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Abstract: Prostatic zinc content is a known biomarker for discriminating normal healthy tissue from benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Given that zinc content is not readily measured without a tissue biopsy, we have been exploring noninvasive imaging methods to detect these diagnostic differences using a zinc-responsive MRI contrast agent. During imaging studies in mice, we observed that a bolus of glucose stimulates secretion of zinc from the prostate of fasted mice. This discovery allowed the use of a Gd-based zinc sensor to detect differential zinc secretion in regions of healthy versus malignant prostate tissue in a transgenic adenocarcinoma mouse model of PCa. Here, we used a zinc-responsive MRI agent to detect zinc release across the prostate during development of malignancy and confirm the loss of total tissue zinc by synchrotron radiation X-ray fluorescence (μSR-XRF). Quantitative μSR-XRF results show that the lateral lobe of the mouse prostate uniquely accumulates high concentrations of zinc, 1.06 ± 0.08 mM, and that the known loss of zinc content in the prostate is only observed in the lateral lobe during development of PCa. Additionally, we confirm that lesions identified by a loss of zinc secretion indeed represent malignant neoplasia and that the relative zinc concentration in the lesion is reduced to 0.370 ± 0.001 mM. The μSR-XRF data also provided insights into the mechanism of zinc secretion by showing that glucose promotes movement of zinc pools (∼1 mM) from the glandular lumen of the lateral lobe of the mouse prostate into the stromal/smooth muscle surrounding the glands. Co-localization of zinc and gadolinium in the stromal/smooth muscle areas as detected by μSR-XRF confirm that glucose initiates secretion of zinc from intracellular compartments into the extracellular spaces of the gland where it binds to the Gd-based agent and albumin promoting MR image enhancement.

Open Access

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Abstract: The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.

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Abstract: Even with the emergence of antibiotic resistance, penicillin and the wider family of beta-lactams have remained the single most important family of antibiotics. The periasmic/extracytoplasmic targets of penicillin are a family of enzymes with a highly conserved catalytic activity involved in the final stage of bacterial cell wall (peptidoglycan, PG) biosynthesis. Named after their ability to bind penicillin, rather than their catalytic activity these key targets are called penicillin-binding proteins (PBPs).Resistance is predominantly mediated by reducing the target drug concentration via beta-lactamases, however, naturally transformable bacteria have also acquired target mediated resistance by inter-species recombination. Here we focus on structural based interpretations of amino acid alterations associated with the emergence of resistance within clinical isolates and include new PBP3 structures along with new, and improved, PBP-beta-lactam co-structures.

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Abstract: Non-graphitic carbons (NGCs) represent the most abundant class of \(sp^2\)-hybridized carbon materials (coal, char coal, activated carbon, etc.). These carbons consist of small graphene layer stacks possessing significant structural disorder both in the single graphene sheets and the stacking. In this study an advanced evaluation approach for the wide-angle neutron scattering (WANS) was developed, based on the method introduced by Ruland and Smarsly (2002). In particular, we elucidated if and how the enhanced WANS data quality and larger values of the modulus of the scattering vector \(s\)-range affect the accuracy and the values of the size and disorder parameters - being fitting parameters by themselves - in comparison to wide-angle x-ray scattering (WAXS), which is usually performed by laboratory equipment. We find a reasonable agreement for the parameters \(L_\mathrm{a}\) and \(L_\mathrm{c}\), i.e. the lateral dimension and stack height, within the error bars, while for the disorder parameters different results for WAXS and WANS were found, the origin of which is discussed. Thus, this study addresses the general issue of how reliably microstructural parameters can be determined from WAXS/WANS, by fitting simulated WAXS and WANS curves, which are quality-impaired by added Gaussion noise at different levels and cut-off at different \(s\)-values. From this analysis we estimated the minimal data quality required for a reliable NGC microstructural analysis based on WAXS/WANS. As an important finding, these simulations show that typical, standard WAXS laboratory setups are sufficient to provide reliable values for the most relevant structural parameters. Furthermore, pair-distribution function (PDF) analyses were performed on WAXS data obtained from a Synchrotron facility. Comparing PDF and WAXS/WANS fitting analysis thus suggests the presence of small highly ordered oligoaromatic domains embedded in the larger graphene sheets, questioning the classical view on the NGC microstructure.

Open Access

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Abstract: Traditionally small-molecule crystallographers have not usually observed or recognized significant radiation damage to their samples during diffraction experiments. However, the increased flux densities provided by third-generation synchrotrons have resulted in increasing numbers of observations of this phenomenon. The diversity of types of small-molecule systems means it is not yet possible to propose a general mechanism for their radiation-induced sample decay, however characterization of the effects will permit attempts to understand and mitigate it. Here, systematic experiments are reported on the effects that sample temperature and beam attenuation have on radiation damage progression, allowing qualitative and quantitative assessment of their impact on crystals of a small-molecule test sample. To allow inter-comparison of different measurements, radiation-damage metrics (diffraction-intensity decline, resolution fall-off, scaling B-factor increase) are plotted against the absorbed dose. For ease-of-dose calculations, the software developed for protein crystallography, RADDOSE-3D, has been modified for use in small-molecule crystallography. It is intended that these initial experiments will assist in establishing protocols for small-molecule crystallographers to optimize the diffraction signal from their samples prior to the onset of the deleterious effects of radiation damage.