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Identification and characterization of a factor Va-binding site on human prothrombin fragment 2
No full textThe fragment 2 domain (F2) of prothrombin and its interaction with factor (F) Va is known to contribute significantly to prothrombinase-catalyzed activation of prothrombin. The extent to which the F2-FVa interaction affects the overall thrombin generation, however, is uncertain. To study this interaction, nuclear magnetic resonance spectroscopy of recombinant F2 was used to identify seven residues within F2 that are significantly responsive to FVa binding. The functional role of this region in interacting with FVa during prothrombin activation was verified by the FVa-dependent inhibition of thrombin generation using peptides that mimic the same region of F2. Because six of the seven residues were within a 9-residue span, these were mutated to generate a prothrombin derivative (PT6). These mutations led to a decreased affinity for FVa as determined by surface plasmon resonance. When thrombin generation by an array of FXa containing prothrombinase components was monitored, a 54% decrease in thrombin generation was observed with PT6 compared with the wild-type, only when FVa was present. The functional significance of the specific low-affinity binding between F2 and FVa is discussed within the context of a dynamic model of molecular interactions between prothrombin and FVa engaging multiple contact sites.Peer reviewed: YesNRC publication: Ye
Impact of N-glycosylation on Fc\u3b3 receptor / IgG interactions: unravelling differences with an enhanced surface plasmon resonance biosensor assay based on coiled-coil interactions
No full textThe N-glycosylation profile of immunoglobulin G (IgG) is considered a critical quality attribute due to its impact on IgG-Fc gamma receptor (Fc\u3b3R) interactions, which subsequently affect antibody-dependent cell-based immune responses. In this study, we investigated the impact of the Fc\u3b3R capture method, as well as Fc\u3b3R N-glycosylation, on the kinetics of interaction with various glycoforms of trastuzumab (TZM) in a surface plasmon resonance (SPR) biosensor assay. More specifically, we developed a novel strategy based on coiled-coil interactions for the stable and oriented capture of coil-tagged Fc\u3b3Rs at the biosensor surface. Coil-tagged Fc\u3b3R capture outperformed all other capture strategies applied to the SPR study of IgG-Fc\u3b3R interactions, as the robustness and reproducibility of the assay and the shelf life of the biosensor chip were excellent (> 1,000 IgG injections with the same biosensor surface). Coil-tagged Fc\u3b3Rs displaying different N-glycosylation profiles were generated either by different expression systems, in vitro glycoengineering or by size-exclusion chromatography, and roughly characterized by lectin blotting. Of salient interest, the overlay of their kinetics of interaction with several TZM glycoforms revealed key differences on both association and dissociation kinetics, confirming a complex influence of the Fc\u3b3R N-glycosylation and its inherent heterogeneity upon receptor interaction with mAbs. This work is thus an important step towards better understanding of the impact of glycosylation upon binding of IgGs, either natural or engineered, to their receptors.Peer reviewed: YesNRC publication: Ye
Calibration process for rechargeable cell and battery test systems
No full textCoulombic efficiency is a powerful metric for evaluating the performance of materials in rechargeable cells and batteries. The ideal Coulombic efficiency, the ratio of charge removed to charge inserted, is unity. Some specialized systems can accurately measure cell capacity and Coulombic efficiency within 0.001%, which requires precise control and measurement of current, voltage, time, and temperature. Most battery electrode and electrolyte research is not performed with such precise but complex systems. The purpose of this paper is to demonstrate a simple, robust procedure to measure and possibly improve the accuracy of capacity and Coulombic efficiency measurements on standard systems in their as-used state. This approach is built on a commercially available thin film rechargeable cell for micro or milliampere currents and can be extended to, e.g., 18 650, cells for higher currents. An improved method to display Coulombic efficiency data is also presented. Regular, consistent calibration of testing systems and reporting of system resolution at specified test conditions is encouraged.Peer reviewed: YesNRC publication: Ye
Implementation of a UAV\u2013Hyperspectral pushbroom imager for ecological monitoring
No full textHyperspectral remote sensing provides a wealth of data essential for vegetation studies encompassing a wide range of applications (e.g., species diversity, ecosystem monitoring, etc.). The development and implementation of UAV-based hyperspectral systems have gained popularity over the last few years with novel efforts to demonstrate their operability. Here we describe the design, implementation, testing, and early results of the UAV-\u3bcCASI system, which showcases a relatively new hyperspectral sensor suitable for ecological studies. The \u3bcCASI (288 spectral bands) was integrated with a custom IMU-GNSS data recorder built in-house and mounted on a commercially available hexacopter platform with a gimbal to maximize system stability and minimize image distortion. We deployed the UAV-\u3bcCASI at three sites with different ecological characteristics across Canada: The Mer Bleue peatland, an abandoned agricultural field on Ile Grosbois, and the Cowichan Garry Oak Preserve meadow. We examined the attitude data from the flight controller to better understand airframe motion and the effectiveness of the integrated Differential Real Time Kinematic (RTK) GNSS. We describe important aspects of mission planning and show the effectiveness of a bundling adjustment to reduce boresight errors as well as the integration of a digital surface model for image geocorrection to account for parallax effects at the Mer Bleue test site. Finally, we assessed the quality of the radiometrically and atmospherically corrected imagery from the UAV-\u3bcCASI and found a close agreement (<2%) between the image derived reflectance and in-situ measurements. Overall, we found that a flight speed of 2.7 m/s, careful mission planning, and the integration of the bundling adjustment were important system characteristics for optimizing the image quality at an ultra-high spatial resolution (3\u20135 cm). Furthermore, environmental considerations such as wind speed (<5 m/s) and solar illumination also play a critical role in determining image quality. With the growing popularity of \u201cturnkey\u201d UAV-hyperspectral systems on the market, we demonstrate the basic requirements and technical challenges for these systems to be fully operational.Peer reviewed: YesNRC publication: Ye
Adhesion strength of titanium particles to alumina substrates: a combined cold spray and LIPIT study
No full textThe cold spray process and laser-induced projectile impact test (LIPIT) are used to deposit Ti powder particles on sintered polycrystalline Al2O3. Whereas LIPIT allows real-time observations of single particle impact and measurement of particle impact velocity, cold spray rapidly and simultaneously deposits particles with a wide range of deposition velocities and sizes. By use of these two techniques, the effect of particle velocity and substrate morphology on adhesion strength of single splats is investigated. The critical velocity for deposition is identified to be approximately 580\u202fm/s for the Ti/Al2O3 system when using LIPIT and particles of 10\u202f\u3bcm. Above the critical velocity, flattening ratio (FR) is also evaluated and observed to be linearly dependent on the particle impact velocity. Splat adhesion testing is performed on LIPIT-deposited as well as on cold spray-deposited powder particles to measure adhesion strength. This analysis shows that adhesion strength is highly affected by local substrate surface morphology, where particles bond more weakly to relatively smooth portions of the substrate. Therefore, mechanical bonding plays a significant role in adhesion. Also, adhesion strength decreases with an increase in FR and therefore velocity. This decrease can be associated with fracture of the ceramic substrate and rebound forces.Peer reviewed: YesNRC publication: Ye
A robust auxin response network controls embryo and suspensor development through a basic Helix Loop Helix transcriptional module
No full textLand plants reproduce sexually by developing an embryo from a fertilized egg cell. However, embryos can also be formed from other cell types in many plant species. Thus, a key question is how embryo identity in plants is controlled, and how this process is modified during nonzygotic embryogenesis. The Arabidopsis (Arabidopsis thaliana) zygote divides to produce an embryonic lineage and an extra-embryonic suspensor. Yet, normally quiescent suspensor cells can develop a second embryo when the initial embryo is damaged, or when response to the signaling molecule auxin is locally blocked. Here we used auxin-dependent suspensor embryogenesis as a model to determine transcriptome changes during embryonic reprogramming. We found that reprogramming is complex and accompanied by large transcriptomic changes before anatomical changes. This analysis revealed a strong enrichment for genes encoding components of auxin homeostasis and response among misregulated genes. Strikingly, deregulation among multiple auxin-related gene families converged upon the re-establishment of cellular auxin levels or response. This finding points to a remarkable degree of feedback regulation to create resilience in the auxin response during embryo development. Starting from the transcriptome of auxin-deregulated embryos, we identified an auxin-dependent basic Helix Loop Helix transcription factor network that mediates the activity of this hormone in suppressing embryo development from the suspensor.Peer reviewed: YesNRC publication: Ye