Computed tomography versus invasive coronary angiography: design and methods of the pragmatic randomised multicentre DISCHARGE trial.

OBJECTIVES: More than 3.5 million invasive coronary angiographies (ICA) are performed in Europe annually. Approximately 2 million of these invasive procedures might be reduced by noninvasive tests because no coronary intervention is performed. Computed tomography (CT) is the most accurate noninvasive test for detection and exclusion of coronary artery disease (CAD). To investigate the comparative effectiveness of CT and ICA, we designed the European pragmatic multicentre DISCHARGE trial funded by the 7th Framework Programme of the European Union (EC-GA 603266). METHODS: In this trial, patients with a low-to-intermediate pretest probability (10-60 %) of suspected CAD and a clinical indication for ICA because of stable chest pain will be randomised in a 1-to-1 ratio to CT or ICA. CT and ICA findings guide subsequent management decisions by the local heart teams according to current evidence and European guidelines. RESULTS: Major adverse cardiovascular events (MACE) defined as cardiovascular death, myocardial infarction and stroke as a composite endpoint will be the primary outcome measure. Secondary and other outcomes include cost-effectiveness, radiation exposure, health-related quality of life (HRQoL), socioeconomic status, lifestyle, adverse events related to CT/ICA, and gender differences. CONCLUSIONS: The DISCHARGE trial will assess the comparative effectiveness of CT and ICA. KEY POINTS: • Coronary artery disease (CAD) is a major cause of morbidity and mortality. • Invasive coronary angiography (ICA) is the reference standard for detection of CAD. • Noninvasive computed tomography angiography excludes CAD with high sensitivity. • CT may effectively reduce the approximately 2 million negative ICAs in Europe. • DISCHARGE addresses this hypothesis in patients with low-to-intermediate pretest probability for CAD.

Rigid DNA beams for high-resolution single-molecule mechanics.

Bridging the gap: Rigid DNA linkers (blue, see picture) between microspheres (green) for high-resolution single-molecule mechanical experiments were constructed using DNA origami. The resulting DNA helical bundles greatly reduce the noise generated in studies of conformation changes using optical tweezers and were applied to study small DNA secondary structures.

Hidden Markov analysis of trajectories in single-molecule experiments and the effects of missed events.

The ever more complex fluctuation patterns discovered by single molecule experiments require statistical methods to analyze multi-state hopping traces of long lengths. Hidden Markov modeling is a statistical tool that offers the scalability to analyze even complex data and extract kinetic information. We give an introduction on how to implement hidden Markov modeling for the analysis of single molecule force spectroscopic traces, deal with missed events, and test the method on a calcium binding protein.

The complex folding network of single calmodulin molecules.

Direct observation of the detailed conformational fluctuations of a single protein molecule en route to its folded state has so far been realized only in silico. We have used single-molecule force spectroscopy to study the folding transitions of single calmodulin molecules. High-resolution optical tweezers assays in combination with hidden Markov analysis reveal a complex network of on- and off-pathway intermediates. Cooperative and anticooperative interactions across domain boundaries can be observed directly. The folding network involves four intermediates. Two off-pathway intermediates exhibit non-native interdomain interactions and compete with the ultrafast productive folding pathway.

Whole-body MRI with assessment of hepatic and extraabdominal enhancement after administration of gadoxetic acid for staging of rectal carcinoma.

BACKGROUND: In TNM staging of rectal cancer by MRI, unspecific extracellular contrast agent Gd-DTPA is established for extrahepatic and vascular enhancement whereas liver-specific gadoxetic acid has proven high accurate detection of liver metastasis. PURPOSE: To compare intraindividually the qualification and quantification of enhancement in liver parenchyma, abdominal, pulmonary, and pelvic vessels between gadoxetic acid and Gd-DTPA. MATERIAL AND METHODS: Sixteen patients with histologically proven rectal carcinoma (mean age 62.9 years) were imaged twice by MRI. For pretherapeutic staging 10 mL gadoxetic acid (mean dose 0.032 mmol Gd/kg body weight) and for restaging after neoadjuvant therapy Gd-DTPA (0.1 mmol Gd/kg body weight) were administered. The liver was acquired in arterial-dominant and portal venous phases, the thorax and pelvis were depicted in venous phases using three-dimensional T1-weighted sequences. Contrast enhancement was rated by two independent readers and compared by means of multinomial regression analysis using generalized estimating equations. Signal-to-noise ratios were compared by two-sided paired t-tests. RESULTS: Overall contrast enhancement was rated sufficient for diagnosis in all examinations and both contrast agents. Vascular enhancement was rated comparable with exception of the aorta, the peripheral intrahepatic veins, and the central lung vessels (p = 0.0182, p = 0.0053, p = 0.0083, in favor of Gd-DTPA). Quantitative evaluation revealed no statistically significant differences in parenchymal and vascular signal-to-noise ratios with exception of the aorta, and the central pulmonary artery (67.4 vs. 89.3; p = 0.0421, 44.5 vs. 59.5; p = 0.0446 respectively, in favor of Gd-DTPA). CONCLUSION: The contrast enhancement after gadoxetic acid is comparable to Gd-DTPA and appears suitable for comprehensive TNM-staging by combining high accurate liver-specific phases with efficacious vascular enhancement in the different anatomic regions.

Designing the folding mechanics of coiled coils.

Naturally occurring coiled coils are often not homogeneous throughout their entire structure but rather interrupted by sequence discontinuities and non-coiled-coil-forming subsegments. We apply atomic force microscopy to locally probe the mechanical folding/unfolding process of a well-understood model coiled coil when unstructured subsegments with different sizes are added. We find that the refolding force decreases from 7.8 pN with increasing size of the added unstructured subsegment, while the unfolding properties of the model coiled coil remain unchanged. We show that this behavior results from the increased size of the nucleation seed which has to form before further coiled-coil folding can proceed. Since the nucleation seed size is linked to the width of the energetic folding barrier, we are able to directly measure the dependence of folding forces on the barrier width. Our results allow the design of coiled coils with designated refolding forces by simply adjusting the nucleation seed size.

Ligand-dependent equilibrium fluctuations of single calmodulin molecules.

Single-molecule force spectroscopy allows superb mechanical control of protein conformation. We used a custom-built low-drift atomic force microscope to observe mechanically induced conformational equilibrium fluctuations of single molecules of the eukaryotic calcium-dependent signal transducer calmodulin (CaM). From this data, the ligand dependence of the full energy landscape can be reconstructed. We find that calcium ions affect the folding kinetics of the individual CaM domains, whereas target peptides stabilize the already folded structure. Single-molecule data of full length CaM reveal that a wasp venom peptide binds noncooperatively to CaM with 2:1 stoichiometry, whereas a target enzyme peptide binds cooperatively with 1:1 stoichiometry. If mechanical load is applied directly to the target peptide, real-time binding/unbinding transitions can be observed.

Single-molecule dynamics of mechanical coiled-coil unzipping.

We use atomic force microscopy (AFM) to mechanically unzip and rezip a double-stranded coiled-coil structure at varying pulling velocities. We find that force-extension traces exhibit hysteresis that grows with increasing pulling velocity. This shows that coiled-coil unzipping and rezipping do not occur in thermal equilibrium on our experimental time scale. We present a nonequilibrium simulation that fully reproduces the hysteresis effects, giving detailed insight into dynamics of coiled-coil folding. Using this model, we find that seed formation is responsible for the hysteresis. The seed consists of four alpha-helical turns on both strands of the coiled coil. To obtain equilibrium information from our nonequilibrium experiments, we used the Crooks fluctuation theorem (CFT) to calculate the equilibrium free energy of folding for all of the different pulling velocities. The paper presented here lays the groundwork for the study of self-assembly properties of many physiologically relevant coiled-coil structures at the single-molecule level.