Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate.

The specific action of serine proteinases on protein substrates is a hallmark of blood coagulation and numerous other physiological processes. Enzymic recognition of substrate sequences preceding the scissile bond is considered to contribute dominantly to specificity and function. We have investigated the contribution of active site docking by unique substrate residues preceding the scissile bond to the function of prothrombinase. Mutagenesis of the authentic P(1)-P(3) sequence in prethrombin 2/fragment 1.2 yielded substrate variants that could be converted to thrombin by prothrombinase. Proteolytic activation was also observed with a substrate variant containing the P(1)-P(3) sequence found in a coagulation zymogen not known to be activated by prothrombinase. Lower rates of activation of the variants derived from a decrease in maximum catalytic rate but not in substrate affinity. Replacement of the P(1) residue with Gln yielded an uncleavable derivative that retained the affinity of the wild type substrate for prothrombinase but did not engage the active site of the enzyme. Thus, active site docking of the substrate contributes to catalytic efficiency, but it is does not determine substrate affinity nor does it fully explain the specificity of prothrombinase. Therefore, extended interactions between prothrombinase and substrate regions removed from the cleavage site drive substrate affinity and enforce the substrate specificity of this enzyme complex

Improving eye care for veterans with diabetes: An example of using the QUERI steps to move from evidence to implementation: QUERI Series

<p>Abstract</p> <p>Background</p> <p>Despite being a critical part of improving healthcare quality, little is known about how best to move important research findings into clinical practice. To address this issue, the Department of Veterans Affairs (VA) developed the Quality Enhancement Research Initiative (QUERI), which provides a framework, a supportive structure, and resources to promote the more rapid implementation of evidence into practice.</p> <p>Methods</p> <p>This paper uses a practical example to demonstrate the use of the six-step QUERI process, which was developed as part of QUERI and provides a systematic approach for moving along the research to practice pipeline. Specifically, we describe a series of projects using the six-step framework to illustrate how this process guided work by the Diabetes Mellitus QUERI (DM-QUERI) Center to assess and improve eye care for veterans with diabetes.</p> <p>Results</p> <p>Within a relatively short time, DM-QUERI identified a high-priority issue, developed evidence to support a change in the diabetes eye screening performance measure, and identified a gap in quality of care. A prototype scheduling system to address gaps in screening and follow-up also was tested as part of an implementation project. We did not succeed in developing a fully functional pro-active scheduling system. This work did, however, provide important information to help us further understand patients' risk status, gaps in follow-up at participating eye clinics, specific considerations for additional implementation work in the area of proactive scheduling, and contributed to a change in the prevailing diabetes eye care performance measure.</p> <p>Conclusion</p> <p>Work by DM-QUERI to promote changes in the delivery of eye care services for veterans with diabetes demonstrates the value of the QUERI process in facilitating the more rapid implementation of evidence into practice. However, our experience with using the QUERI process also highlights certain challenges, including those related to the hybrid nature of the research-operations partnership as a mechanism for promoting rapid, system-wide implementation of important research findings. In addition, this paper suggests a number of important considerations for future implementation work, both in the area of pro-active scheduling interventions, as well as for implementation science in general.</p

Kinetic mechanism of controlled Fab-arm exchange for the formation of bispecific immunoglobulin G1 antibodies

Bispecific antibodies (bsAbs) combine the antigen specificities of two distinct Abs and demonstrate therapeutic promise based on novel mechanisms of action. Among the many platforms for creating bsAbs, controlled Fab-arm exchange (cFAE) has proven useful based on minimal changes to native Ab structure and the simplicity with which bsAbs can be formed from two parental Abs. Despite a published protocol for cFAE and its widespread use in the pharmaceutical industry, the reaction mechanism has not been determined. Knowledge of the mechanism could lead to improved yields of bsAb at faster rates as well as foster adoption of process control. In this work, a combination of Forster resonance energy transfer (FRET), nonreducing SDS-PAGE, and strategic mutation of the Ab hinge region was employed to identify and characterize the individual steps of cFAE. Fluorescence correlation spectroscopy (FCS) was used to determine the affinity of parental (homodimer) and bispecific (heterodimer) interactions within the C(H)3 domain, further clarifying the thermodynamic basis for bsAb formation. The result is a clear sequence of events with rate constants that vary with experimental conditions, where dissociation of the K409R parental Ab into half-Ab controls the rate of the reactio

Microsimulation for Local Impact Analysis: an Application to Plant Shutdown

Microsimulation was introduced nearly 50 years ago but has recently experienced a revival in the social sciences. Its use in regional science, however, has been limited, although it offers some advantages over common regional analytic methods. This paper describes a microsimulation model that can be used to analyze the impact of a regional economic event. The model incorporates spatial, social, and economic factors and allows outcomes to be aggregated at different geographical scales, for different cohorts, and for variables not ordinarily considered in impact analysis. The model is used to simulate the effects of a plant shutdown on workers. Copyright Blackwell Publishers, 2005

High speed analog-to-digital conversion with silicon photonics

Sampling rates of high-performance electronic analog-to-digital converters (ADC) are fundamentally limited by the timing jitter of the electronic clock. This limit is overcome in photonic ADC's by taking advantage of the ultra-low timing jitter of femtosecond lasers. We have developed designs and strategies for a photonic ADC that is capable of 40 GSa/s at a resolution of 8 bits. This system requires a femtosecond laser with a repetition rate of 2 GHz and timing jitter less than 20 fs. In addition to a femtosecond laser this system calls for the integration of a number of photonic components including: a broadband modulator, optical filter banks, and photodetectors. Using silicon-on-insulator (SOI) as the platform we have fabricated these individual components. The silicon optical modulator is based on a Mach-Zehnder interferometer architecture and achieves a V[subscript pi]L of 2 Vcm. The filter banks comprise 40 second-order microring-resonator filters with a channel spacing of 80 GHz. For the photodetectors we are exploring ion-bombarded silicon waveguide detectors and germanium films epitaxially grown on silicon utilizing a process that minimizes the defect density.Defense Advanced Research Projects Agency (contracts W911NF-04-1-0431 and HR0011-05-C-0155)Department of the Air Force (Air Force Contract FA8721-05-C-0002