Grafted fluoropolymers as supports for solid-phase organic chemistry: Preparation and characterization

Solid-phase organic chemistry has rapidly expanded in the last decade, and, as a consequence, so has the need for the development of supports that can withstand the extreme conditions required to facilitate some reactions. The authors here prepare a thermally stable, grafted fluoropolymer support (see Figure for an example) in three solvents, and found that the penetration of the graft was greatest in dichloromethane.

Specificity of PCR and in situ hybridization assays designed for detection of Marteilia sydneyi and M-refringens

Primers and DNA probes designed for use in the specific detection of the paramyxean parasites Marteilia sydneyi and Marteilia refringens were tested for their potential to cross-react with closely related species in Polymerase Chain Reaction (PCR) and in situ hybridization. PCR primers and a DNA probe designed within the ITS1 rRNA of M. sydneyi were specific for M. sydneyi when compared with related species of Marteilia and Marteilioides. PCR primers designed within the 18S rRNA of M. refringens were specific in the detection of this species in PCR while a DNA probe (named Smart 2) designed on the same gene cross-reacted with M. sydneyi in tissue sections of Saccostrea glomerata as well as Marteilioides sp. infecting Striostrea mytiloides. Though not species specific, the Smart 2 probe provided a stronger signal in detection of all stages of M. sydneyi than the ITS1 probe. The ITS probe is proposed for use as a confirmatory diagnostic too] for M. sydneyi.

Detection of the initial infective stages of the protozoan parasite Marteilia sydneyi in Saccostrea glomerata and their development through to sporogenesis

DNA probes were used in in situ hybridisation on histological sections of oysters exposed for defined intervals to Marteilia sydneyi infection to reveal the early development of the parasite in the oyster host, Saccostrea glomerata. The initial infective stages enter through the palps and gills whereupon extrasporogonic proliferation results in the liberation of cells into surrounding connective tissue and haemolymph spaces. Following systemic dissemination, the parasite infiltrates the digestive gland and becomes established as a nurse cell beneath the epithelial cells ill a digestive tubule. Here, cell-within-cell proliferation results in the eventual liberation of daughter cells from the nurse cell into spaces between adjacent epithelial cells. None of these stages had previously been described. Proliferation is associated with host responses, including haemocytic infiltration of the connective tissue and diapedesis across tubule epithelia. The responses cease as sporogenesis begins. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

Quantification and stability of everolimus (SDZ RAD) in human blood by high-performance liquid chromatography-electrospray tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug, everolimus (SDZ RAD), using HPLC-tandem mass spectrometry. Whole blood samples (500 mul) were prepared by protein precipitation, followed by C-18 solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface operating in positive ionization mode. The assay was linear from 0.5 to 100 mug/l (r(2) > 0.996, n = 9). The analytical recovery and inter-day imprecision, determined using whole blood quality control samples (n = 5) at 0.5, 1.2, 20.0, and 75.0 mug/l, was 100.3-105.4% and less than or equal to7.6%, respectively. The assay had a mean relative recovery of 94.8 +/- 3.8%. Extracted samples were stable for up to 24 h. Fortified everolimus blood samples were stable at -80 degreesC for at least 8 months and everolimus was found to be stable in blood when taken through at least three freeze-thaw cycles. The reported method provides accurate, precise and specific measurement of everolimus in blood over a wide analytical range and is currently supporting phase 11 and III clinical trials. (C) 2002 Elsevier Science B.V. All rights reserved.

Phase variation in meningococcal lipooligosaccharide biosynthesis genes

Neisseria meningitidis expresses a range of lipooligosaccharide (LOS) structures, comprising of at least 13 immunotypes (ITs). Meningococcal LOS is subject to phase variation of its terminal structures allowing switching between ITs, which is proposed to have functional significance in disease. The objectives of this study were to investigate the repertoire of structures that can be expressed in clinical isolates, and to examine the role of phase-variable expression of LOS genes during invasive disease. Southern blotting was used to detect the presence of LOS biosynthetic genes in two collections of meningococci, a global set of strains previously assigned to lineages of greater or lesser virulence, and a collection of local clinical isolates which included paired throat and blood isolates from individual patients. Where the phase-variable genes lgtA, lgtC or IgtG were identified, they were amplified by PCR and the homopolymeric tracts, responsible for their phase-variable expression, were sequenced. The results revealed great potential for variation between alternate LOS structures in the isolates studied, with most strains capable of expressing several alternative terminal structures. The structures predicted to be currently expressed by the genotype of the strains agreed well with conventional immunotyping. No correlation was observed between the structural repertoire and virulence of the isolate. Based on the potential for LOS phase variation in the clinical collection and observations with the paired patient isolates, our data suggest that phase variation of LOS structures is not required for translocation between distinct compartments in the host. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

Phase variable restriction-modification systems in Moraxella catarrhalis

A repetitive DNA motif was used as a marker to identify novel genes in the mucosal pathogen Moraxella catarrhalis. There is a high prevalence of such repetitive motifs in virulence genes that display phase variable expression. Two repeat containing loci were identified using a digoxigenin-labelled 5'-(CAAC)(6)-3' oligonucleotide probe. The repeats are located in the methylase components of two distinct type III restriction-modification (R-M) systems. We suggest that the phase variable nature of these R-M systems indicates that they have an important role in the biology of M. catarrhalis. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

The development of a positive non-infectious control for the detection of Perkinsus using the Ray test

To establish a noncontagious control for the Ray thioglycollate test for the detection of Perkinsus in mollusks we evaluated nonviable stages of P. olseni for enlargement of hypnospores and blue/black iodine stain. Trophozoites made nonviable with formalin, irradiation or colchicine failed to swell in thioglycollate. They remained small and did not differentially stain in iodine. Trophozoites that had already developed into hypnospores in thioglycollate were rendered inactive by freezing, ethanol or formalin immersion. They retained their iodinophilic properties and thus could provide a partial control for the Ray Test.

Cloud-point extraction and preconcentration of cyanobacterial toxins (microcystins) from natural waters using a cationic surfactant

A new cloud-point extraction and preconcentration method, using a cationic, surfactant, Aliquat-336 (tricaprylyl-methy;ammonium chloride), his-been developed for the determination of cyanobacterial toxins, microcystins, in natural waters. Sodium sulfate was used to induce phase separation at 25 degreesC. The phase behavior of Aliquat-336 with respect to concentration of Na2SO4 was studied. The cloud-point system revealed a very high phase volume ratio compared to other established systems of nonionic, anionic, and cationic surfactants: At pH 6-7, it showed an outstanding selectivity in ahalyte extraction for anionic species. Only MC-LR and MC-YR, which are known to be predominantly anionic, were extracted (with averaged recoveries of 113.9 +/- 9% and 87.1 +/- 7%, respectively). MC-RR, which is likely to be amphoteric at the above pH range, was. not cle tectable in.the extract. Coupled to HPLC/UV separation and detection, the cloud-point extraction method (with 2.5 mM Aliquat-336 and 75 mM Na2SO4 at 25 degreesC) offered detection limits of 150 +/- 7 and 470 +/- 72 pg/mL for MC-LR and MC-YR, respectively, in 25 mL of deionized water. Repeatability of the method was 7.6% for MC-LR and 7.3% for MC-YR: The cloud-point extraction process can be. completed within 10-15 min with no cleanup steps required. Applicability of the new method to the determination of microcystins in real samples was demonstrated using natural surface waters, collected from a local river and a local duck pond spiked with realistic. concentrations of microcystins. Effects of salinity and organic matter (TOC) content in the water sample on the extraction efficiency were also studied.

Detection of swallowing sounds: Methodology revisited

Cervical auscultation is in the process of gaining clinical credibility. In order for it to be accepted by the clinical community, the procedure and equipment used must first be standardized. Takahashi et al. [Dysphagia 9:54-62, 1994] attempted to provide benchmark methodology for administering cervical auscultation. They provided information about the acoustic detector unit best suited to picking up swallowing sounds and the best cervical site to place it. The current investigation provides contrasting results to Takahashi et al. with respect to the best type of acoustic detector unit to use for detecting swallowing sounds. Our study advocates an electret microphone as opposed to an accelerometer for recording swallowing sounds. However, we agree on the optimal placement site. We conclude that cervical auscultation is within reach of the average dysphagia clinic.

Phase equilibria and thermodynamics of zinc fuming slags

A range of materials is treated in zinc fuming processes to recover metal values and produce benign slag waste products. The selection of the optimum process conditions in these various technologies can be greatly assisted by the use of a chemical thermodynamic model of the system. In this paper the effects of slag chemistry on the liquidus temperatures, subliquidus phase equilibria and thermodynamic properties are described by the F*A*C*T computer package with the new thermodynamic database of the ZnO-PbO-FeO-Fe2O3-CaO-SiO2 system. The implications of these findings for plant practice are discussed.

Experimental study of phase equilibria in the "FeO"-ZNO-(CaO+SiO2) system with the CaO/SiO2 weight ratio of 0.71 at metallic iron saturation

The pseudoternary section FeO-ZnO-(CaO + SiO2) with a CaO/SiO2 weight ratio of 0.71 in equilibrium with metallic iron has been experimentally investigated in the temperature range from 1000 degreesC to 1300 degreesC (1273 to 1573 K). The liquidus surface in this pseudoternary. section has been determined in the composition range of 0 to 33 wt pct ZnO and 30 to 70 wt pct (CaO + SiO2)The system contains primary-phase fields of wustite (FexZn1-xO1+y), zincite (ZnzFe1-zO), fayalite (FewZn2-wSiO4), melilite (Ca2ZnuFe1-uSi2O7), and pseudowollastonite (CaSiO3). The phase equilibria involving the liquid phase and the solid solutions have also been measured.

Phase equilibrium studies in the "MnO"-Al2O3-SiO2 system

Phase relations and the liquidus surface in the system "MnO"-Al2O3-SiO2 at manganese-rich alloy saturation have been investigated in the temperature range from 1373 to 1773 K. This system contains the primary-phase fields of tridymite and cristobalite (SiO2); mullite (3Al(2)O(3).2SiO(2)); corundum (Al2O3); galaxite (MnO.Al2O3); manganosite (MnO); tephroite (2MnO.SiO2); rhodonite (MnO.SiO2); spessartine (3MnO.Al2O3.SiO2); and the compound MnO.Al2O3.2SiO(2).

Phase equilibrium data and liquidus for the system "MnO"-CaO-(Al2O3+SiO2) at Al2O3/SiO2 of 0.41

Phase-equilibrium data and the liquidus for the system. "MnO"-CaO-(Al2O3-SiO2) at a manganese-rich alloy saturation have been determined in the temperature range from 1423 to 1723 K. The results are presented in the form of a pseudoternary section "MnO"-CaO-(Al2O3 + SiO2) with an Al2O3/SiO2 weight ratio of 0.41. The following primary phases are present in the range of conditions investigated:, 3Al(2)O(3).2SiO(2); SiO2; MnO.Al2O3-2SiO(2); (Mn,Ca)O.SiO2; 2(Mn,Ca)O.SiO2; MnO.Al2O3; (Mn,Ca)O; alpha-2CaO.SiO2; alpha'-2CaO.SiO2; 2CaO.Al2O3.SiO2; CaO.SiO2, and CaO.Al2O3.2SiO(2). The presence of alumina in this system is shown to have a significant effect on the liquidus compared to the system "MnO"-CaO-SiO2, leading to, the stabilization of the anorthite and gehlenite phases.