Probing the Reaction Mechanism of the D-ala-D-ala Dipeptidase, VanX, by Using Stopped-Flow Kinetic and Rapid-Freeze Quench EPR Studies on the Co(II)-Substituted Enzyme

In an effort to probe the reaction mechanism of VanX, the D-ala-D-ala dipeptidase required for high-level vancomycin resistance in bacteria, stopped-flow kinetic and rapid-freeze quench EPR studies were conducted on the Co(II)-substituted enzyme when reacted with d-ala-d-ala. The intensity of the Co(II) ligand field band at 550 nm decreased (ε550 = 140 to 18 M-1 cm-1) when VanX was reacted with substrate, suggesting that the coordination number of the metal increases from 5 to 6 upon substrate binding. The stopped-flow trace was fitted to a kinetic mechanism that suggests the presence of an intermediate whose breakdown is rate-limiting. Rapid-freeze quench EPR studies verified the presence of a reaction intermediate that exhibits an unusually low hyperfine constant (33 G), which suggests a bidentate coordination of the intermediate to the metal center. The EPR studies also identified a distinct enzyme product complex. The results were used to offer a detailed reaction mechanism for VanX that can be used to guide future inhibitor design efforts

A Five-coordinate Metal Center in Co(II)-substituted VanX

In an effort to structurally probe the metal binding site in VanX, electronic absorption, EPR, and extended x-ray absorption fine structure (EXAFS) spectroscopic studies were conducted on Co(II)-substituted VanX. Electronic spectroscopy revealed the presence of Co(II) ligand field transitions that had molar absorptivities of ∼100 m–1 cm–1, which suggests that Co(II) is five-coordinate in Co(II)-substituted VanX. Low temperature EPR spectra of Co(II)-substituted VanX were simulated using spin Hamiltonian parameters of M = |±½〉, E/D = 0.14, greal(x,y) = 2.37, and grealS(z) = 2.03. These parameters lead to the prediction that Co(II) in the enzyme is five-coordinate and that there may be at least one solvent-derived ligand. Single scattering fits of EXAFS data indicate that the metal ions in both native Zn(II)-containing and Co(II)-substituted VanX have the same coordination number and that the metal ions are coordinated by 5 nitrogen/oxygen ligands at ∼ 2.0 Å. These data demonstrate that Co(II) (and Zn(II) from EXAFS studies) is five-coordinate in VanX in contrast to previous crystallographic studies (Bussiere, D. E., Pratt, S. D., Katz, L., Severin, J. M., Holzman, T., and Park, C. H. (1998) Mol. Cell 2, 75–84). These spectroscopic studies also demonstrate that the metal ion in Co(II)-substituted VanX when complexed with a phosphinate analog of substrate d-Ala-d-Ala is also five-coordinate

Evaluation of variables for the communication of uncertainties using peripheral awareness displays

The communication of system uncertainties may be key for overcoming challenges related to overtrust in automated driving. Existing approaches are limited to conveying uncertainties using visual displays in the instrument cluster. This requires operators to regularly monitor the display in order to perceive changes which impedes the execution of nondriving related tasks and thereby degrades the user experience. This study evaluates variables for the communication of uncertainties using peripheral awareness displays, considering changes in brightness, hue, position, size, pulse frequency, and movement speed. All variables were assessed in terms of how well participants can distinguish different instances, how logical they are, and how interrupting to a secondary task. With the exception of changes in position, all variables were ranked highly in terms of logic while changes in pulse frequency were perceived as most interrupting. The results inform the development of unobtrusive interfaces for uncertainty communication

SoK: Safer Digital-Safety Research Involving At-Risk Users

Research involving at-risk users -- that is, users who are more likely to experience a digital attack or to be disproportionately affected when harm from such an attack occurs -- can pose significant safety challenges to both users and researchers. Nevertheless, pursuing research in computer security and privacy is crucial to understanding how to meet the digital-safety needs of at-risk users and to design safer technology for all. To standardize and bolster safer research involving such users, we offer an analysis of 196 academic works to elicit 14 research risks and 36 safety practices used by a growing community of researchers. We pair this inconsistent set of reported safety practices with oral histories from 12 domain experts to contribute scaffolded and consolidated pragmatic guidance that researchers can use to plan, execute, and share safer digital-safety research involving at-risk users. We conclude by suggesting areas for future research regarding the reporting, study, and funding of at-risk user researchComment: 13 pages, 3 table

Where to place emergency ambulance vehicles: use of a capacitated maximum covering location model with real call data

This study integrates geographical information systems (GIS) with a mathematical optimization technique to enhance emergency medical services (EMS) coverage in a county in the northeast of Iran. EMS demand locations were determined through one-year EMS call data analysis. We formulated a maximal covering location problem (MCLP) as a mixed-integer linear programming model with a capacity threshold for vehicles using the CPLEX optimizer, an optimization software package from IBM. To ensure applicability to the EMS setting, we incorporated a constraint that maintains an acceptable level of service for all EMS calls. Specifically, we implemented two scenarios: a relocation model for existing ambulances and an allocation model for new ambulances, both using a list of candidate locations. The relocation model increased the proportion of calls within the 5-minute coverage standard from 69% to 75%. With the allocation model, we found that the coverage proportion could rise to 84% of total calls by adding ten vehicles and eight new stations. The incorporation of GIS techniques into optimization modelling holds promise for the efficient management of scarce healthcare resources, particularly in situations where time is of the essence

Archaeobotany in Australia and New Guinea: practice, potential and prospects

Archaeobotany is the study of plant remains from archaeological contexts. Despite Australasian research being at the forefront of several methodological innovations over the last three decades, archaebotany is now a relatively peripheral concern to most archaeological projects in Australia and New Guinea. In this paper, many practicing archaeobotanists working in these regions argue for a more central role for archaeobotany in standard archaeological practice. An overview of archaeobotanical techniques and applications is presented, the potential for archaeobotany to address key historical research questions is indicated, and initiatives designed to promote archaeobotany and improve current practices are outlined

Archaeobotany in Australia and New Guinea: practice, potential and prospects

Archaeobotany is the study of plant remains from archaeological contexts. Despite Australasian research being at the forefront of several methodological innovations over the last three decades, archaebotany is now a relatively peripheral concern to most archaeological projects in Australia and New Guinea. In this paper, many practicing archaeobotanists working in these regions argue for a more central role for archaeobotany in standard archaeological practice. An overview of archaeobotanical techniques and applications is presented, the potential for archaeobotany to address key historical research questions is indicated, and initiatives designed to promote archaeobotany and improve current practices are outlined