Short, synthetic and selectively 13C-labeled RNA sequences for the NMR structure determination of protein-RNA complexes

We report an optimized synthesis of all canonical 2′-O-TOM protected ribonucleoside phosphoramidites and solid supports containing [13C5]-labeled ribose moieties, their sequence-specific introduction into very short RNA sequences and their use for the structure determination of two protein-RNA complexes. These specifically labeled sequences facilitate RNA resonance assignments and are essential to assign a high number of sugar-sugar and intermolecular NOEs, which ultimately improve the precision and accuracy of the resulting structures. This labeling strategy is particularly useful for the study of protein-RNA complexes with single-stranded RNA in solution, which is rapidly an increasingly relevant research area in biolog

Short, synthetic and selectively 13C-labeled RNA sequences for the NMR structure determination of protein–RNA complexes

We report an optimized synthesis of all canonical 2′-O-TOM protected ribonucleoside phosphoramidites and solid supports containing [13C5]-labeled ribose moieties, their sequence-specific introduction into very short RNA sequences and their use for the structure determination of two protein–RNA complexes. These specifically labeled sequences facilitate RNA resonance assignments and are essential to assign a high number of sugar–sugar and intermolecular NOEs, which ultimately improve the precision and accuracy of the resulting structures. This labeling strategy is particularly useful for the study of protein–RNA complexes with single-stranded RNA in solution, which is rapidly an increasingly relevant research area in biology

Association of smoking and physical inactivity with MRI derived changes in cardiac function and structure in cardiovascular healthy subjects

We aimed to investigate the association of smoking and physical exercise on ventricular function and structure, determined by cardiac magnetic resonance imaging (CMR), in subjects without known cardiovascular diseases. A total of 381 participants (median age 57 years) of the Cooperative Health Research in the Region of Augsburg (KORA) FF4 cohort underwent CMR. The participants' smoking and sporting habits were measured by a questionnaire. Physical inactivity was associated with a reduction of left ventricular ejection fraction (LV-EF), stroke volume, early diastolic peak filling rate and peak ejection rate of the left ventricle as well as right ventricular stroke volume. LV-EF was reduced in subjects with almost no physical activity compared to subjects with regular physical activity (68.4%, 95%CI 66.8-70.1% vs. 70.8%, 95%CI 69.2-72.3%, p < 0,05). Smokers had lower right ventricular end-diastolic volumes (80.6 ml/m(2), 95%CI 76.7-84.5 ml/m(2);never-smokers: 85.5 ml/m(2), 95%CI 82.6-88.3 ml/m(2);p < 0.05) but higher extracellular volume fractions (ECV) and fibrosis volumes (34.3 ml, 95%CI 32.5-36.0 ml, vs. 31.0 ml, 95%CI 29.6-32.3 ml, p < 0.01). We conclude that asymptomatic individuals without known cardiovascular diseases show differences in cardiac function and structure depending on their physical activity and smoking habits. This underlines the importance of prevention and health education

Underneath the Arches, no. 61, October 24, 1968

Underneath the Arches was Grand Valley State\u27s faculty and staff newsletter, published from 1963 to 1971. It was a precursor to the Forum

Visualizing Typical Features of Breast Fibroadenomas Using Phase-Contrast CT: An Ex-Vivo Study

Background: Fibroadenoma is the most common benign solid breast lesion type and a very common cause for histologic assessment. To justify a conservative therapy, a highly specific discrimination between fibroadenomas and other breast lesions is crucial. Phase-contrast imaging offers improved soft-tissue contrast and differentiability of fine structures combined with the potential of 3-dimensional imaging. In this study we assessed the potential of grating-based phase-contrast CT imaging for visualizing diagnostically relevant features of fibroadenomas. Materials and Methods: Grating-based phase-contrast CT was performed on six ex-vivo formalin-fixed breast specimens containing a fibroadenoma and three samples containing benign changes that resemble fibroadenomas using Talbot Lau interferometry and a polychromatic X-ray source. Phase-contrast and simultaneously acquired absorption-based 3D-datasets were manually matched with corresponding histological slices. The visibility of diagnostically valuable features was assessed in comparison with histology as the gold-standard. Results: In all cases, matching of grating-based phase-contrast CT images and histology was successfully completed. Grating-based phase-contrast CT showed greatly improved differentiation of fine structures and provided accurate depiction of strands of fibrous tissue within the fibroadenomas as well as of the diagnostically valuable dilated, branched ductuli of the fibroadenomas. A clear demarcation of tumor boundaries in all cases was provided by phase- but not absorption-contrast CT. Conclusions: Pending successful translation of the technology to a clinical setting and considerable reduction of the required dose, the data presented here suggest that grating-based phase- contrast CT may be used as a supplementary non-invasive diagnostic tool in breast diagnostics. Phase-contrast CT may thus contribute to the reduction of false positive findings and reduce the recall and core biopsy rate in population-based screening. Phase-contrast CT may further be used to assist during histopathological workup, offering a 3D view of the tumor and helping to identify diagnostically valuable tissue sections within large tumors

Dose-compatible grating-based phase-contrast mammography on mastectomy specimens using a compact synchrotron source

With the introduction of screening mammography, the mortality rate of breast cancer has been reduced throughout the last decades. However, many women undergo unnecessary subsequent examinations due to inconclusive diagnoses from mammography. Two pathways appear especially promising to reduce the number of false-positive diagnoses. In a clinical study, mammography using synchrotron radiation was able to clarify the diagnosis in the majority of inconclusive cases. The second highly valued approach focuses on the application of phase-sensitive techniques such as grating-based phasecontrast and dark-field imaging. Feasibility studies have demonstrated a promising enhancement of diagnostic content, but suffer from dose concerns. Here we present dose-compatible grating-based phase-contrast and dark-field images as well as conventional absorption images acquired with monochromatic x-rays from a compact synchrotron source based on inverse Compton scattering. Images of freshly dissected mastectomy specimens show improved diagnostic content over ex-vivo clinical mammography images at lower or equal dose. We demonstrate increased contrast-to-noise ratio for monochromatic over clinical images for a well-defined phantom. Compact synchrotron sources could potentially serve as a clinical second level examination

Sequence-specific binding of single-stranded RNA: is there a code for recognition?

A code predicting the RNA sequence that will be bound by a certain protein based on its amino acid sequence or its structure would provide a useful tool for the design of RNA binders with desired sequence-specificity. Such de novo designed RNA binders could be of extraordinary use in both medical and basic research applications. Furthermore, a code could help to predict the cellular functions of RNA-binding proteins that have not yet been extensively studied. A comparative analysis of Pumilio homology domains, zinc-containing RNA binders, hnRNP K homology domains and RNA recognition motifs is performed in this review. Based on this, a set of binding rules is proposed that hints towards a code for RNA recognition by these domains. Furthermore, we discuss the intermolecular interactions that are important for RNA binding and summarize their importance in providing affinity and specificity

Structure and function of PTB and Fox, two regulators of alternative splicing

When the human genome was initially sequenced and analyzed, the alignment of expressed sequence tags with the genomic DNA sequence revealed that about 60% of all human genes are alternatively spliced (Lander et al., 2001). Hence, alternative splicing is a major mechanism to increase proteome diversity and to achieve genetic regulation in man. Misregulation of alternative splicing is implicated in many human diseases like certain types of cancer, cystic fibrosis, or neurological disorders. Frequently, mutations that affect alternative splicing cause more severe defects than mutations that merely change the identity of individual amino acids, as they can disrupt inclusion of entire exons or activate cryptic splice sites (Blcncowe, 2006; Licatalosi and Darnell, 2006). Alternative splicing is regulated by numerous protein factors that recognize specific sequences within immature messenger RNAs (prc-mRNAs). The aim of this thesis was to gain a more detailed understanding of splicing regulation by studying the structure and function of two splicing factors, the polypyrimidine tract binding protein (PTB) and the protein Fox. These proteins are key factors in controlling splicing regulation of the c-src pre-mRNA, which encodes a receptor tyrosine kinase in which a short exon is selectively included in neurons but excluded in all other cell types. The structures of the RNA recognition motif domains (RRMs) of PTB and Fox were solved in complex with their target RNA sequences using NMR spectroscopy. The structure of the Fox RRM/RNA complex reveals a novel mode of RNA recognition by an RRM and the structure of PTB in complex with polypyrimidine tracts suggests a new model for splicing repression. Furthermore, the RNA binding properties of PTB and Fox were analyzed and evidence that argues for a model that explains c-src splicing regulation by competition of PTB and Fox for overlapping binding sites was collected. Im Zuge der Sequenzierung des menschlichen Genoms ergab eine vergleichende Sequenzanalyse von zytoplasmatischen RNAs mit der genomischen DNA Sequenz, dass etwa 60%) aller menschlichen Gene alternativ gespleisst werden (Lander et al., 2001). Dies bedeutet, dass alternatives Spleissen einen der wichtigsten Mechanismen zur Erweiterung der Diversität des Proteoms und zur Erreichung genetischer Regulation im Menschen darstellt. Die Misregulierung von alternativem Spleissen steht in Zusammenhang mit verschiedenen menschlichen Krankheiten, wie bestimmten Krebsarten, zystischer Fibrose, oder neurologischen Störungen. Oft verursachen Mutationen, die alternatives Spleissen beeinflussen, schwerere Defekte als Mutationen, die lediglich die die Identität einzelner Aminosäuren verändern, weil sie das Einschliessen ganzer Exons verhindern oder kryptische Spleiss-Stellen akitvieren können (Blencowe, 2006; Licatalosi and Darneil, 2006). Alternatives Spleissen wird von unzähligen Proteinfaktoren reguliert, die spezifische Sequenzen innerhalb der unreifen messenger RNA (prämRNA) erkennen. Das Ziel dieser Arbeit war es, durch die Untersuchung von Struktur und Funktion zweier Spleissfaktorcn, dem Polypyrimidin-Trakt bindenden Protein (PTB) und dem Protein Fox, ein detaillierteres Verständnis der Regulation des RNA Spleissens zu erlangen. Diese Proteine sind Schlüsselfaktoren für die Kontrolle des Spleissens der c-src prä-mRNA, die für eine Rezeptor-Tyrosinkinase kodiert und in die ein kurzes Exon selektiv in Neuronen eingeschlossen wird, während es in allen anderen Zelltypen ausgeschlossen wird. Die Strukturen der RNA-bindenden Domänen („RNA recognition motif domains", RRMs) von PTB und Fox wurden im Komplex mit ihren RNA Zielsequenzen und mit Hilfe von NMR Spektroskopie gelöst. Die Struktur des Fox RRM/RNA Komplexes offenbart eine neuartige Form der RNA Erkennung durch eine RRM Domäne und die Struktur von PTB im Komplex mit Polypyrimidin Sequenzen impliziert ein neues Modell der Spleiss-Unterdrückung. Ausserdem wurden die RNA Bindungseigenschaften von PTB und Fox analysiert und Hinweise, die für ein Modell argumentieren, das die Regulation des c-src Nl Exons durch Kompetition von PTB und Fox um überlappende Bindestcllen erklärt, wurden gesammelt

Structure-function relationships of the polypyrimidine tract binding protein

ISSN:1420-682XISSN:1420-907