Experimentelle Charakterisierung von Aortenaneurysmen mittels Magnetic Particle Imaging unter Verwendung von 3D-gedruckten Modellen

Einleitung Die Magnetpartikelbildgebung (Magnetic Particle Imaging, MPI) ist eine neuartige, strahlenfreie, dreidimensionale bildgebende Modalität wobei magnetische Nanopartikel (Tracer) in hoher Auflösung dargestellt werden können. Wir untersuchten hierbei das Potenzial der ersten kommerziell erhältlichen MPI-Scanner für weitere Experimente und mögliche klinische Anwendungen zur Darstellung von Aortenaneurysmen. Methodik Mehrere in-vitro Experimente wurden mit zwei verschiedenen MPI Geräten (Scannern) durchgeführt. In einem Scanner wurde eine separate Empfängerspule eingebaut. Wir untersuchten die Bildgebung der Scanner. Gerade Silikonschläuche mit Innendurchmessern von 1 oder 2 mm wurden als Modelle verwendet und mit Resovist® (Ferucarbotran), magnetischen Nanopartikeln in unterschiedlichen Konzentrationen von 1, 5, 25, 250 und 500 mmol/l, gefüllt. Zur Darstellung von Aortenaneurysmen im Tiermodell wurden 3D-gedruckte Kaninchen- (ohne Aneurysma) und Mäusemodelle (mit Aortenaneurysma) erstellt. Die Mäusemodelle wurden in Originalskalierung und vergrößert (Faktor x1,5 und x2) hergestellt und mit Resovist-Konzentrationen gefüllt. Ein erstes in-vivo Experiment mit diesem Scanner wurde an einer Maus durchgeführt, um die Verteilung des Tracers im zeitlichen Verlauf und die Möglichkeit der Darstellung der Aorta zu untersuchen. Rekonstruktionen wurden mit dem Kaczmarz-Algorithmus durchgeführt. Resultate In den geraden Silikonschläuchen mit Innendurchmesser von 1 und 2 mm war die Morphologie ab einer Konzentration von 25 mmol/l deutlich erkenntlich. Für den Scanner mit separater Empfängerspule waren Konzentrationen von 5 mmol/ml 13 teilweise erkenntlich. Zwei anliegende Schläuche mit Innendurchmesser von 1 und 2 mm Wanddicke konnten bei 5 mmol/l unterschieden werden. Die Darstellung der Kaninchenaorta war möglich, die Morphologie war ersichtlich. Die Darstellung der Mäusaortenmodelle in Skalierungen x1, x1,5 und x2 war ab einer Konzentration von 25 mmol/l oder höher deutlich ersichtlich. Das in-vivo Experiment erlaubte die problemlose zeitlich und räumliche Darstellung der Mäuseaorta. Diskussion Unsere Ergebnisse zeigen die vergleichbare und adäquate Leistung der untersuchten präklinischen MPI Geräte und moderate Verbesserung der Bildgebung mit separater Empfängerspule. Sie zeigen die notwendigen Voraussetzungen für weitere in-vivo Experimente zur Erforschung von medizinischen Anwendungen wie der Darstellung von Aortenaneurysmen.Magnetic Particle Imaging (MPI) is a novel radiation-free, three-dimensional imaging modality that visualizes magnetic nanoparticles (tracers) with a high temporal resolution. We examined the suitability of the first commercially available MPI-scanners for experiments and clinical applications, particularly the imaging of aortic aneurysms. Methods A series of in-vitro experiments were performed with two preclinical MPI scanners. In one scanner, a prototype signal receiver coil was installed. We compare the imaging performance offered by the old and new hardware designs. Straight silicone tubes with internal diameters of either 1 mm or 2 mm were used as imaging phantoms. These were filled with Resovist® (Ferucarbotran) nanoparticles at concentrations of 1, 5, 25, 50, 250 and 500 mmol/l. To demonstrate the capabilities of the studied MPI scanners for use in animal models, 3D-printed models of rabbit aortas without aneurysms and mice aortas with abdominal aneurysms were produced. Mice models were printed in their original size and scaled by factors of 1.5 and 2.0. A first in-vivo experiment with a mouse on this scanner was performed to evaluate the temporal distribution of the tracer and to assess its ability to visualize the aorta. Reconstructions were performed using the Kaczmarz algorithm. Results For the straight-tube models of both 1 and 2 mm internal diameter, morphology was clearly visible for reconstructed MPI images at concentrations of 25 mmol/l or higher. For the upgraded scanner, concentrations as low as 5 mmol/l were suitable for imaging, depending on model sizes. It was possible to distinguish twoadjacent tubes of 1 mm internal diameter and 1 or 2 mm wall-thickness each, at tracer concentrations of 5 mmol/l. Imaging the rabbit aortic model served as a proof of concept, morphology was easily visible. Imaging the aortic aneurysm models at magnifications of 1.0, 1.5 and 2.0, all scanners were able to visualize the vessel clearly at concentrations of 25 mmol/l and higher. The in-vivo experiment allowed the temporal and spatial visualization of the mice aorta without difficulty. Discussion These results show comparable and adequate performance of these available preclinical MPI scanners and moderately improved visualizations after modification with a separate receiver-coil. They also demonstrate the required capabilities of existing MPI systems for in-vivo experiments examining clinical problems such as visualizing aortic aneurysms

Structural Studies of Two Enzymes of Pantothenate Biosynthesis in Escherichia Coli

Pantothenate (vitamin B5), which is the invariable metabolic precursor to coenzyme A, is synthesized from L-aspartate and alpha-ketoisovalerate in a converging four-step process in bacteria. Here, structural studies of two enzymes of pantothenate biosynthesis in Escherichia coli, L-aspartate-alpha-decarboxylase and ketopantoate hydroxymethyltransferase, are described. Ketopantoate hydroxymethyltransferase catalyzes the transfer of a hydroxymethyl group on to alpha-ketoisovalerate, assisted by the cofactor 5,10-methylene-5,6,7,8-tetrahydrofolate. In order to determine the mode of cofactor binding to the protein, ketopantoate hydroxymethyltransferase was crystallized in the presence of two 5,10-methylene-5,6,7,8-tetrahydrofolate analogues and alpha-ketoisovalerate. X-ray diffraction patterns, collected on the in-house X-ray diffraction data collection facility, extended to 4.0 Angstroem. Unit cell dimensions derived from these diffraction patterns indicate an asymmetric unit with one decameric enzyme. A detailed comparative structural analysis of the fold of ketopantoate hydroxymethyltransferase was carried out. Based on this investigation it was possible to assign the enzyme to the phosphoenolpyruvate/pyruvate enzyme superfamily. Furthermore, similarities in the mode of ligand binding to the catalytic magnesium, as well as differences in the mechanisms between the enzymes within this superfamily could be delineated. In common with a small, but widely distributed, group of mechanistically-related enzymes, L-aspartate-alpha-decarboxylase is translated as an inactive pro-enzyme, which self-processes at a specific site. In this process of intra-molecular protein maturation a covalently bound pyruvoyl cofactor is formed. A fast purification system for eight L-aspartate-alpha-decarboxylase mutants was established that allows production of large amounts of enzyme. In order to gain insights into the molecular mechanism of self-processing, crystallographic studies were carried out. Several of the purified mutants have been crystallized. X-ray diffraction data from glycine 24 to serine and serine 25 to threonine mutants were collected, to a maximum resolution of 1.26 Angstroem. The respective crystal structures were solved by molecular replacement. Along with the structures of an unprocessed, native precursor form of L-aspartate-alpha-decarboxylase and a serine 25 to alanine mutation, the structure models were refined and evaluated, and the models deposited in the Protein Data Bank. Analysis of these four structures together with four other L-aspartate-alpha-decarboxylase mutant structures revealed specific conformational constraints on the self-processing mechanism. Threonine 57 and a water molecule could be identified as catalytic elements, most likely essential for acid-base catalysis, and stabilization of the oxyoxazolidine intermediate in the self-processing reaction. A molecular mechanism for self-processing in L-aspartate-alpha-decarboxylase, largely based on the threonine 57 and a water molecule, is proposed. The differences in the structures of the cleavage site of the serine 25 to alanine and serine 25 to threonine mutants, relative to the structure of the unprocessed native precursor, suggest that molecular models of the cleavage site and mechanisms, based solely on serine to alanine and serine to threonine mutants, may lead to erroneous interpretations of the mechanism. On comparison with other self-processing systems, particularly, glycosylasparaginase, remarkable parallels in the structural features of the environment of the cleavage site were identified.Cambridge European Trust, University of Vienna, Sidney Sussex Colleg

HF-EPR, Raman, UV/VIS Light Spectroscopic, and DFT Studies of the Ribonucleotide Reductase R2 Tyrosyl Radical from Epstein-Barr Virus

Epstein-Barr virus (EBV) belongs to the gamma subfamily of herpes viruses, among the most common pathogenic viruses in humans worldwide. The viral ribonucleotide reductase small subunit (RNR R2) is involved in the biosynthesis of nucleotides, the DNA precursors necessary for viral replication, and is an important drug target for EBV. RNR R2 generates a stable tyrosyl radical required for enzymatic turnover. Here, the electronic and magnetic properties of the tyrosyl radical in EBV R2 have been determined by X-band and high-field/high-frequency electron paramagnetic resonance (EPR) spectroscopy recorded at cryogenic temperatures. The radical exhibits an unusually low g1-tensor component at 2.0080, indicative of a positive charge in the vicinity of the radical. Consistent with these EPR results a relatively high C-O stretching frequency associated with the phenoxyl radical (at 1508 cm−1) is observed with resonance Raman spectroscopy. In contrast to mouse R2, EBV R2 does not show a deuterium shift in the resonance Raman spectra. Thus, the presence of a water molecule as a hydrogen bond donor moiety could not be identified unequivocally. Theoretical simulations showed that a water molecule placed at a distance of 2.6 Å from the tyrosyl-oxygen does not result in a detectable deuterium shift in the calculated Raman spectra. UV/VIS light spectroscopic studies with metal chelators and tyrosyl radical scavengers are consistent with a more accessible dimetal binding/radical site and a lower affinity for Fe2+ in EBV R2 than in Escherichia coli R2. Comparison with previous studies of RNR R2s from mouse, bacteria, and herpes viruses, demonstrates that finely tuned electronic properties of the radical exist within the same RNR R2 Ia class

Evolution of pathogenicity and sexual reproduction in eight Candida genomes

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.publishe

Molecular basis for inner kinetochore configuration through RWD domain–peptide interactions

Abstract Kinetochores are dynamic cellular structures that connect chromosomes to microtubules. They form from multi‐protein assemblies that are evolutionarily conserved between yeasts and humans. One of these assemblies—COMA—consists of subunits Ame1CENP‐U, Ctf19CENP‐P, Mcm21CENP‐O and Okp1CENP‐Q. A description of COMA molecular organization has so far been missing. We defined the subunit topology of COMA, bound with inner kinetochore proteins Nkp1 and Nkp2, from the yeast Kluyveromyces lactis, with nanoflow electrospray ionization mass spectrometry, and mapped intermolecular contacts with hydrogen‐deuterium exchange coupled to mass spectrometry. Our data suggest that the essential Okp1 subunit is a multi‐segmented nexus with distinct binding sites for Ame1, Nkp1‐Nkp2 and Ctf19‐Mcm21. Our crystal structure of the Ctf19‐Mcm21 RWD domains bound with Okp1 shows the molecular contacts of this important inner kinetochore joint. The Ctf19‐Mcm21 binding motif in Okp1 configures a branch of mitotic inner kinetochores, by tethering Ctf19‐Mcm21 and Chl4CENP‐N‐Iml3CENP‐L. Absence of this motif results in dependence on the mitotic checkpoint for viability

Retraction of Mechanistic Basis for Epstein–Barr Virus Ribonucleotide-reductase Small-Subunit Function

Retraction of Mechanistic Basis for Epstein–Barr Virus Ribonucleotide-reductase Small-Subunit Functio

CT screening and follow-up of lung nodules: effects of tube current-time setting and nodule size and density on detectability and of tube current-time setting on apparent size

The purpose of the study was to quantify and compare the effect of CT dose and of size and density of nodules on the detectability of lung nodules and to quantify the influence of CT dose on the size of the nodules