Biosensor based on enzyme-catalysed degradation of thin polymer films

A biosensor based on the enzyme-catalysed dissolution of biodegradable polymer films has been developed. Three polymer-enzyme systems were investigated for use in the sensor: a poly(ester amide), which is degraded by the proteolytic enzyme alpha-chymotrypsin; a dextran hydrogel, which is degraded by dextranase; and poly(trimethylene) succinate, which is degraded by a lipase. Dissolution of the polymer films was monitored by Surface Plasmon Resonance (SPR). The rate of degradation was directly related to enzyme concentration for each polymer/enzyme couple. The poly(ester amide)/alpha-chymotrypsin couple proved to be the most sensitive over a concentration range from 4 x 10(-11) to 4 x 10(-7) mol l(-1) of enzyme. The rate of degradation was shown to be independent of the thickness of the poly(ester amide) films. The dextran hydrogel/dextranase couple was less sensitive than the poly(ester amide)/alpha-chymotrypsin couple but showed greater degradation rates at low enzyme concentrations. Enzyme concentrations as low as 2 x 10(-11) mol l(-1) were detected in less than 20 min. Potential fields of application of such a sensor system are the detection of enzyme concentrations and the construction of disposable enzyme based immunosensors, which employ the polymer-degrading enzyme as an enzyme label.     

Prezygotic reproductive isolation between Saccharomyces cerevisiae and Saccharomyces paradoxus

Background  

Matings between different Saccharomyces sensu stricto yeast species produce sexually sterile hybrids, so individuals should avoid mating with other species. Any mechanism that reduces the frequency of interspecific matings will confer a selective advantage. Here we test the ability of two closely-related Saccharomyces sensu stricto species to select their own species as mates and avoid hybridisation.     

GSK-3 phosphorylation of the Alzheimer epitope within collapsin response mediator proteins regulates axon elongation in primary neurons

Elevated glycogen synthase kinase-3 (GSK-3) activity is associated with Alzheimer disease. We have found that collapsin response mediator proteins (CRMP) 2 and 4 are physiological substrates of GSK-3. The amino acids targeted by GSK-3 comprise a hyperphosphorylated epitope first identified in plaques isolated from Alzheimer brain. Expression of wild type CRMP2 in primary hippocampal neurons or SH-SY5Y neuroblastoma cells promotes axon elongation. However, a GSK-3-insensitive CRMP2 mutant has dramatically reduced ability to promote axon elongation, a similar effect to pharmacological inhibition of GSK-3. Hence, we propose that phosphorylation of CRMP proteins by GSK-3 regulates axon elongation. This work provides a direct connection between hyperphosphorylation of these residues and elevated GSK-3 activity, both of which are observed in Alzheimer brain.     

Human behaviour and the epidemiology of parasitic zoonoses

The behaviour of Homo sapiens has a pivotal role to play in the macro and microepidemiology of emerging or re-emerging parasitic zoonoses. Changing demographics and the concomitant alterations to the environment, climate, technology, land use and changes in human behavior, converge to favour the emergence and spread of parasitic zoonoses. The recent unprecedented movements of people, their animals and their parasites around the world, introduce and mix genes, cultural preferences, customs, and behavioral patterns. The increasing proclivity for eating meat, fish, crabs, shrimp, molluscs raw, undercooked, smoked, pickled or dried facilitates a number of protozoan (Toxoplasma), trematode (Fasciola sp., Paragonimus spp., Clonorchis sp., Opisthorchis spp., Heterophyes sp., Metagonimus sp., Echinostoma spp., Nanophyetus sp.) cestode (Taenia spp, Diphyllobothrum sp.) and nematode (Trichinella spp., Capillaria spp., Gnathostoma spp., Anisakis sp., Parastrongylus spp.) caused zoonoses. The increasing world population and the inability to keep pace with the provision of adequate sanitation and clean, safe drinking water, has led to an increased importance of waterborne zoonoses, such as those caused by Giardia, Cryptosporidium and Toxoplasma. Our close relationship with and the numerous uses to which we put companion animals and their ubiquitous distribution has resulted in dogs and cats unwitting participation in sharing over 60 parasite species including: Giardia, Cryptosporidium, Toxoplasma, most foodborne trematode species, Diphyllobothrum, Echinococcus spp., Ancylostoma and Toxocara. Changing human behaviour through education, to encourage the proper cooking of food, which may have cultural and social significance, will remain as challenging as controlling stray and feral pet populations, improving hygiene levels and the provision of safe drinking water and the proper use of sanctuary facilities. Long pre-patent periods and the normally insidious sub-clinical nature of most zoonoses makes advice requiring behavioural change for their control a difficult task. Our clearer understanding of the heterogeneity of susceptibility to infection, the complex genetic variations of people and parasite species and the development of molecular epidemiological tools is shedding more light on transmission routes and the spectrum of disease that is observed. Improved and new serological, molecular and imaging diagnostic tests and the development of broad spectrum chemotherapeutic agents has led to the attenuation of morbidity and mortality due to parasitic zoonoses in economically advantaged regions. Such advancements, in partnership with supportive behavioural change, has the potential for a sustainable global reduction in the burden of ill health due to parasitic zoonoses. Whether this will materialise is a challenge for us all.     

The genetic architecture of resistance to virus infection in Drosophila

Variation in susceptibility to infection has a substantial genetic component in natural populations, and it has been argued that selection by pathogens may result in it having a simpler genetic architecture than many other quantitative traits. This is important as models of host–pathogen co‐evolution typically assume resistance is controlled by a small number of genes. Using the Drosophila melanogaster multiparent advanced intercross, we investigated the genetic architecture of resistance to two naturally occurring viruses, the sigma virus and DCV (Drosophila C virus). We found extensive genetic variation in resistance to both viruses. For DCV resistance, this variation is largely caused by two major‐effect loci. Sigma virus resistance involves more genes – we mapped five loci, and together these explained less than half the genetic variance. Nonetheless, several of these had a large effect on resistance. Models of co‐evolution typically assume strong epistatic interactions between polymorphisms controlling resistance, but we were only able to detect one locus that altered the effect of the main effect loci we had mapped. Most of the loci we mapped were probably at an intermediate frequency in natural populations. Overall, our results are consistent with major‐effect genes commonly affecting susceptibility to infectious diseases, with DCV resistance being a near‐Mendelian trait.     

Mechanisms of thaxtomin A-induced root toxicity revealed by a thaxtomin A sensitive <Emphasis Type="Italic">Arabidopsis</Emphasis> mutant (<Emphasis Type="Italic">ucu2</Emphasis>-<Emphasis Type="Italic">2/gi</Emphasis>-<Emphasis Type="Italic">2</Emphasis>)

Key message

The Arabidopsis mutant ( ucu2 - 2/gi - 2 ) is thaxtomin A, isoxaben and NPA-sensitive indicated by root growth and ion flux responses providing new insights into these compounds mode of action and interactions.

Abstract

Thaxtomin A (TA) is a cellulose biosynthetic inhibitor (CBI) that promotes plant cell hypertrophy and cell death. Electrophysiological analysis of steady-state K⁺ and Ca²⁺ fluxes in Arabidopsis thaliana roots pretreated with TA for 24 h indicated a disturbance in the regulation of ion movement across the plant cell membrane. The observed inability to control solute movement, recorded in rapidly growing meristematic and elongation root zones, may partly explain typical root toxicity responses to TA treatment. Of note, the TA-sensitive mutant (ucu2-2/gi-2) was more susceptible with K⁺ and Ca²⁺ fluxes altered between 1.3 and eightfold compared to the wild-type control where fluxes altered between 1.2 and threefold. Root growth inhibition assays showed that the ucu2-2/gi-2 mutant had an increased sensitivity to the auxin 2,4-D, but not IAA or NAA; it also had increased sensitivity to the auxin efflux transport inhibitor, 1-naphthylphthalamic acid (NPA), but not 2,3,5- Triiodobenzoic acid (TIBA), when compared to the WT. The NPA sensitivity data were supported by electrophysiological analysis of H⁺ fluxes in the mature (but not elongation) root zone. Increased sensitivity to the CBI, isoxaben (IXB), but not dichlobenil was recorded. Increased sensitivity to both TA and IXB corresponded with higher levels of accumulation of these toxins in the root tissue, compared to the WT. Further root growth inhibition assays showed no altered sensitivity of ucu2-2/gi-2 to two other plant pathogen toxins, alternariol and fusaric acid. Identification of a TA-sensitive Arabidopsis mutant provides further insight into how this CBI toxin interacts with plant cells.