Template-free 13-protofilament microtubule–MAP assembly visualized at 8 A resolution

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX’s unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin–tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX’s exquisite binding selectivity uncovers important insights into regulation of cellular MTs

Validation of lethality processes for products with slow come up time: Bacon and bone-in ham

Pork bellies and boneless hams were smoked or cooked using unusually long processes to determine the impact of extended come-up times on the populations of Clostridium perfringens, Salmonella enterica, Staphylococcus aureus and Listeria monocytogenes. The products were formulated using brine formulations representative of what might be used in commercial production, and the thermal processes were more than doubled in length. Pork bellies and boneless hams were inoculated on the surface as well as 1 cm below the surface, and samples were collected every 3 h. The populations of C. perfringens (spores and vegetative cells) at internal locations of pork bellies increased by less than 1 log10 and declined significantly (approximately 3 log10/cm2) on the surface of the bellies during an extended bacon process. The populations of S. enterica, L. monocytogenes and S. aureusdid not increase during the extended bacon process. The populations of C. perfringens (spores and vegetative cells), S. aureus, S. enterica and L. monocytogenesdeclined significantly over an extended ham process. There were significant population reductions (\u3e2 log10/cm2) at 7 h (surface) and 12 h (\u3e5 log10/g; internal) for the hams. Populations of both surface and internal locations of the hams declined to a point approaching the limit of detection of the assays within 17 h

Structural basis for the clamping and Ca²⁺ activation of SNARE-mediated fusion by synaptotagmin

Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to calcium (Ca2+) influx. Here we report the cryo-electron microscopy structure of the Syt1–SNARE complex on anionic-lipid containing membranes. Under resting conditions, the Syt1 C2 domains bind the membrane with a magnesium (Mg2+)-mediated partial insertion of the aliphatic loops, alongside weak interactions with the anionic lipid headgroups. The C2B domain concurrently interacts the SNARE bundle via the ‘primary’ interface and is positioned between the SNAREpins and the membrane. In this configuration, Syt1 is projected to sterically delay the complete assembly of the associated SNAREpins and thus, contribute to clamping fusion. This Syt1–SNARE organization is disrupted upon Ca2+-influx as Syt1 reorients into the membrane, likely displacing the attached SNAREpins and reversing the fusion clamp. We thus conclude that the cation (Mg2+/Ca2+) dependent membrane interaction is a key determinant of the dual clamp/activator function of Synaptotagmin-1

CONTAINER MATERIALS, FABRICATION AND ROBUSTNESS

The multi-barrier 3013 container used to package plutonium-bearing materials is robust and thereby highly resistant to identified degradation modes that might cause failure. The only viable degradation mechanisms identified by a panel of technical experts were pressurization within and corrosion of the containers. Evaluations of the container materials and the fabrication processes and resulting residual stresses suggest that the multi-layered containers will mitigate the potential for degradation of the outer container and prevent the release of the container contents to the environment. Additionally, the ongoing surveillance programs and laboratory studies should detect any incipient degradation of containers in the 3013 storage inventory before an outer container is compromised

DEVELOPMENT AND APPLICATION OF MATERIALS PROPERTIES FOR FLAW STABILITY ANALYSIS IN EXTREME ENVIRONMENT SERVICE

Discovery of aging phenomena in the materials of a structure may arise after its design and construction that impact its structural integrity. This condition can be addressed through a demonstration of integrity with the material-specific degraded conditions. Two case studies of development of fracture and crack growth property data, and their application in development of in-service inspection programs for nuclear structures in the defense complex are presented. The first case study covers the development of fracture toughness properties in the form of J-R curves for rolled plate Type 304 stainless steel with Type 308 stainless steel filler in the application to demonstrate the integrity of the reactor tanks of the heavy water production reactors at the Savannah River Site. The fracture properties for the base, weld, and heat-affected zone of the weldments irradiated at low temperatures (110-150 C) up to 6.4 dpa{sub NRT} and 275 appm helium were developed. An expert group provided consensus for application of the irradiated properties for material input to acceptance criteria for ultrasonic examination of the reactor tanks. Dr. Spencer H. Bush played a lead advisory role in this work. The second case study covers the development of fracture toughness for A285 carbon steel in high level radioactive waste tanks. The approach in this case study incorporated a statistical experimental design for material testing to address metallurgical factors important to fracture toughness. Tolerance intervals were constructed to identify the lower bound fracture toughness for material input to flaw disposition through acceptance by analysis

A seesaw model for intermolecular gating in the kinesin motor protein

Recent structural observations of kinesin-1, the founding member of the kinesin group of motor proteins, have led to substantial gains in our understanding of this molecular machine. Kinesin-1, similar to many kinesin family members, assembles to form homodimers that use alternating ATPase cycles of the catalytic motor domains, or “heads”, to proceed unidirectionally along its partner filament (the microtubule) via a hand-over-hand mechanism. Cryo-electron microscopy has now revealed 8-Å resolution, 3D reconstructions of kinesin-1•microtubule complexes for all three of this motor’s principal nucleotide-state intermediates (ADP-bound, no-nucleotide, and ATP analog), the first time filament co-complexes of any cytoskeletal motor have been visualized at this level of detail. These reconstructions comprehensively describe nucleotide-dependent changes in a monomeric head domain at the secondary structure level, and this information has been combined with atomic-resolution crystallography data to synthesize an atomic-level "seesaw" mechanism describing how microtubules activate kinesin’s ATP-sensing machinery. The new structural information revises or replaces key details of earlier models of kinesin’s ATPase cycle that were based principally on crystal structures of free kinesin, and demonstrates that high-resolution characterization of the kinesin–microtubule complex is essential for understanding the structural basis of the cycle. I discuss the broader implications of the seesaw mechanism within the cycle of a fully functional kinesin dimer and show how the seesaw can account for two types of "gating" that keep the ATPase cycles of the two heads out of sync during processive movement

Proton radiotherapy at PTC Czech in Prague

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Prime movers : mechanochemistry of mitotic kinesins

Mitotic spindles are self-organizing protein machines that harness teams of multiple force generators to drive chromosome segregation. Kinesins are key members of these force-generating teams. Different kinesins walk directionally along dynamic microtubules, anchor, crosslink, align and sort microtubules into polarized bundles, and influence microtubule dynamics by interacting with microtubule tips. The mechanochemical mechanisms of these kinesins are specialized to enable each type to make a specific contribution to spindle self-organization and chromosome segregation

Comparing Skill Acquisition Under Varying Onsets of Differential Reinforcement: A Preliminary Analysis

The purpose of the current study was to evaluate the effect of implementing differential reinforcement at different times relative to the onset of teaching new skills to learners with autism spectrum disorder. Specifically, we first determined the most efficient differential reinforcement arrangement for each participant. Using the most efficient arrangement, we evaluated if differential reinforcement from the immediate onset, early onset, or late onset is the most efficient for learners to acquire a new skill. Three children diagnosed with autism spectrum disorder who have a history of receiving intervention based on the principles of applied behavior analysis participated in this study. The immediate onset of differential reinforcement resulted in the most efficient instruction in 6 of 7 comparisons. The results are discussed in light of previous studies and suggestions for future research are provided