Structure of isolated Z-disks from honeybee flight muscle

The Z-disk is a complex structure comprising some 40 proteins that are involved in the transmission of force developed during muscle contraction and in important signalling pathways that govern muscle homeostasis. In the Z-disk the ends of antiparallel thin filaments from adjacent sarcomeres are crosslinked by α-actinin. The structure of the Z-disk lattice varies greatly throughout the animal kingdom. In vertebrates the thin filaments form a tetragonal lattice, whereas invertebrate flight muscle has a hexagonal lattice. The width of the Z-disk varies considerably and correlates with the number of α-actinin bridges. A detailed description at a high resolution of the Z-disk lattice is needed in order to better understand muscle function and disease. The molecular architecture of the Z-disk lattice in honeybee (Apis mellifera) is known from plastic embedded thin sections to a resolution of 7 nm, which is not sufficient to dock component protein crystal structures. It has been shown that sectioning is a damaging process that leads to the loss of finer details present in biological specimens. However, the Apis Z-disk is a thin structure (120 nm) suitable for cryo EM. We have isolated intact honeybee Z-disks from indirect flight muscle, thus obviating the need of plastic sectioning. We have employed cryo electron tomography and image processing to investigate the arrangement of proteins within the hexagonal lattice of the Apis Z-disk. The resolution obtained, ~6 nm, was probably limited by damage caused by the harshness of the conditions used to extract the myofibrils and isolate the Z-disks

Structure of the Vacuolar H⁺-ATPase Rotary Motor Reveals New Mechanistic Insights

Vacuolar H+-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases

Subunit positioning and stator filament stiffness in regulation and power transmission in the V1 motor of the manduca sexta V-ATpase

The vacuolar H(+)-ATPase (V-ATPase) is an ATP-driven proton pump essential to the function of eukaryotic cells. Its cytoplasmic V1 domain is an ATPase, normally coupled to membrane-bound proton pump Vo via a rotary mechanism. How these asymmetric motors are coupled remains poorly understood. Low energy status can trigger release of V1 from the membrane and curtail ATP hydrolysis. To investigate the molecular basis for these processes, we have carried out cryo-electron microscopy three-dimensional reconstruction of deactivated V1 from Manduca sexta. In the resulting model, three peripheral stalks that are parts of the mechanical stator of the V-ATPase are clearly resolved as unsupported filaments in the same conformations as in the holoenzyme. They are likely therefore to have inherent stiffness consistent with a role as flexible rods in buffering elastic power transmission between the domains of the V-ATPase. Inactivated V1 adopted a homogeneous resting state with one open active site adjacent to the stator filament normally linked to the H subunit. Although present at 1:1 stoichiometry with V1, both recombinant subunit C reconstituted with V1 and its endogenous subunit H were poorly resolved in three-dimensional reconstructions, suggesting structural heterogeneity in the region at the base of V1 that could indicate positional variability. If the position of H can vary, existing mechanistic models of deactivation in which it binds to and locks the axle of the V-ATPase rotary motor would need to be re-evaluated

Making Muscle Elastic: The Structural Basis of Myomesin Stretching

The muscle M-band protein myomesin comprises a 36-nm long filament made of repetitive immunoglobulin–helix modules that can stretch to 2.5-fold this length, demonstrating substantial molecular elasticity

Post-construction thermal testing: Some recent measurements

In the UK, it has become apparent in recent years that there is often a discrepancy between the steady-state predicted and the measured in situ thermal performance of the building fabric, with the measured in situ performance being greater than that predicted. This discrepancy or gap in the thermal performance of the building fabric is commonly referred to as the building fabric 'performance gap'. This paper presents the results and key messages obtained from undertaking a whole-building heat loss test (a coheating test) on seven new-build dwellings as part of the Technology Strategy Board's Building Performance Evaluation Programme. While the total number of dwellings involved in the work reported here is small, the results illustrate that a wide range of discrepancies in thermal performance was measured for the tested dwellings. Despite this, the results also indicate that it is possible to construct dwellings where the building fabric performs thermally more or less as predicted, thus effectively bridging the traditional building fabric performance gap that exists in mainstream housing in the UK

Enhanced case management can be delivered for patients with EVD in Africa : experience from a UK military ebola treatment centre in Sierra Leone

TF is funded by the Wellcome Trust (104480/Z/14/Z) and the UK Ministry of Defence.Background:  Limited data exist describing supportive care management, laboratory abnormalities and outcomes in patients with EVD (Ebola virus disease) in West Africa. We report data which constitute the first description of the provision of enhanced EVD case management protocols in a West African setting. Methods:   Demographic, clinical and laboratory data were collected by retrospective review of clinical and laboratory records of patients with confirmed EVD admitted between 5 November 2014 and 30 June 2015. Results:  A total of 44 EVD cases were admitted (median age 37 years (range 17-63), 32/44 healthcare workers), and excluding those evacuated, the case fatality rate was 49% (95% CI 33-65%). No pregnant women were admitted. At admission 9/44 had stage 1 disease (fever and constitutional symptoms only), 12/44 stage 2 disease (presence of diarrhoea and/or vomiting) and 23/44 had stage 3 disease (presence of diarrhoea and/or vomiting with organ failure), with case fatality rates of 11% (95% CI 1-58%), 27% (95% CI 6-61%), and 70% (95% CI 47-87%) respectively (p=0.009). Haemorrhage occurred in 17/41 (41%) patients. The majority (21/40) of patients had hypokalaemia with hyperkalaemia occurring in 12/40 patients. Acute Kidney Injury (AKI) occurred in 20/40 patients, with 14/20 (70%, 95% CI 46-88%) dying, compared to 5/20 (25%, 95% CI 9-49%) dying who did not have AKI (p=0.01). Ebola virus (EBOV) PCR cycle threshold value at baseline was mean 20.3 (SD 4.3) in fatal cases and 24.8 (SD 5.5) in survivors (p=0.007). Mean National Early Warning Score (NEWS) at admission was 5.5 (SD 4.4) in fatal cases and 3.0 (SD 1.9) in survivors (p=0.02). Central venous catheters were placed in 37/41 patients and intravenous fluid administered to 40/41 patients (median duration of 5 days). Faecal management systems were inserted in 21/41 patients, urinary catheters placed in 27/41 and blood component therapy administered to 20/41 patients. Conclusions:  EVD is commonly associated life-threatening electrolyte imbalance and organ dysfunction. We believe that the enhanced levels of protocolized care, scale and range of medical interventions we report, offers a blueprint for the future management of EVD in resource-limited settings.Publisher PDFPeer reviewe