Burton RA, Tanner NA, Alhassan A, Guelig D, Poole CB, Carlow CK, Zhang Y, LaBarre P, Wanji S, Li Z, Diesburg S, Evans TC
[
PLoS One,
2017]
Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP) tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA) device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus) or repetitive DNA (40 min, B. malayi and W. bancrofti) was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs.
Marc De Raeymaeker, Nina Cromheecke, Marleen Brunain, Marc Van de Craen, Christ Platteeuw, Joel Vandekerckhove, Walter Luyten, Peter Verhasselt, Luc Maertens, Johan Geysen, Thierry Bogaert
[
International C. elegans Meeting,
1999]
Growth cone steering is a cyclic process which starts with filopodia outgrowth at the leading edge of the cell followed by the reading and integration of multiple directional signals that are translated into local stabilisation of the F-actin cytoskeleton in one of the growth cone spikes. Subsequent interaction of the F-actin cytoskeleton with microtubule plus-ends leads to permanent stabilisation of a part of the growth cone in the new direction. Finally, a new cycle of filopodia outgrowth is initiated in the new direction. UNC-53 (Steerin) is a novel nematode signal transduction molecule that integrates extracellular signals into cellular responses which determine the direction of migration of hypodermal cells, neurons, myoblasts and the excretory cell in C. elegans. Based on homology, Steerin is predicted to contain an N-terminal alpha-actinin-like F-actin binding domain and a C-terminal nucleotide-binding domain. The middle region predominantly consists of alpha helical structures comprising two proline rich helices and three coiled coils. A family of structurally and functionally related human Steerins has been cloned in a collaborative effort. Recombinant Steerin was shown to bind recombinant GST-SEM-5 and GST-GRB-2, suggesting that Steerin converts receptor tyosine signalling into directional signals. Steerin can also physically associate with the cytoskeleton via F-actin and microtubule plus-ends. Biochemical studies illustrated that Steerin can directly interact with F-actin. Steerin was shown to co-localise with microtubules with preference for the microtubule (+)-ends using monoclonal antibody post-fixation immunofluorescence and transient overexpression of GFP-Steerin fusion proteins in living mammalian cells. Overexpression of Steerin and a number of deletion mutants in mammalian cells has a positive effect on neuronal outgrowth, filopodia production, lamellipodia formation and cell motility.