RNA-Puzzles Round II: assessment of RNA structure prediction programs applied to three large RNA structures

This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5–3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson–Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/.     

Fast backward steps and detachment of single kinesin molecules measured under a wide range of loads

To understand force generation under a wide range of loads, the stepping of single kinesin molecules was measured at loads from −20 to 42 pN by optical tweezers with high temporal resolution. The optical trap has been improved to halve positional noise and increase bandwidth by using 200-nm beads. The step size of the forward and backward steps was 8.2 nm even over a wide range of loads. Histograms of the dwell times of backward steps and detachment fit well to two independent exponential equations with fast (~0.4 ms) and slow (>3 ms) time constants, indicating the existence of a fast step in addition to the conventional slow step. The dwell times of the fast steps were almost independent of the load and ATP concentration, while those of the slow backward steps and detachment depended on those. We constructed the kinetic model to explain the fast and slow steps under a wide range of loads.     

区域农业综合生产力的AHP-Fuzzy综合评判

根据系统分析原理,在确定区域农业综合生产力系统的层次结构基础上,用层次分析法确定各因素的权重,用模糊多级综合评判法对各区域综合生产力进行综合的评价．具有一定的科学性和实用价格．     

AFM nano‐mechanical study of the beating profile of hiPSC‐derived cardiomyocytes beating bodies WT and DM1

Myotonic Dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults, characterized by a variety of multisystemic features and associated with cardiac anomalies. Among cardiac phenomena, conduction defects, ventricular arrhythmias, and dilated cardiomyopathy represent the main cause of sudden death in DM1 patients. Patient‐specific induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) represent a powerful in vitro model for molecular, biochemical, and physiological studies of disease in the target cells. Here, we used an Atomic Force Microscope (AFM) to measure the beating profiles of a large number of cells, organized in CM clusters (Beating Bodies, BBs), obtained from wild type (WT) and DM1 patients. We monitored the evolution over time of the frequency and intensity of the beating. We determined the variations between different BBs and over various areas of a single BB, caused by morphological and biomechanical variations. We exploited the AFM tip to apply a controlled force over the BBs, to carefully assess the biomechanical reaction of the different cell clusters over time, both in terms of beating frequency and intensity. Our measurements demonstrated differences between the WT and DM1 clusters highlighting, for the DM1 samples, an instability which was not observed in WT cells. We measured differences in the cellular response to the applied mechanical stimulus in terms of beating synchronicity over time and cell tenacity, which are in good agreement with the cellular behavior in vivo. Overall, the combination of hiPSC‐CMs with AFM characterization can become a new tool to study the collective movements of cell clusters in different conditions and can be extended to the characterization of the BB response to chemical and pharmacological stimuli.     

Scaling of feeding biomechanics in the horn shark Heterodontus francisci: ontogenetic constraints on durophagy

Organismal performance changes over ontogeny as the musculoskeletal systems underlying animal behavior grow in relative size and shape. As performance is a determinant of feeding ecology, ontogenetic changes in the former can influence the latter. The horn shark Heterodontus francisci consumes hard-shelled benthic invertebrates, which may be problematic for younger animals with lower performance capacities. Scaling of feeding biomechanics was investigated in H. francisci (n=16, 19–59 cm standard length (SL)) to determine the biomechanical basis of allometric changes in feeding performance and whether this performance capacity constrains hard-prey consumption over ontogeny. Positive allometry of anterior (8–163 N) and posterior (15–382 N) theoretical bite force was attributed to positive allometry of cross-sectional area in two jaw adducting muscles and mechanical advantage at the posterior bite point (0.79–1.26). Mechanical advantage for anterior biting scaled isometrically (0.52). Fracture forces for purple sea urchins Strongylocentrotus purpuratus consumed by H. francisci ranged from 24 to 430 N. Comparison of these fracture forces to the bite force of H. francisci suggests that H. francisci is unable to consume hard prey early in its life history, but can consume the majority of S. purpuratus by the time it reaches maximum size. Despite this constraint, positive allometry of biting performance appears to facilitate an earlier entry into the durophagous niche than would an isometric ontogenetic trajectory. The posterior gape of H. francisci is significantly smaller than the urchins capable of being crushed by its posterior bite force. Thus, the high posterior bite forces of H. francisci cannot be fully utilized while consuming prey of similar toughness and size to S. purpuratus, and its potential trophic niche is primarily determined by anterior biting capacity.     

Studies on well-coupled Photosystem I-enriched subchloroplast vesicles — energy-dependent switching between two different active states of the proton-translocation adenosine triphosphatase

Single-turnover flash-induced ATP synthesis coupled to natural cyclic electron flow in Photosystem I-enriched subchloroplast vesicles (from spinach) was continuously followed by the luciferin-luciferase luminescence. The ATP yield per flash was maximal (1 ATP per s per 1000 Chl) around a flash frequency of 0.5–2 Hz. It decreased both at lower and higher flash frequencies. The decrease at high flash frequency was due to limitation by the electron-transfer rate, while the decrease at low flash frequency was directly due to intrinsic properties of the ATPase itself. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) decreased the yield at low frequency more than at high frequency. The same behaviour was observed if electron transfer was artificially mediated by pyocyanin. If the ADP concentration was increased from 40 to at least 80 μM, or if the vesicles were preincubated with 5 mM dithiothreitol (DTT), the decrease of the yield at flash frequencies below 0.5 Hz was no longer observed. Incubation with DTT increased the rates of ATP hydrolysis and synthesis at any flash frequency. The decrease of the yield could be elicited again by addition of 50 nM FCCP. It is concluded that at low levels of the protonmotive force (Δ gm_H⁺), the ATPase is converted into an active ATP-hydrolyzing state in which ATP synthesis activity is decreased due to a decreased affinity towards ADP and/or to a decreased release of newly synthesized ATP, that can be cancelled by increasing the ADP concentration or by addition of DTT in the absence of uncoupler.     

Free-energy landscapes of the coupled conformational transition and inclusion processes of altro-cyclodextrins

Abstract

Mono-altro-cyclodextrin (altro-CD) may undergo a conformational change of its altropyranose unit when encapsulating guest molecules of different sizes. This conformational transition is found to be coupled to the inclusion processes. In the present contribution, the possible conformational transition pathways in the four (self-)inclusion processes of altro-α and -β-CDs with moieties of variant shapes are explored from the insights of free-energy calculations. The two-dimensional free-energy landscapes characterising the coupled (self-)inclusion and isomerisation processes are determined, and the lowest free-energy pathways (LFEP) connecting the minima of the landscapes are located. The conformational statistics of the altropyranose units along the LFEPs reveal different transition pathways in the four (self-)inclusion processes. It can be concluded that when accommodating a free bulky guest molecule, the altropyranose unit will adjust its conformation to match the guest. However, such induced fit effect in the self-complexation of altro-CD derivatives will be weakened. The conformation of the altropyranose unit changes accompanying the self-complexation, but always adopts the ⁴C₁ one in the self-inclusion complex, irrespective of the shape of the guest moieties. The present results help determine the transition states of the (self-)inclusion processes of CDs and further improve the understanding of the mechanical properties of CD-based molecular shuttles.     

Roughness of the plasma membrane as an independent morphological parameter to study RBCs: A quantitative atomic force microscopy investigation

A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane-skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.     

High-resolution atomic force microscopy visualization of metalloproteins and their complexes

Background

Metalloproteins myeloperoxidase (MPO), ceruloplasmin (CP) and lactoferrin (LF) play an important role in regulation of inflammation and oxidative stress in vertebrates. It was previously shown that these proteins may work synergetically as antimicrobial and anti-inflammatory agents by forming complexes, such as MPO-CP and LF-CP. However, interaction of metalloprotein molecules with each other has never been characterized at a single-molecule level.