Evidence for a Dinuclear Active Site in the Metallo-β-lactamase BcII with Substoichiometric Co(II): A New Model for Uptake

Metallo-β-lactamases are zinc-dependent enzymes that constitute one of the main resistance mechanisms to β-lactam antibiotics. Metallo-β-lactamases have been characterized both in mono- and dimetallic forms. Despite many studies, the role of each metal binding site in substrate binding and catalysis is still unclear. This is mostly due to the difficulties in assessing the metal content and site occupancy in solution. For this reason, Co(II) has been utilized as a useful probe of the active site structure. We have employed UV-visible, EPR, and NMR spectroscopy to study Co(II) binding to the metallo-β-lactamase BcII from Bacillus cereus. The spectroscopic features were attributed to the two canonical metal binding sites, the 3H (His116, His118, and His196) and DCH (Asp120, Cys221, and His263) sites. These data clearly reveal the coexistence of mononuclear and dinuclear Co(II)-loaded forms at Co(II)/enzyme ratios as low as 0.6. This picture is consistent with the macroscopic dissociation constants here determined from competition binding experiments. A spectral feature previously assigned to the DCH site in the dinuclear species corresponds to a third, weakly bound Co(II) site. The present work emphasizes the importance of using different spectroscopic techniques to follow the metal content and localization during metallo-β-lactamase turnover

Asp-120 Locates Zn2 for Optimal Metallo-β-lactamase Activity

Metallo-β-lactamases are zinc-dependent hydrolases that inactivate β-lactam antibiotics, rendering bacteria resistant to them. Asp-120 is fully conserved in all metallo-β-lactamases and is central to catalysis. Several roles have been proposed for Asp-120, but so far there is no agreed consensus. We generated four site-specifically substituted variants of the enzyme BcII from Bacillus cereus as follows: D120N, D120E, D120Q, and D120S. Replacement of Asp-120 by other residues with very different metal ligating capabilities severely impairs the lactamase activity without abolishing metal binding to the mutated site. A kinetic study of these mutants indicates that Asp-120 is not the proton donor, nor does it play an essential role in nucleophilic activation. Spectroscopic and crystallographic analysis of D120S BcII, the least active mutant bearing the weakest metal ligand in the series, reveals that this enzyme is able to accommodate a dinuclear center and that perturbations in the active site are limited to the Zn2 site. It is proposed that the role of Asp-120 is to act as a strong Zn2 ligand, locating this ion optimally for substrate binding, stabilization of the development of a partial negative charge in the β-lactam nitrogen, and protonation of this atom by a zinc-bound water molecule

Policy responses to invasive native species: issues of social and private benefits and costs

Farm and catchment managers in Australia face decisions about controlling invasive native species (or scrub) which may infest agricultural land. The treatment of this land to remove the infestation and re-establish native pastures is likely to be expensive for landholders. Yet there are potential social benefits from such remediation and so a policy question arises of what to do to about facilitating such change. New South Wales state government legislation addresses this issue through regulations, and the Catchment Management Authorities are responsible for administering public funds to achieve associated natural resource improvements. However, the extent of the private costs and social benefits associated with such changes are not known, which precludes benefit-cost analyses using the traditional welfare economics framework. This paper reports results of a social and private economic analysis of the impacts of a typical infestation remediation decision. We show that for the landholder the private costs exceed the benefits achieved from increased livestock productivity. However, there are social benefits expressed by the willingness to pay by members of the local catchment community for improvements in native vegetation and biodiversity. When these social benefits are included, the economic analysis shows a positive social net benefit. This raises questions of how to reconcile the public and private accounting, and whether any changes to policies, regulations or procedures for natural resource management in New South Wales are warranted.Invasive native scrub, environmental values, choice modelling, financial, economic, Namoi catchment,

Monolayer MoS2 strained to 1.3% with a microelectromechanical system

We report on a modified transfer technique for atomically thin materials integrated onto microelectromechanical systems (MEMS) for studying strain physics and creating strain-based devices. Our method tolerates the non-planar structures and fragility of MEMS, while still providing precise positioning and crack free transfer of flakes. Further, our method used the transfer polymer to anchor the 2D crystal to the MEMS, which reduces the fabrication time, increases the yield, and allowed us to exploit the strong mechanical coupling between 2D crystal and polymer to strain the atomically thin system. We successfully strained single atomic layers of molybdenum disulfide (MoS2) with MEMS devices for the first time and achieved greater than 1.3% strain, marking a major milestone for incorporating 2D materials with MEMS We used the established strain response of MoS2 Raman and Photoluminescence spectra to deduce the strain in our crystals and provide a consistency check. We found good comparison between our experiment and literature.Published versio

Inversion Leads to Quantitative, Not Qualitative, Changes in Face Processing

AbstractHumans are remarkably adept at recognizing objects across a wide range of views. A notable exception to this general rule is that turning a face upside down makes it particularly difficult to recognize [1–3]. This striking effect has prompted speculation that inversion qualitatively changes the way faces are processed. Researchers commonly assume that configural cues strongly influence the recognition of upright, but not inverted, faces [3–5]. Indeed, the assumption is so well accepted that the inversion effect itself has been taken as a hallmark of qualitative processing differences [6]. Here, we took a novel approach to understand the inversion effect. We used response classification [7–10] to obtain a direct view of the perceptual strategies underlying face discrimination and to determine whether orientation effects can be explained by differential contributions of nonlinear processes. Inversion significantly impaired performance in our face discrimination task. However, surprisingly, observers utilized similar, local regions of faces for discrimination in both upright and inverted face conditions, and the relative contributions of nonlinear mechanisms to performance were similar across orientations. Our results suggest that upright and inverted face processing differ quantitatively, not qualitatively; information is extracted more efficiently from upright faces, perhaps as a by-product of orientation-dependent expertise

Band Gap Engineering with Ultralarge Biaxial Strains in Suspended Monolayer MoS2

We demonstrate the continuous and reversible tuning of the optical band gap of suspended monolayer MoS2 membranes by as much as 500 meV by applying very large biaxial strains. By using chemical vapor deposition (CVD) to grow crystals that are highly impermeable to gas, we are able to apply a pressure difference across suspended membranes to induce biaxial strains. We observe the effect of strain on the energy and intensity of the peaks in the photoluminescence (PL) spectrum, and find a linear tuning rate of the optical band gap of 99 meV/%. This method is then used to study the PL spectra of bilayer and trilayer devices under strain, and to find the shift rates and Gr\"uneisen parameters of two Raman modes in monolayer MoS2. Finally, we use this result to show that we can apply biaxial strains as large as 5.6% across micron sized areas, and report evidence for the strain tuning of higher level optical transitions.Comment: Nano Lett., Article ASA