Negative Staining and Image Classification – Powerful Tools in Modern Electron Microscopy

Vitrification is the state-of-the-art specimen preparation technique for molecular electron microscopy (EM) and therefore negative staining may appear to be an outdated approach. In this paper we illustrate the specific advantages of negative staining, ensuring that this technique will remain an important tool for the study of biological macromolecules. Due to the higher image contrast, much smaller molecules can be visualized by negative staining. Also, while molecules prepared by vitrification usually adopt random orientations in the amorphous ice layer, negative staining tends to induce preferred orientations of the molecules on the carbon support film. Combining negative staining with image classification techniques makes it possible to work with very heterogeneous molecule populations, which are difficult or even impossible to analyze using vitrified specimens

Mice with Mutation in Dynein Heavy Chain 1 Do Not Share the Same Tau Expression Pattern with Mice with SOD1-Related Motor Neuron Disease

Due to controversy about the involvement of Dync1h1 mutation in pathogenesis of motor neuron disease, we investigated expression of tau protein in transgenic hybrid mice with Dync1h1 (so-called Cra1/+), SOD1G93A (SOD1/+), double (Cra1/SOD1) mutations and wild-type controls. Total tau-mRNA and isoforms 0, 1 and 2 N expression was studied in frontal cortex, hippocampus, spinal cord and cerebellum of presymptomatic and symptomatic animals (age 70, 140 and 365 days). The most significant differences were found in brain cortex and cerebellum, but not in hippocampus and spinal cord. There were less changes in Cra1/SOD1 double heterozygotes compared to mice harboring single mutations. The differences in total tau expression and in profile of its isoforms between Cra1/+ and SOD1/+ transgenics indicate a distinct pathogenic entity of these two conditions

Modelling the long-term dynamics of the energy transition accounting for socioeconomic behaviour and biophysical constraints: overview of the Wiliam Energy Module

WILIAM (Within Limit Integrated Assessment Model) is a global multiregional IAM that combines economic, social, demographic, environmental, energy and material related aspects into one system dynamics model. It aims to provide stakeholders with an open source, welldocumented model to assess the feasibility, effectiveness, costs and impacts of different sustainability policy options. The adequate representation of energy production is key to assess future sustainability pathways. The main function of the developed energy module is to estimate the primary energy requirements and related GHG emissions for satisfying the economic demand. This goal was achieved by 7 major sub-modules: (1) End-use: translates the economic demand into final energy demand through a hybrid approach combining bottom-up with energy intensities for different sectors. (2) Energy transformation: maps the entire energy conversion chain from final to primary energy, including intermediary energy commodities and an allocation function for power plant utilization. (3) Energy capacity: keeps track of the current power plant capacity stock, decommissioning of expired capacities, as well as the build-up of new capacities. An allocation function for choosing the suitable technology types for new capacities stands at the core of this sub-module. (4) Computation of the EROI of green technologies (5) Variability and storage: keeps track of sub-annual time scale effects on annual energy balances depending on the current power system setup (DSM, Storage, sector coupling). (6) Consideration of techno-sustainable potentials of RES considering geographical, resource and Energy Return on Energy Investment (EROI) constraints. (7) Computation of the energy-related GHG emissions

FKBP12 Activates the Cardiac Ryanodine Receptor Ca2+-Release Channel and Is Antagonised by FKBP12.6

Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca2+-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca2+-induced Ca2+-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca2+, whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 µM) increased Ca2+-wave frequency and decreased the SR Ca2+-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12

Triadin/Junctin Double Null Mouse Reveals a Differential Role for Triadin and Junctin in Anchoring CASQ to the jSR and Regulating Ca2+ Homeostasis

Triadin (Tdn) and Junctin (Jct) are structurally related transmembrane proteins thought to be key mediators of structural and functional interactions between calsequestrin (CASQ) and ryanodine receptor (RyRs) at the junctional sarcoplasmic reticulum (jSR). However, the specific contribution of each protein to the jSR architecture and to excitation-contraction (e-c) coupling has not been fully established. Here, using mouse models lacking either Tdn (Tdn-null), Jct (Jct-null) or both (Tdn/Jct-null), we identify Tdn as the main component of periodically located anchors connecting CASQ to the RyR-bearing jSR membrane. Both proteins proved to be important for the structural organization of jSR cisternae and retention of CASQ within them, but with different degrees of impact. Our results also suggest that the presence of CASQ is responsible for the wide lumen of the jSR cisternae. Using Ca2+ imaging and Ca2+ selective microelectrodes we found that changes in e-c coupling, SR Ca2+content and resting [Ca2+] in Jct, Tdn and Tdn/Jct-null muscles are directly correlated to the effect of each deletion on CASQ content and its organization within the jSR. These data suggest that in skeletal muscle the disruption of Tdn/CASQ link has a more profound effect on jSR architecture and myoplasmic Ca2+ regulation than Jct/CASQ association

Structural insights into Ca2+-activated long-range allosteric channel gating of RyR1

Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca2+-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 angstrom and a resolution of 4.2 angstrom for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca2+ activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family.Strategic Priority Research Program of Chinese Academy of Sciences [XDB08030202]; National Basic Research Program (973 Program); Ministry of Science & Technology of China [2012CB917200, 2014CB910700]; National Natural Science Foundation of China [31270768]; Ministry of Education of China (111 Program China)SCI(E)PubMed中国科技核心期刊(ISTIC)[email protected]; [email protected]

Small scale structures: the fabrication of polymeric nanostructures for biomedical applications using pattern replication techniques

Polymers are excellent candidates for the production of biomedical devices incorporating nanometric structures. Good optical transparency and sealing properties, low fabrication costs, fast design realization times, and, crucially, biocompatibility are all advantages that can be exploited by scientists for the production of such devices. Here, we review some of the methods and techniques used in the fabrication of polymeric nanostructures by pattern replication techniques that may be of relevance in the production of biomedical devices. Emphasis is placed on imprint production of polymeric replicas, with master fabrication using focussed ion-beam technology, as a relatively simple method for reproducibly obtaining large numbers of nanostructures. The use of these structures in polymercasting techniques is also described, together with some specific fabrication considerations. The maturity reached by polymer-based nanotechnologies, together with the first polymer-based applications for single-cell analysis and for counting single DNA molecules, demonstrates that polymers constitute a viable alternative to silicon-based nanotechnologies for biomedical applications