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

Production and separation of 43Sc for radiopharmaceutical purposes

Abstract Background The favorable decay properties of 43Sc and 44Sc for PET make them promising candidates for future applications in nuclear medicine. An advantage 43Sc (T1/2 = 3.89 h, Eβ+ av = 476 keV [88%]) exhibits over 44Sc, however, is the absence of co-emitted high energy γ-rays. While the production and application of 44Sc has been comprehensively discussed, research concerning 43Sc is still in its infancy. This study aimed at developing two different production routes for 43Sc, based on proton irradiation of enriched 46Ti and 43Ca target material. Results 43Sc was produced via the 46Ti(p,α)43Sc and 43Ca(p,n)43Sc nuclear reactions, yielding activities of up to 225 MBq and 480 MBq, respectively. 43Sc was chemically separated from enriched metallic 46Ti (97.0%) and 43CaCO3 (57.9%) targets, using extraction chromatography. In both cases, ~90% of the final activity was eluted in a small volume of 700 μL, thereby, making it suitable for direct radiolabeling. The prepared products were of high radionuclidic purity, i.e. 98.2% 43Sc were achieved from the irradiation of 46Ti, whereas the product isolated from irradiated 43Ca consisted of 66.2% 43Sc and 33.3% 44Sc. A PET phantom study performed with 43Sc, via both nuclear reactions, revealed slightly improved resolution over 44Sc. In order to assess the chemical purity of the separated 43Sc, radiolabeling experiments were performed with DOTANOC, attaining specific activities of 5–8 MBq/nmol, respectively, with a radiochemical yield of >96%. Conclusions It was determined that higher 43Sc activities were accessible via the 43Ca production route, with a comparatively less complex target preparation and separation procedure. The product isolated from irradiated 46Ti, however, revealed purer 43Sc with minor radionuclidic impurities. Based on the results obtained herein, the 43Ca route features some advantages (such as higher yields and direct usage of the purchased target material) over the 46Ti path when aiming at 43Sc production on a routine basis

47Sc as useful β–-emitter for the radiotheragnostic paradigm: a comparative study of feasible production routes

Abstract Background Radiotheragnostics makes use of the same molecular targeting vectors, labeled either with a diagnostic or therapeutic radionuclide, ideally of the same chemical element. The matched pair of scandium radionuclides, 44Sc and 47Sc, satisfies the desired physical aspects for PET imaging and radionuclide therapy, respectively. While the production and application of 44Sc was extensively studied, 47Sc is still in its infancy. The aim of the present study was, therefore, to investigate and compare two different methods of 47Sc production, based on the neutron irradiation of enriched 46Ca and 47Ti targets, respectively. Methods 47Sc was produced by thermal neutron irradiation of enriched 46Ca targets via the 46Ca(n,γ)47Ca → 47Sc nuclear reaction and by fast neutron irradiation of 47Ti targets via the 47Ti(n,p)47Sc nuclear reaction, respectively. The product was compared with regard to yield and radionuclidic purity. The chemical separation of 47Sc was optimized in order to obtain a product of sufficient quality determined by labeling experiments using DOTANOC. Finally, preclinical SPECT/CT experiments were performed in tumor-bearing mice and compared with the PET image of the 44Sc labeled counterpart. Results Up to 2 GBq 47Sc was produced by thermal neutron irradiation of enriched 46Ca targets. The optimized chemical isolation of 47Sc from the target material allowed formulation of up to 1.5 GBq 47Sc with high radionuclidic purity (>99.99%) in a small volume (~700 μL) useful for labeling purposes. Three consecutive separations were possible by isolating the in-grown 47Sc from the 46/47Ca-containing fraction. 47Sc produced by fast neutron irradiated 47Ti targets resulted in a reduced radionuclidic purity (99.95–88.5%). The chemical purity of the separated 47Sc was determined by radiolabeling experiments using DOTANOC achievable at specific activities of 10 MBq/nmol. In vivo the 47Sc-DOTANOC performed equal to 44Sc-DOTANOC as determined by nuclear imaging. Conclusion The production of 47Sc via the 46Ca(n,γ)47Ca nuclear reaction demonstrated significant advantages over the 47Ti production route, as it provided higher quantities of a radionuclidically pure product. The subsequent decay of 47Ca enabled the repeated separation of the 47Sc daughter nuclide from the 47Ca parent nuclide. Based on the results obtained from this work, 47Sc shows potential to be produced in suitable quality for clinical application