Specific and genotypic identification of Cryptosporidium from a broad range of host species by nonisotopic SSCP analysis of nuclear ribosomal DNA

The accurate identification of Cryptosporidium (Protozoa: Apicomplexa) species and genotypes is central to the understanding of the transmission and to the diagnosis and control of cryptosporidiosis. In this study, we demonstrate the effectiveness of nonisotopic SSCP analysis of a approximately 300 bp region of the small subunit (pSSU) of ribosomal DNA for the specific identification of and delineation among 18 different Cryptosporidium species and genotypes from a wide range of hosts. This mutation scanning approach allowed the rapid and reliable differentiation between species/genotypes differing by as little as 1.3% in the pSSU sequence, with the capacity to detect point mutations. The present findings confirm the usefulness of this tool for the rapid genetic screening of Cryptosporidium samples from any host species, providing a foundation for detailed systematic, epidemiological and ecological studies. Although applied herein to pSSU, this low cost approach should be applicable to a wide range of genetic loci for population genetic investigations of Cryptosporidium.     

Binding of nitrate to a CuII-cyclen complex bearing a ferrocenyl pendant: synthesis, solid-state X-ray structure, and solution-phase electrochemical and spectrophotometric studies

The reaction of Cu(NO3)2.3H2O with the ligand 1-(ferrocenemethyl)-1,4,7,10-tetraazacyclododecane (L) in acetonitrile leads to the formation of a blue complex, [Cu(L)(NO3)][NO3] (C1). The X-ray structure determination shows an unexpected binding of a nitrate anion in that the CuII center is surrounded by four N atoms of the 1,4,7,10-tetraazacyclododecane (cyclen) macrocycle and two O atoms from a chelating nitrate anion, both Cu-O distances being below the sums of the van de Waals radii. Hydrogen-bonding interactions in the crystal lattice and a weak interaction between a second nitrate O and the CuII center in C1 give rise to a highly distorted CuII geometry relative to that found in the known structure of [Cu(cyclen)(NO3)][NO3] (C5). Electrochemical studies in acetonitrile containing 0.1 M [Bu4N][NO3] as the supporting electrolyte showed that oxidation of C1 in this medium exhibits a single reversible one-electron step with a formal potential E degrees f of +85 mV vs Fc0/+ (Fc = ferrocene). This process is associated with oxidation of the ferrocenyl pendant group. Additionally, a reversible one-electron reduction reaction with an E degrees f value of -932 mV vs Fc0/+, attributed to the CuII/I redox couple, is detected. Gradual change of the supporting electrolyte from 0.1 M [Bu4N][NO3] to the poorly coordinating [Bu4N][PF6] electrolyte, at constant ionic strength, led to a positive potential shift in E degrees f values by +107 and +39 mV for the CuII/I(C1) and Fc0/+(C1) redox couples, respectively. Analysis of these electrochemical data and UV-vis spectra is consistent with the probable presence of the complexes C1, [Cu(L)(CH3CN)2]2+ (C2), [Cu(L)(CH3CN)(NO3)]+ (C3), and [Cu(L)(NO3)2] (C4) as the major species in nitrate-containing acetonitrile solutions. In weakly solvating nitromethane, the extent of nitrate complexation remains significant even at low nitrate concentrations, due to the lack of solvent competition.     

Characterization and modelling of a carbon ramming mix used in high-temperature industry

This paper is devoted to the modelling of a specific ramming mix mainly used in the high-temperature industry due to its high-compacting behaviour. This material has the ability to absorb the deformation of parts submitted to high thermal loads. Triaxial and instrumented die compaction tests were carried out in order to identify the shear and hardening behaviours, respectively. Tests on the ramming mix were led for a temperature range between 20 °C and 80 °C. The temperature effect is particularly observed on the material response when it is compacted. The main features of the behaviour of the ramming mix can be represented by the theoretical framework of the Modified Cam-Clay model. A single variable allows to accurately reproduce the hardening behaviour depending on the temperature. Moreover, an extension of the model for the hardening behaviour at high pressures is proposed. A good agreement between the experimental data and numerical tests is reached with this model.     

Genotyping Taenia tapeworms by single-strand conformation polymorphism of mitochondrial DNA.

To overcome limitations in identifying tapeworms of the genus Taenia by traditional approaches, we have established a single-strand conformation polymorphism (SSCP) method utilizing two different regions of mitochondrial (mt) DNA as targets. The NADH dehydrogenase 1 and the cytochrome c oxidase subunit I genes were amplified from genomic DNA by polymerase chain reaction (PCR), denatured and subjected to electrophoresis in mutation detection enhancement gels. SSCP analysis achieved delineation among eight different species of Taenia from different hosts based on characteristic profiles and enabled the detection of intraspecific variability in profiles for some taxa. This SSCP-based typing method has important implications for taxonomy, diagnosis and for studying the genetic structure of Taenia populations.     

One-channel Roy equations revisited

The Roy equation in the single-channel case is a nonlinear, singular integral equation for the phase shift in the low-energy region. We first investigate the infinitesimal neighborhood of a given solution, and then present explicit expressions for amplitudes that satisfy the nonlinear equation exactly. These amplitudes contain free parameters that render the non-uniqueness of the solution manifest. They display, however, an unphysical singularity at the upper end of the interval considered. This singularity disappears and uniqueness is achieved if one uses analyticity properties of the amplitudes that are not encoded in the Roy equation. Received: 25 March 1999 / Published online: 15 July 1999     

Mechanical homogenisation of masonry wall without mortar

Some refractory linings of metallurgical vessels consist of masonry without mortar. To describe the mechanical behaviour of these large-sized structures, it is necessary to use an equivalent material instead of a model that comprises all the bricks and joints involved. The properties of the equivalent material depend on the opening and closure mechanism of joints. In this paper, four joint states which are the combination of open/closed states of bed and head joints are identified, and the corresponding equivalent elastic properties are determined accordingly using homogenisation techniques. The transition criterion between these joint states is based on the unilateral contact conditions written in terms of macroscopic strain. The developed model is then compared to an in-plane biaxial compression test. The numerical and experimental results are in good agreement.     

Modelling Fire Events in Long Tunnels

We consider a fluiddynamic model derived to describe tunnel fires. The derivation of the model and results obtained by the model are discussed.     

Mutation scanning-based analysis of anisakid larvae from Sillago flindersi from Bass Strait, Australia

Anisakidosis is an important fish-borne disease caused by the larvae of anisakid nematodes, which affects humans and a range of other animals. The accurate identification of members of this nematode group is central to investigating the epidemiology of the parasites and in the surveillance and control of anisakidosis. It is now well known that morphological identification alone does not allow specific identification, particularly of larval stages. To better understand the epidemiology of anisakid nematodes in southern Australian fishes and the potential risks posed to human health, a survey of 50 specimens of the commercially important fish, Sillago flindersi, from Bass Strait, Australia was conducted. We characterised anisakid larvae by PCR-coupled mutation scanning, sequencing and phylogenetic analyses of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA. This study revealed that 92% of the S. flindersi examined were infected with anisakids (n=194), which were represented by seven genotypes. Phylogenetic analyses of the genotypes defined herein, together with reference sequence for Anisakis pegreffii and Hysterothylacium sp. from public databases (i.e. GenBank), revealed the presence of A. pegreffii (n=24), Hysterothylacium larval type IV (n=90) and Hysterothylacium larval type VIII (n=80) in S. flindersi. Thus, the PCR-coupled mutation scanning approach employed herein is an effective tool for the genetic characterisation of anisakid nematodes for diagnostic and analytical purposes (nucleotide sequences reported in this paper are available in the GenBank database under accession nos. JN631796-809).     

Electrochemiluminescent monomers for solid support syntheses of Ru(II)-PNA bioconjugates: multimodal biosensing tools with enhanced duplex stability

The feasibility of devising a solid support mediated approach to multimodal Ru(II)-peptide nucleic acid (PNA) oligomers is explored. Three Ru(II)-PNA-like monomers, [Ru(bpy)(2)(Cpp-L-PNA-OH)](2+) (M1), [Ru(phen)(2)(Cpp-L-PNA-OH)](2+) (M2), and [Ru(dppz)(2)(Cpp-L-PNA-OH)](2+) (M3) (bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine, Cpp-L-PNA-OH = [2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-[6-(2-(pyridin-2yl)pyrimidine-4-carboxamido)hexanoyl]-glycine), have been synthesized as building blocks for Ru(II)-PNA oligomers and characterized by IR and (1)H NMR spectroscopy, mass spectrometry, electrochemistry and elemental analysis. As a proof of principle, M1 was incorporated on the solid phase within the PNA sequences H-g-c-a-a-t-a-a-a-a-Lys-NH(2) (PNA1) and H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-lys-NH(2) (PNA4) to give PNA2 (H-g-c-a-a-t-a-a-a-a-M1-lys-NH(2)) and PNA3 (H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-M1-lys-NH(2)), respectively. The two Ru(II)-PNA oligomers, PNA2 and PNA3, displayed a metal to ligand charge transfer (MLCT) transition band centered around 445 nm and an emission maximum at about 680 nm following 450 nm excitation in aqueous solutions (10 mM PBS, pH 7.4). The absorption and emission response of the duplexes formed with the cDNA strand (DNA: 5'-T-T-T-T-T-T-T-A-T-T-G-C-T-T-T-3') showed no major variations, suggesting that the electronic properties of the Ru(II) complexes are largely unaffected by hybridization. The thermal stability of the PNA·DNA duplexes, as evaluated from UV melting experiments, is enhanced compared to the corresponding nonmetalated duplexes. The melting temperature (T(m)) was almost 8 °C higher for PNA2·DNA duplex, and 4 °C for PNA3·DNA duplex, with the stabilization attributed to the electrostatic interaction between the cationic residues (Ru(II) unit and positively charged lysine/arginine) and the polyanionic DNA backbone. In presence of tripropylamine (TPA) as co-reactant, PNA2, PNA3, PNA2·DNA and PNA3·DNA displayed strong electrochemiluminescence (ECL) signals even at submicromolar concentrations. Importantly, the combination of spectrochemical, thermal and ECL properties possessed by the Ru(II)-PNA sequences offer an elegant approach for the design of highly sensitive multimodal biosensing tools.