Kinesin Moving through the Spotlight: Single-Motor Fluorescence Microscopy with Submillisecond Time Resolution

AbstractKinesin-1 is one of the motor proteins that drive intracellular transport in eukaryotes. This motor makes hundreds of 8-nm steps along a microtubule before releasing. Kinesin-1 can move at velocities of up to ∼800nm/s, which means that one turnover on average takes 10ms. Important details, however, concerning the coordination between the two motor domains have not been determined due to limitations of the techniques used. In this study, we present an approach that allows the observation of fluorescence intensity changes on individual kinesins with a time resolution far better than the duration of a single step. In our approach, the laser focus of a confocal fluorescence microscope is pointed at a microtubule and the photons emitted by fluorescently labeled kinesin motors walking through the spot are detected with submicrosecond accuracy. We show that the autocorrelation of a fluorescence time trace of an individual kinesin motor contains information at time lags down to 0.1ms. The quality and time resolution of the autocorrelation is primarily determined by the amount of signal photons used. By adding the autocorrelations of several tens of kinesins, fluorescence intensity changes can be observed at a timescale below 100μs

Creep-rupture behavior of candidate Stirling engine iron supperalloys in high-pressure hydrogen. Volume 2: Hydrogen creep-rupture behavior

The creep rupture behavior of nine iron base and one cobalt base candidate Stirling engine alloys is evaluated. Rupture life, minimum creep rate, and time to 1% strain data are analyzed. The 3500 h rupture life stress and stress to obtain 1% strain in 3500 h are also estimated

Grenville Foreland Deformation and Sedimentation in Southwest Ohio Indicated by Reprocessed Seismic Reflection Profiles near Middletown, Ohio, USA

The late Mesoproterozoic to early Neoproterozoic Middle Run Formation contains vital information about the crustal evolution of the North American Craton. Four reprocessed seismic reflection lines in the vicinity of the AK Steel facility in Middletown, Ohio, provide new insights into the structural and depositional setting of the Middle Run Formation in this region. A residual statics solution improved the resolution and coherency of reflections in these profiles that underlie the Cambrian Mount Simon Sandstone. Reprocessing revealed gently inclined, west-southwest-dipping reflectors and the occurrence of an angular unconformity between the Middle Run Formation and the overlying Paleozoic strata. The weak and discontinuous seismic reflection character of the Middle Run Formation in these seismic lines overlies a sequence of stronger parallel reflections that are like those observed on the eastward ODNR-1-88 seismic line located near core hole DGS 2627, the stratotype of the Middle Run Formation. This inferred thickness indicates that the basin in which the Middle Run Formation was deposited ranges from at least 670 to 1,128 m (2,200 to 3,700 ft) deep at the AK Steel area and dips gently west-southwest, which is in contrast with the moderate easterly dip observed on the ODNR-1-88 seismic line to the northeast. Correlation of these features across the 10 km (approximately 6 mi) cross-strike gap between the AK Steel lines and the ODNR-1-88 seismic line suggests the presence of a reverse fault with approximately 792 m (2,600 ft) of estimated vertical displacement. A regional cross section—including the WSU 1990 seismic line eastward of the ODNR-1-88 line—exhibits a faulted west-verging asymmetric syncline in near proximity to the Grenville Front. This cross section also shows that deformation of the Middle Run Formation and the underlying layered sequence exhibits a consistent tectonic style of reverse faulting and folding that developed in response to Grenville Front tectonism

Characterizaiton of Cold-formed Steel Shear Wall Behavior under Cyclic Loading for the CFS-NEES Building

The objective of this paper is to provide a full hysteretic characterization of OSB sheathed cold-formed steel (CFS) shear walls designed for use in the National Science Foundation funded Network for Earthquake Engineering Simulation (NEES) project: CFS-NEES (www.ce.jhu.edu/cfsnees). The shear walls were designed for a two-story ledger-framed building (i.e., the CFS-NEES building) that will undergo full-scale shake table testing at the University of Buffalo NEES site. Shear walls in real construction, such as the CFS-NEES building, have details that differ from the shear walls tested and provided for strength prediction in standards such as AISI-S213-07. Differences include: (a) ledger (rim track) members are attached across the interior face of the studs, (b) OSB panel seams, both horizontal and vertical, may not be aligned with the chord studs or only blocked with strap, (c) interior gypsum board is in place, (d) field studs may have a different thickness or grade from the chord studs, and other differences. In this work, these four highlighted differences (a-d) are specifically explored in a series of shear walls tests loaded via cyclic (CUREE) protocols to determine their hysteretic performance. The test results are compared with AISIS213-07 and hysteretic material characterizations utilizing an elastic-plastic model (EEEP) and a model capable of exhibiting pinching in the hysteretic loops (Pinching4). Recommendations are made with respect to modeling the shear walls

Optical properties of TiCx (0.64≤x≤0.90) from 0.1 to 30 eV

The stoichiometry-dependent optical properties of bulk samples of TiCx have been determined for four samples in the range 0.64≤x≤0.90. Reflectance and absorptance data taken in the range 0.1-30 eV have been Kramers-Kronig analyzed to obtain the dielectric function and related functions. Interband absorption begins at 0.1 eV or less. The observed interband transitions are interpreted on the basis of existing energy-band calculations. Comparison of optical structure with joint-density-of-states calculations shows that the rigid-band model cannot be applied strictly to explain the x-dependent structure, especially in the 5-10 eV region. The electron-energy-loss functions exhibit two peaks, one near the free-electron plasmon energy and one near 10 eV, both peaks shifting to higher energy as x increases

Calibrating bead displacements in optical tweezers using acousto-optic deflectors

Displacements of optically trapped particles are often recorded using back-focal-plane interferometry. In order to calibrate the detector signals to displacements of the trapped object, several approaches are available. One often relies either on scanning a fixed bead across the waist of the laser beam or on analyzing the power spectrum of movements of the trapped bead. Here, we introduce an alternative method to perform this calibration. The method consists of very rapidly scanning the laser beam across the solvent-immersed, trapped bead using acousto-optic deflectors while recording the detector signals. It does not require any knowledge of solvent viscosity and bead diameter, and works in all types of samples, viscous or viscoelastic. Moreover, it is performed with the same bead as that used in the actual experiment. This represents marked advantages over established methods. © 2006 American Institute of Physics

Quantum Electrodynamics of the Helium Atom

Using singlet S states of the helium atom as an example, I describe precise calculation of energy levels in few-electron atoms. In particular, a complete set of effective operators is derived which generates O(m*alpha^6) relativistic and radiative corrections to the Schr"odinger energy. Average values of these operators can be calculated using a variational Schr"odinger wave function.Comment: 23 pages, revte

Rapid interrogation of the physical and chemical characteristics of salbutamol sulphate aerosol from a pressurised metered-dose inhaler (pMDI)

Individual micron-sized solid particles from a Salamols pharmaceutical inhaler are stably captured in air using an optical trap for the first time. Raman spectroscopy of the levitated particles allows online interrogation of composition and deliquescent phase change within a high humidity environment that mimics the particle’s travel from inhaler to lun

Microtubule cross-linking triggers the directional motility of kinesin-5

Although assembly of the mitotic spindle is known to be a precisely controlled process, regulation of the key motor proteins involved remains poorly understood. In eukaryotes, homotetrameric kinesin-5 motors are required for bipolar spindle formation. Eg5, the vertebrate kinesin-5, has two modes of motion: an adenosine triphosphate (ATP)–dependent directional mode and a diffusive mode that does not require ATP hydrolysis. We use single-molecule experiments to examine how the switching between these modes is controlled. We find that Eg5 diffuses along individual microtubules without detectable directional bias at close to physiological ionic strength. Eg5's motility becomes directional when bound between two microtubules. Such activation through binding cargo, which, for Eg5, is a second microtubule, is analogous to known mechanisms for other kinesins. In the spindle, this might allow Eg5 to diffuse on single microtubules without hydrolyzing ATP until the motor is activated by binding to another microtubule. This mechanism would increase energy and filament cross-linking efficiency

Evaluation of Rapid Syphilis Testing Using the Syphilis Health Check in Florida, 2015–2016

The Syphilis Health Check (SHC) had low estimated specificity (91.5%) in one Florida county. We investigated use of SHC by a range of Florida publicly-funded programs between 2015 and 2016 to estimate specificity, positive predictive value (PPV), field staff acceptance, and impacts on programmatic outcomes. All reported SHC results were extracted from routinely collected program data. Field staff were surveyed about SHC’s utility. Analyses investigated differences between SHC and traditional syphilis testing outcomes. Of 3,630 SHC results reported, 442 were reactive; 92 (20.8%) had prior diagnoses of syphilis; 7 (1.6%) had no further testing. Of the remaining 343; 158 (46.0%) were confirmed cases, 168 (49.0%) were considered false-positive, and 17 (5.0%) were not cases but not clearly false-positive. Estimated specificity of SHC was 95.0%. Overall, 48.5% of positives became confirmed cases (PPV). PPV varied according to prevalence of syphilis in populations tested. Staff (90%) thought SHC helped identify new cases but expressed concern regarding discordance between reactive SHC and lab-based testing. Programmatic outcomes assessment showed shorter time to treatment and increased numbers of partners tested for the SHC group; these enhanced outcomes may better mitigate the spread of syphilis compared to traditional syphilis testing alone, but more research is needed