Precision and accuracy of single-molecule FRET measurements - a multi-laboratory benchmark study

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods

Engineered heart muscle allografts for heart repair in primates and humans

Cardiomyocytes can be implanted to remuscularize the failing heart. Challenges include sufficient cardiomyocyte retention for a sustainable therapeutic impact without intolerable side effects, such as arrhythmia and tumour growth. We investigated the hypothesis that epicardial engineered heart muscle (EHM) allografts from induced pluripotent stem cell-derived cardiomyocytes and stromal cells structurally and functionally remuscularize the chronically failing heart without limiting side effects in rhesus macaques. After confirmation of in vitro and in vivo (nude rat model) equivalence of the newly developed rhesus macaque EHM model with a previously established Good Manufacturing Practice-compatible human EHM formulation, long-term retention (up to 6 months) and dose-dependent enhancement of the target heart wall by EHM grafts constructed from 40 to 200 million cardiomyocytes/stromal cells were demonstrated in macaques with and without myocardial infarction-induced heart failure. In the heart failure model, evidence for EHM allograft-enhanced target heart wall contractility and ejection fraction, which are measures for local and global heart support, was obtained. Histopathological and gadolinium-based perfusion magnetic resonance imaging analyses confirmed cell retention and functional vascularization. Arrhythmia and tumour growth were not observed. The obtained feasibility, safety and efficacy data provided the pivotal underpinnings for the approval of a first-in-human clinical trial on tissue-engineered heart repair. Our clinical data confirmed remuscularization by EHM implantation in a patient with advanced heart failure

Design Considerations for Haptic-Enabled Virtual Reality Simulation for Interactive Learning of Nanoscale Science in Schools

This paper reports on a study which investigated whether the addition of haptics (virtual touch) to a 3D virtual reality (VR) simulation promotes understanding of key nanoscale concepts in membrane systems for students aged 12 to 13. We developed a virtual model of a section of the cell membrane and a haptic enabled interface that enables students to interact with the model and to manipulate objects in the model. Students, in two schools in England, worked collaboratively in pairs on activities designed to develop their understanding of key concepts of cell membrane function. Results of pre-and post-tests of conceptual knowledge and understanding showed significant knowledge gains but there were no significant differences between the haptic and non-haptic condition. However, findings from observation of the activities and student interviews revealed that students were very positive about using the system and believed that being able to feel structures and manipulate objects within the model assisted their learning. We examine some of the design challenges and issues affecting the perception of haptic feedback.</p

Thermoswitchable Nanoparticles Based on Elastin-like Polypeptides

The design of biocompatible particles with defined size on the nanometer scale has proven to be a challenging task in current biomedical research. Here we present an approach toward temperature-responsive nanoparticles by covalently cross-linking micelles based on trimeric constructs of elastin-like polypeptides. These trimers can be triggered to assemble into micelles by heating the solution above a specific transition temperature (<i>T</i>_t) which was shown in previous studies. Here we show that the disassembly of the micelles below the <i>T</i>_t can be prevented by the incorporation of covalent cross-links in the core of the micelles. This facilitates a temperature-triggered swelling and collapsing by around 35% in diameter, as determined by dynamic light scattering. Size distribution was confirmed by fluorescence correlation spectroscopy, atomic force microscopy, and transmission electron microscopy. We show switchable nanoparticles with reversible volume changes in the temperature region between 30 and 40 °C, making these particles promising candidates for switchable drug delivery carriers

Dynamics of heat shock protein 90 C-terminal dimerization is an important part of its conformational cycle

The molecular chaperone heat shock protein 90 (Hsp90) is an important and abundant protein in eukaryotic cells, essential for the activation of a large set of signal transduction and regulatory proteins. During the functional cycle, the Hsp90 dimer performs large conformational rearrangements. The transient N-terminal dimerization of Hsp90 has been extensively investigated, under the assumption that the C-terminal interface is stably dimerized. Using a fluorescence-based single molecule assay and Hsp90 dimers caged in lipid vesicles, we were able to separately observe and kinetically analyze N- and C-terminal dimerizations. Surprisingly, the C-terminal dimer opens and closes with fast kinetics. The occupancy of the unexpected C-terminal open conformation can be modulated by nucleotides bound to the N-terminal domain and by N-terminal deletion mutations, clearly showing a communication between the two terminal domains. Moreover our findings suggest that the C- and N-terminal dimerizations are anticorrelated. This changes our view on the conformational cycle of Hsp90 and shows the interaction of two dimerization domains