Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis

Pseudomonas aeruginosa genotyping relies mainly upon DNA fingerprinting methods, which can be subjective, expensive and time-consuming. The detection of at least three different clonal P. aeruginosa strains in patients attending two cystic fibrosis (CF) centres in a single Australian city prompted the design of a non-gel-based PCR method to enable clinical microbiology laboratories to readily identify these clonal strains. We designed a detection method utilizing heat-denatured P. aeruginosa isolates and a ten-single-nucleotide polymorphism (SNP) profile. Strain differences were detected by SYBR Green-based real-time PCR and high-resolution melting curve analysis (HRM10SNP assay). Overall, 106 P. aeruginosa sputum isolates collected from 74 patients with CF, as well as five reference strains, were analysed with the HRM10SNP assay, and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE). The HRM10SNP assay accurately identified all 45 isolates as members of one of the three major clonal strains characterized by PFGE in two Brisbane CF centres (Australian epidemic strain-1, Australian epidemic strain-2 and P42) from 61 other P. aeruginosa strains from Australian CF patients and two representative overseas epidemic strain isolates. The HRM10SNP method is simple, is relatively inexpensive and can be completed in <3 h. In our setting, it could be made easily available for clinical microbiology laboratories to screen for local P. aeruginosa strains and to guide infection control policies. Further studies are needed to determine whether the HRM10SNP assay can also be modified to detect additional clonal strains that are prevalent in other CF centres.

Genetically monomorphic brown trout (Salmo trutta L.) populations as revealed by mitochondrial DNA, multi-locus and single-locus minisatellite (VNTR) analyses.

Normally, populations of brown trout are genetically highly variable. Two adjacent populations from NW Scotland, which had previously been found to be monomorphic for 46 protein-coding loci, were studied by higher resolution techniques. Analyses of mitochondrial DNA, multilocus DNA fingerprints and eight specific minisatellite loci revealed no genetic variation among individuals or genetic differences between the two populations. Continual low effective population sizes or severe repeated bottlenecks, as a result of low or variable recruitment, probably explain the atypical absence of genetic variation in these trout populations. Growth data do not provide any evidence of a reduction in fitness in trout from these populations.

Differences in expression of junctional adhesion molecule-A and beta catenin in multiple sclerosis brain tissue: increasing evidence for the role of tight junction pathology

Previously we have employed antibodies to the tight junction (TJ)-associated proteins ZO-1 and occludin to describe endothelial tight junction abnormalities, in lesional and normal appearing white matter, in primary and secondary progressive multiple sclerosis (MS). This work is extended here by use of antibodies to the independent TJ-specific proteins and junctional adhesion molecule A & B (JAM-A, JAM-B). We have also assessed the expression in MS of ß-catenin, a protein specific to the TJ-associated adherens junction. Immunocytochemistry and semiquantitative confocal microscopy for JAM-A and ß-catenin was performed on snap-frozen sections from MS cases (n = 11) and controls (n = 6). Data on 1,443 blood vessels was acquired from active lesions (n = 13), inactive lesions (n = 13), NAWM (n = 20) and control white matter (n = 13). In MS abnormal JAM-A expression was found in active (46%) and inactive lesions (21%), comparable to previous data using ZO-1. However, a lower level of TJ abnormality was found in MS NAWM using JAM-A (3%) compared to ZO-1 (13%). JAM-B was strongly expressed on a small number of large blood vessels in control and MS tissues but at too low a level for quantitative analysis. By comparison with the high levels of abnormality observed with the TJ proteins, the adherens junction protein ß-catenin was normally expressed in all MS and control tissue categories. These results confirm, by use of the independent marker JAM-A, that TJ abnormalities are most frequent in active white matter lesions. Altered expression of JAM-A, in addition to affecting junctional tightness may also both reflect and affect leukocyte trafficking, with implications for immune status within the diseased CNS. Conversely, the adherens junction component of the TJ, as indicated by ß-catenin expression is normally expressed in all MS and control tissue categories.

Triclabendazole-resistant Fasciola hepatica: β-tubulin and response to in vitro treatment with triclabendazole

Resistance in Fasciola hepatica to triclabendazole (Fasinex) has emerged in several countries. Benzimidazole resistance in parasitic nematodes has been linked to a single amino acid substitution (phenylalanine to tyrosine) at position 200 on the [beta]-tubulin molecule. Sequencing of [beta]-tubulin cDNAs from triclabendazole-susceptible and triclabendazole-resistant flukes revealed no amino acid differences between their respective primary amino acid sequences. In order to investigate the mechanism of triclabendazole resistance, triclabendazole-susceptible and triclabendazole-resistant flukes were incubated in vitro with triclabendazole sulphoxide (50 [mu]g/ml). Scanning and transmission electron microscopy revealed extensive damage to the tegument of triclabendazole-susceptible F. hepatica, whereas triclabendazole-resistant flukes showed only localized and relatively minor disruption of the tegument covering the spines. Immunocytochemical studies, using an anti-tubulin antibody, showed that tubulin organization was disrupted in the tegument of triclabendazole-susceptible flukes. No such disruption was evident in triclabendazole-resistant F. hepatica. The significance of these findings is discussed with regard to the mechanism of triclabendazole resistance in F. hepatica.

Measurement and interpretation of the mid-infrared properties of single crystal and polycrystalline gold

The contribution of electron-phonon scattering and grain boundary scattering to the mid-IR (lambda = 3.392 mum) properties of An has been assessed by examining both bulk, single crystal samples-Au(1 1 1) and Au(1 1 0)-and thin film, polycrystalline An samples at 300 K and 100 K by means of surface plasmon polariton excitation. The investigation constitutes a stringent test for the in-vacuo Otto-configuration prism coupler used to perform the measurements, illustrating its strengths and limitations. Analysis of the optical response is guided by a physically based interpretation of the Drude model. Relative to the reference case of single crystal Au at 100 K (epsilon = - 568 + i17.5), raising the temperature to 300 K causes increased electron-phonon scattering that accounts for a reduction of similar to40 nm in the electron mean free path. Comparison of a polycrystalline sample to the reference case determines a mean free path due to grain boundary scattering of similar to 17 nm, corresponding to about half the mean grain size as determined from atomic force microscopy and indicating a high reflectance coefficient for the An grain boundaries. An analysis combining consideration of grain boundary scattering and the inclusion of a small percentage of voids in the polycrystalline film by means of an effective medium model indicates a value for the grain boundary reflection coefficient in the range 0.55-0.71. (C) 2005 Elsevier B.V. All rights reserved.

Scaling of domain periodicity with thickness measured in BaTiO3 single crystal lamellae and comparison with other ferroics

The focused ion beam microscope has been used to cut parallel-sided {100}-oriented thin lamellae of single crystal barium titanate with controlled thicknesses, ranging from 530 nm to 70 nm. Scanning transmission electron microscopy has been used to examine domain configurations. In all cases, stripe domains were observed with {011}-type domain walls in perovskite unit-cell axes, suggesting 90 degrees domains with polarization in the plane of the lamellae. The domain widths were found to vary as the square root of the lamellar thickness, consistent with Kittel's law, and its later development by Mitsui and Furuichi and by Roytburd. An investigation into the manner in which domain period adapts to thickness gradient was undertaken on both wedge-shaped lamellae and lamellae with discrete terraces. It was found that when the thickness gradient was perpendicular to the domain walls, a continuous change in domain periodicity occurred, but if the thickness gradient was parallel to the domain walls, periodicity changes were accommodated through discrete domain bifurcation. Data were then compared with other work in literature, on both ferroelectric and ferromagnetic systems, from which conclusions on the widespread applicability of Kittel's law in ferroics were made.

The use of Raman microscopy to determine and localize vitamin E in biological samples

Alpha-tocopherol (aT), the predominant form of vitamin E in mammals, is thought to prevent oxidation of polyunsaturated fatty acids. In the lung, aT is perceived to be accumulated in alveolar type II cells and secreted together with surfactant into the epithelial lining fluid. Conventionally, determination of aT and related compounds requires extraction with organic solvents. This study describes a new method to determine and image the distribution of aT and related compounds within cells and tissue sections using the light-scattering technique of Raman microscopy to enable high spatial as well as spectral resolution. This study compared the nondestructive analysis by Raman microscopy of vitamin E, in particular aT, in biological samples with data obtained using conventional HPLC analysis. Raman spectra were acquired at spatial resolutions of 2-0.8 microm. Multivariate analysis techniques were used for analyses and construction of corresponding maps showing the distribution of aT, alpha-tocopherol quinone (aTQ), and other constituents (hemes, proteins, DNA, and surfactant lipids). A combination of images enabled identification of colocalized constituents (heme/aTQ and aT/surfactant lipids). Our data demonstrate the ability of Raman microscopy to discriminate between different tocopherols and oxidation products in biological specimens without sample destruction. By enabling the visualization of lipid-protein interactions, Raman microscopy offers a novel method of investigating biological characterization of lipid-soluble compounds, including those that may be embedded in biological membranes such as aT.

Safe fabrication of sharp gold tips for light emission in scanning tunnelling microscopy

Gold is the optimal tip metal for light emission in scanning tunnelling microscopy (LESTM) under ambient conditions. Sharp Au-tips of similar to 10nm radius were produced reliably using a safe, two-step etching method in 20% (w/w) CaCl2 solution. Previous CaCl2-based methods have tended to produce blunter tips, while other etching techniques that do produce sharp Au-tips, do so with the use of toxic or hazardous electrolytes. The tips are characterised using scanning electron microscopy and their efficacy in LESTM is evidenced by high-resolution, simultaneous topographic and photon mapping of Au(1 1 1)- and polycrystalline Au-surfaces. Spectra of the optical emission exhibit only one or two peaks with etched tips in contrast to the more complex spectra typical of cut tips; this feature, together with the highly symmetric geometry of the tips, facilitates a definitive analysis of the light emission process. (c) 2007 Elsevier B. V.. All rights reserved.

Scanning electron microscopy study of microstructural evolution of electroless nickel-phosphorus deposits with heat treatment

This paper follows previous X-ray diffraction work on crystallisation and phase transformation of electroless nickel–phosphorus deposits, concentrating on microstructural changes. Amorphous or nanocrystalline coatings, depending on their phosphorus content, were heat treated at temperatures between 100 and 500 °C for 1 h. Changes in microstructure after the heat treatment were examined using high-resolution field emission scanning electron microscope. Crystallisation and grain growth effects are observed, as well as some inherent defect structures in the coatings and their changes. These are compared with the previous X-ray diffraction work and in general, good agreement is observed. The complementary strength and weakness of the different characterisation techniques are discussed.

The persistence of the HgCl2 molecule in five new compounds in the system (NH4)(w)HgxCly(H2O)(z) with w : x : y : z=1 : 5 : 11 : 0, 2 : 3 : 8 : 1, 4 : 3 : 10 : 2, 2 : 1 : 4 : 1, and 10 : 3 : 16 : 0

Five new compounds in the system (NH4)Cl/HgCl2/H2O have been obtained as colourless single crystals, (NH4)Hg5Cl11, (NH4)(2)Hg3Cl8(H2O), (NH4)(4)Hg3Cl10(H2O)(2), (NH4)(2)HgCl4(H2O), and (NH4)(10)Hg3Cl16. In all of these, as in HgCl2 itself, (almost) linear HgCl2 molecules persist with Hg-Cl distances varying from 229 to 236 pm. In (NH4)(10)Hg3Cl16 there are also tetrahedra [HgCl4] with d(Hg-Cl) = 247 pm present. If larger Hg-Cl distances (of up to 340 pm) are considered as belonging to the coordination sphere of Hg-II, the structures may be described as consisting of isolated octahedra and tetrahedra as in (NH4)(10)Hg3Cl16, edge-connected chains as in (NH4)(2)HgCl4(H2O), edge-connected chains and layers of octahedra as in (NH4)(4)Hg3Cl10(H2O)(2), corrugated layers of edge-connected octahedra as in (NH4)(2)Hg3Cl8(H2O), and, finally, a three-dimensional network of connected six- and seven-coordinate Hg-Cl polyhedra as in (NH4)Hg5Cl11. The water molecules are never attached to Hg-II. The (NH4)(+) cations, and sometimes Cl- anions, play a role for electroneutrality only.

Resolution of a taxonomic conundrum: an ultrastructural and molecular description of the life cycle of Pleistophora mulleri (Pfeiffer 1895; Georgevitch 1929)

The classification of a microsporidian parasite observed in the abdominal muscles of amphipod hosts has been repeatedly revised but still remains inconclusive. This parasite has variable spore numbers within a sporophorous vesicle and has been assigned to the genera Glugea, Pleistophora, Stempellia, and Thelohania. We used electron microscopy and molecular evidence to resolve the previous taxonomic confusion and confirm its identification as Pleistophora mulleri. The life cycle of P. mulleri is described from the freshwater amphipod host Gammarus duebeni celticus. Infection appeared as white tubular masses within the abdominal muscle of the host. Light and transmission electron microscope examination revealed the presence of an active microsporidian infection that was diffuse within the muscle block with no evidence of xenoma formation. Paucinucleate merogonial plasmodia were surrounded by an amorphous coat immediately external to the plasmalemma. The amorphous coat developed into a merontogenetic sporophorous vesicle that was present throughout sporulation. Sporogony was polysporous resulting in uninucleate spores, with a bipartite polaroplast, an anisofilar polar filament and a large posterior vacuole. SSU rDNA analysis supported the ultrastructural evidence clearly placing this parasite within the genus Pleistophora. This paper indicates that Pleistophora species are not restricted to vertebrate hosts.

Domain Bundle Boundaries in Single Crystal BaTiO3 Lamellae: Searching for Naturally Forming Dipole Flux-Closure / Quadrupole Chains

Naturally occurring boundaries between bundles of 90o stripe domains, which form in BaTiO3 lamellae on cooling through the Curie Temperature, have been characterised using both piezoresponse force microscopy (PFM) and scanning transmission electron microscopy (STEM). Detailed interpretation of the dipole configurations present at these boundaries (using data taken from PFM) shows that, in the vast majority of cases, they are composed of simple zigzag 180° domain walls. Topological information from STEM shows that, occasionally, domain bundle boundaries can support chains of dipole flux closure and quadrupole nanostructures, but these kinds of boundaries are comparatively rare; when such chains do exist, it is notable that singularities at the cores of the dipole structures are avoided. The symmetry of the boundary shows that diads and centres of inversion exist at positions where core singularities should have been expected.

Active Phase Conjugating Lens With Sub-Wavelength Resolution Capability

Experimental results are presented for the focusing capability of an active phase conjugating lens for a single and a dipole source pair and these are compared with predictions. In addition for a single source we illustrate the ability of the lens to project a null at the lens focus instead of a peak. A scheme is also presented such that when a source or pair of sources is imaged through an identical pair of passive scatterers located symmetrically about the lens that imaging with sub-wavelength resolution is possible. The rationale for the operation of the lens and aberrations observed due to its finite array size is discussed and is supported throughout by means of numerical simulation.

Aligning conservation priorities across taxa in Madagascar with high-resolution planning tools

Globally, priority areas for biodiversity are relatively well known, yet few detailed plans exist to direct conservation action within them, despite urgent need. Madagascar, like other globally recognized biodiversity hot spots, has complex spatial patterns of endemism that differ among taxonomic groups, creating challenges for the selection of within-country priorities. We show, in an analysis of wide taxonomic and geographic breadth and high spatial resolution, that multitaxonomic rather than single-taxon approaches are critical for identifying areas likely to promote the persistence of most species. Our conservation prioritization, facilitated by newly available techniques, identifies optimal expansion sites for the Madagascar government's current goal of tripling the land area under protection. Our findings further suggest that high-resolution multitaxonomic approaches to prioritization may be necessary to ensure protection for biodiversity in other global hot spots.