Toward Quantification and Visualization of Active Stress Waves for Myocardial Biomechanical Function Assessment.

Estimating and visualizing myocardial active stress wave patterns is crucial to understanding the mechanical activity of the heart and provides a potential non-invasive method to assess myocardial function. These patterns can be reconstructed by analyzing 2D and/or 3D tissue displacement data acquired using medical imaging. Here we describe an application that utilizes a 3D finite element formulation to reconstruct active stress from displacement data. As a proof of concept, a simple cubic mesh was used to represent a myocardial tissue "sample" consisting of a 10 × 10 × 10 lattice of nodes featuring different fiber directions that rotate with depth, mimicking cardiac transverse isotropy. In the forward model, tissue deformation was generated using a test wave with active stresses that mimic the myocardial contractile forces. The generated deformation field was used as input to an inverse model designed to reconstruct the original active stress distribution. We numerically simulated malfunctioning tissue regions (experiencing limited contractility and hence active stress) within the healthy tissue. We also assessed model sensitivity by adding noise to the deformation field generated using the forward model. The difference image between the original and reconstructed active stress distribution suggests that the model accurately estimates active stress from tissue deformation data with a high signal-to-noise ratio.

Broccoli Fluorets: Split Aptamers as a User-Friendly Fluorescent Toolkit for Dynamic RNA Nanotechnology.

RNA aptamers selected to bind fluorophores and activate their fluorescence offer a simple and modular way to visualize native RNAs in cells. Split aptamers which are inactive until the halves are brought within close proximity can become useful for visualizing the dynamic actions of RNA assemblies and their interactions in real time with low background noise and eliminated necessity for covalently attached dyes. Here, we design and test several sets of F30 Broccoli aptamer splits, that we call fluorets, to compare their relative fluorescence and physicochemical stabilities. We show that the splits can be simply assembled either through one-pot thermal annealing or co-transcriptionally, thus allowing for direct tracking of transcription reactions via the fluorescent response. We suggest a set of rules that enable for the construction of responsive biomaterials that readily change their fluorescent behavior when various stimuli such as the presence of divalent ions, exposure to various nucleases, or changes in temperature are applied. We also show that the strand displacement approach can be used to program the controllable fluorescent responses in isothermal conditions. Overall, this work lays a foundation for the future development of dynamic systems for molecular computing which can be used to monitor real-time processes in cells and construct biocompatible logic gates.

Dynamic Behavior of RNA Nanoparticles Analyzed by AFM on a Mica/Air Interface.

RNA is an attractive biopolymer for engineering self-assembling materials suitable for biomedical applications. Previously, programmable hexameric RNA rings were developed for the controlled delivery of up to six different functionalities. To increase the potential for functionalization with little impact on nanoparticle topology, we introduce gaps into the double-stranded regions of the RNA rings. Molecular dynamic simulations are used to assess the dynamic behavior and the changes in the flexibility of novel designs. The changes suggested by simulations, however, cannot be clearly confirmed by the conventional techniques such as nondenaturing polyacrylamide gel electrophoresis (native-PAGE) and dynamic light scattering (DLS). Also, an in vitro analysis in primary cultures of human peripheral blood mononuclear cells does not reveal any discrepancy in the immunological recognition of new assemblies. To address these deficiencies, we introduce a computer-assisted quantification strategy. This strategy is based on an algorithmic atomic force microscopy (AFM)-resolved deformation analysis of the RNA nanoparticles studied on a mica/air interface. We validate this computational method by manual image analysis and fitting it to the simulation-predicted results. The presented nanoparticle modification strategy and subsequent AFM-based analysis are anticipated to provide a broad spectrum approach for the future development of nucleic acid-based nanotechnology.