The \u3cem\u3edapE\u3c/em\u3e-encoded \u3cem\u3eN\u3c/em\u3e-Succinyl-l,l-Diaminopimelic Acid Desuccinylase from \u3cem\u3eHaemophilus influenzae\u3c/em\u3e Is a Dinuclear Metallohydrolase

The Zn K-edge extended X-ray absorption fine structure (EXAFS) spectra, of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae have been recorded in the presence of one or two equivalents of Zn(II) (i.e. [Zn_(DapE)] and [ZnZn(DapE)]). The Fourier transforms of the Zn EXAFS are dominated by a peak at ca. 2.0 Å, which can be fit for both [Zn_(DapE)] and [ZnZn(DapE)], assuming ca. 5 (N,O) scatterers at 1.96 and 1.98 Å, respectively. A second-shell feature at ca. 3.34 Å appears in the [ZnZn(DapE)] EXAFS spectrum but is significantly diminished in [Zn_(DapE)]. These data show that DapE contains a dinuclear Zn(II) active site. Since no X-ray crystallographic data are available for any DapE enzyme, these data provide the first glimpse at the active site of DapE enzymes. In addition, the EXAFS data for DapE incubated with two competitive inhibitors, 2-carboxyethylphosphonic acid and 5-mercaptopentanoic acid, are also presented

Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM-1

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data

Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

Background: State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings: The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance: The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution fo

Plant-mediated effects on mosquito capacity to transmit human malaria

The ecological context in which mosquitoes and malaria parasites interact has received little attention, compared to the genetic and molecular aspects of malaria transmission. Plant nectar and fruits are important for the nutritional ecology of malaria vectors, but how the natural diversity of plant-derived sugar sources affects mosquito competence for malaria parasites is unclear. To test this, we infected Anopheles coluzzi, an important African malaria vector, with sympatric field isolates of Plasmodium falciparum, using direct membrane feeding assays. Through a series of experiments, we then examined the effects of sugar meals from Thevetia neriifolia and Barleria lupilina cuttings that included flowers, and fruit from Lannea microcarpa and Mangifera indica on parasite and mosquito traits that are key for determining the intensity of malaria transmission. We found that the source of plant sugar meal differentially affected infection prevalence and intensity, the development duration of the parasites, as well as the survival and fecundity of the vector. These effects are likely the result of complex interactions between toxic secondary metabolites and the nutritional quality of the plant sugar source, as well as of host resource availability and parasite growth. Using an epidemiological model, we show that plant sugar source can be a significant driver of malaria transmission dynamics, with some plant species exhibiting either transmission-reducing or -enhancing activities

Photophysical properties of C76

International audienc

Lasalocid (X537A) as a selective carrier for Cd(II) in supported liquid membranes

International audienceThe carboxylic antibiotic lasalocid was shown to facilitate the transport of Cd(II) cations through supported liquid membranes, the driving force being the proton gradient between the two aqueous phases. With hydrocarbons as solvents inside the porous support, the initial metal fluxes were linear versus the inverses of the viscosity values, in accordance with a diffusion limited mechanism. With the polar solvent nitrophenyl octyl ether, the initial flux was six times higher than expected from the viscosity value, and up to 95% of the metal ions were transported. In competitive experiments with equal initial Cd(II) and Zn(II) concentrations, after 94% of Cd(II) had been transported the ratio of Cd(II) to Zn(II) in the receiving phase was 3.8 (pH 8.2 in the source phase, pH 2 in the receiving phase, membrane impregnated with lasalocid 5×10−3 M in nitrophenyl octyl ether)

Low-energy proton calibration and energy-dependence linearization of EBT-XD radiochromic films

In this work, we calibrate the newly developed EBT-XD radiochromic films (RCFs) manufactured by Gafchromictm using protons in the energy range of 4–10 MeV. Irradiation was performed on the 2 × 6 MV tandem linear accelerator located at the Université de Montréal. The RCFs were digitized using an Epson Perfection V700 flatbed scanner using both the red-green-blue and grayscale channels. The proton fluences were measured with Faraday cups calibrated in absolute terms. The linear energy transfer function within the active layer of the films was calculated using the mass stopping power tables coming from the PSTAR database from the National Institute of Standards and Technology (NIST) to allow retrieval of the deposited dose. We find that the calibration curves for 7 and 10 MeV protons are nearly equivalent. The 4 MeV calibration curves exhibit a quenching effect due to the Bragg peak that falls close to the active layer. A linearization of this energy dependence was developed using a semiempirical parametric model to allow the generation of calibration curves for any incident proton energy within the present range. Excellent correspondence (<5% dose difference for the same netOD) of the 10 MeV calibration curves was noted when compared to existing high-energy proton (148.2 MeV) calibration curves reported in the literature. Our calibration extends the range of operation of EBT-XD films to low-energy proton beam dosimetry