an. Schließlich werden in dieser Arbeit Metall-Affinitäts-Slides präsentiert, die auf Derivaten von Aminosäuren basieren. Diese Substrate ermöglichen die Immobilisierung von poly-Histidin-markierten Proteinen mittels Nickel-Chelat-Komplexbildung und erleichtern so den Aufbau von Assays mit gleichmäßig orientierten Proteinen. Die Slides wurden mit Hilfe der Photoelektronenspektroskopie charakterisiert und wiesen Selektivitäten >95% für hexa-Histidin-markierte Proteine auf. Außerdem waren die Signalstärken bis zu sechsmal so hoch wie auf kommerziellen Substraten.Protein microarrays are an emerging technology in clinical diagnostics. They permit the analysis of an entire set of biomarkers reflecting disease stage and likelihood of progression. Thus they could give raise to new, less toxic therapies on an individual patient's basis.Melanoma, a form of skin cancer, features a highly variable clinical course that is not predictable by current staging criteria. To facilitate establishment of a prognosis a protein microarray for the simultaneous and highly parallel analysis of five melanoma biomarkers in patients' blood serum was developed. The chip quantifies specific prognostic and staging factors S100B and Vascular Endothelial Growth Factor A (VEGF-A) as well as disease-related inflammatory and immunomodulatory parameters - C-reactive Protein (CRP), interleukins IL6 and IL10. Individual tests are implemented as sandwich immunoassays with streptavidin-biotin chemistry and fluorescence detection. Optimization of parameters such as print buffer composition, antibody concentrations, assay binding buffer and incubation time led to high reproducibility (coefficient of variation While interleukins have to be detected at the pg/mL scale, CRP serum concentrations are normally below 3 mg/L and rise by a factor of 1000 in response to an inflammatory stimulus. The CRP antibody sandwich assay features a working range of 0.4 µg/L - 0.2 mg/L CRP. To render possible the parallel analyte determination, in this thesis RNA aptamers were investigated as alternative binding elements. In fact aptamer-antibody sandwiches yielded broader working ranges (10 µg/L - 100 mg/L) and showed improved coverage of elevated physiological CRP concentrations. To furthermore enhance the sensitivity for low abundant target proteins which only produce low fluorescence signals strengths, a reflective gold chip coated with xanthan chitosan polyelectrolyte multilayers is introduced. On this amplification substrate fluorescence signals are up to 50 times higher than on glass slides and in comparison to commercial substrates the signal to noise ratio is enhanced by up to factor 11.Also sensitivity increases up to 38 fold.Finally this thesis reports on metal affinity slides based on amino acid derived ligands. These substrates enable the immobilisation of poly-histidine-tagged proteins via nickel-chelate complexes and thus facilitate the set-up of assays with uniformly oriented probe molecules.The slides are characterized by X-ray photoelectron spectroscopy (XPS) and show a selectivity >95% for tagged proteins in functional tests.Signal strengths are up to six times higher than on commercial products.11

44Sc for labeling of DOTA- and NODAGA-functionalized peptides: preclinical in vitro and in vivo investigations

Abstract Background Recently, 44Sc (T1/2 = 3.97 h, Eβ+ av = 632 keV, I = 94.3 %) has emerged as an attractive radiometal candidate for PET imaging using DOTA-functionalized biomolecules. The aim of this study was to investigate the potential of using NODAGA for the coordination of 44Sc. Two pairs of DOTA/NODAGA-derivatized peptides were investigated in vitro and in vivo and the results obtained with 44Sc compared with its 68Ga-labeled counterparts. DOTA-RGD and NODAGA-RGD, as well as DOTA-NOC and NODAGA-NOC, were labeled with 44Sc and 68Ga, respectively. The radiopeptides were investigated with regard to their stability in buffer solution and under metal challenge conditions using Fe3+ and Cu2+. Time-dependent biodistribution studies and PET/CT imaging were performed in U87MG and AR42J tumor-bearing mice. Results Both RGD- and NOC-based peptides with a DOTA chelator were readily labeled with 44Sc and 68Ga, respectively, and remained stable over at least 4 half-lives of the corresponding radionuclide. In contrast, the labeling of NODAGA-functionalized peptides with 44Sc was more challenging and the resulting radiopeptides were clearly less stable than the DOTA-derivatized matches. 44Sc-NODAGA peptides were clearly more susceptible to metal challenge than 44Sc-DOTA peptides under the same conditions. Instability of 68Ga-labeled peptides was only observed if they were coordinated with a DOTA in the presence of excess Cu2+. Biodistribution data of the 44Sc-labeled peptides were largely comparable with the data obtained with the 68Ga-labeled counterparts. It was only in the liver tissue that the uptake of 68Ga-labeled DOTA compounds was markedly higher than for the 44Sc-labeled version and this was also visible on PET/CT images. The 44Sc-labeled NODAGA-peptides showed a similar tissue distribution to those of the DOTA peptides without any obvious signs of in vivo instability. Conclusions Although DOTA revealed to be the preferred chelator for stable coordination of 44Sc, the data presented in this work indicate the possibility of using NODAGA in combination with 44Sc. In view of a clinical study, thorough investigations will be necessary regarding the labeling conditions and storage solutions in order to guarantee sufficient stability of 44Sc-labeled NODAGA compounds