Browsing by Author "Egan, Timothy"
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- ItemMetadata onlyIn Situ Growth Measurements of Sodium Sulfate during Cooling Crystallization(Wiley-VCH Verlag, 2014) Egan, Timothy; Rodriguez Pascual, Marcos; Lewis, Alison EmslieCombined rainbow Schlieren deflectometry (RSD) and liquid crystal thermography (LCT) served as in situ non-intrusive techniques to determine local concentration and temperature fields based on refractive index gradients in solution during crystallization of sodium sulfate from aqueous solution. The combination of RSD with LCT allowed the decoupling of concentration and temperature effects. Convective mass transport happened during periods of fast, dendritic growth, but even higher levels occurred during the densification of the dendritic crystals, indicating significant mass deposition in this phase. The convection developed mainly as a result of concentration gradients as opposed to temperature gradients. A supersaturation field map was generated and growth kinetics of the sodium sulfate salt was determined. The kinetics followed a power law relationship, with constants in line with those in the literature.
- ItemOpen AccessNeutral lipids associated with haemozoin mediate efficient and rapid beta-haematin formation at physiological pH, temperature and ionic composition(BioMed Central Ltd, 2012) Ambele, Melvin; Egan, TimothyBACKGROUND: The malaria parasite disposes of host-derived ferrihaem (iron(III)protoporphyrin IX, Fe(III)PPIX) by conversion to crystalline haemozoin in close association with neutral lipids. Lipids mediate synthetic haemozoin (beta-haematin) formation very efficiently. However, the effect on reaction rates of concentrations of lipid, Fe(III)PPIX and physiologically relevant ions and biomolecules are unknown. METHODS: Lipid emulsions containing Fe(III)PPIX were prepared in aqueous medium (pH 4.8, 37degreesC) to mediate beta-haematin formation. The reaction was quenched at various times and free Fe(III)PPIX measured colorimetrically as a pyridine complex and the kinetics and yields analysed. Products were also characterized by FTIR, TEM and electron diffraction. Autofluorescence was also used to monitor beta-haematin formation by confocal microscopy. RESULTS: At fixed Fe(III)PPIX concentration, beta-haematin yields remained constant with decreasing lipid concentration until a cut-off ratio was reached whereupon efficiency decreased dramatically. For the haemozoin-associated neutral lipid blend (NLB) and monopalmitoylglycerol (MPG), this occurred below a lipid/Fe(III)PPIX (L/H) ratio of 0.54. Rate constants were found to increase with L/H ratio above the cut-off. At 16 muM MPG, Fe(III)PPIX concentration could be raised until the L/H ratio reached the same ratio before a sudden decline in yield was observed. MPG-mediated beta-haematin formation was relatively insensitive to biologically relevant cations (Na+, K+, Mg2+, Ca2+), or anions (H2PO4, HCO3, ATP, 2,3-diphosphoglycerate, glutathione). Confocal microscopy demonstrated beta-haematin formation occurs in association with the lipid particles. CONCLUSIONS: Kinetics of beta-haematin formation have shown that haemozoin-associated neutral lipids alone are capable of mediating beta-haematin formation at adequate rates under physiologically realistic conditions of ion concentrations to account for haemozoin formation.
- ItemOpen AccessOptimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials(BioMed Central Ltd, 2015) Combrinck, Jill; Fong, Kim; Gibhard, Liezl; Smith, Peter; Wright, David; Egan, TimothyBACKGROUND: The activity of several well-known anti-malarials, including chloroquine (CQ), is attributed to their ability to inhibit the formation of haemozoin (Hz) in the malaria parasite. The formation of inert Hz, or malaria pigment, from toxic haem acquired from the host red blood cell of the parasite during haemoglobin digestion represents a pathway essential for parasite survival. Inhibition of this critical pathway therefore remains a desirable target for novel anti-malarials. A recent publication described the results of a haem fractionation assay used to directly determine haemoglobin, free haem and Hz in Plasmodium falciparum inoculated with CQ. CQ was shown to cause a dose-dependent increase in cellular-free haem that was correlated with decreased parasite survival. The method provided valuable information but was limited due to its low throughput and high demand on parasite starting material. Here, this haem fractionation assay has been successfully adapted to a higher throughput method in 24-well plates, significantly reducing lead times and starting material volumes. METHODS: All major haem species in P. falciparum trophozoites, isolated through a series of cellular fractionation steps were determined spectrophotometrically in aqueous pyridine (5%v/v, pH7.5) as a low spin complex with haematin. Cell counts were determined using a haemocytometer and a rapid novel fluorescent flow cytometry method. RESULTS: A higher throughput haem fractionation assay in 24-well plates, containing at most ten million trophozoites was validated against the original published method using CQ and its robustness was confirmed. It provided a minimum six-fold improvement in productivity and 24-fold reduction in starting material volume. The assay was successfully applied to amodiaquine (AQ), which was shown to inhibit Hz formation, while the antifolate pyrimethamine (PYR) and the mitochondrial electron transporter inhibitor atovaquone (Atov) demonstrated no increase in toxic cellular free haem. CONCLUSIONS: This higher throughput cellular haem fractionation assay can easily be applied to novel anti-malarials with a significantly decreased lead time, providing a valuable tool with which to probe the mechanisms of action of both new and established anti-malarials.