Demonstration of RNAi Yeast Insecticide Activity in Semi-Field Larvicide and Attractive Targeted Sugar Bait Trials Conducted on Aedes and Culex Mosquitoes.

Eco-friendly new mosquito control innovations are critical for the ongoing success of global mosquito control programs. In this study, Sh.463_56.10R, a robust RNA interference (RNAi) yeast insecticide strain that is suitable for scaled fermentation, was evaluated under semi-field conditions. Inactivated and dried Sh.463_56.10R yeast induced significant mortality of field strain Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus larvae in semi-field larvicide trials conducted outdoors in St. Augustine, Trinidad, where 100% of the larvae were dead within 24 h. The yeast was also stably suspended in commercial bait and deployed as an active ingredient in miniature attractive targeted sugar bait (ATSB) station sachets. The yeast ATSB induced high levels of Aedes and Culex mosquito morbidity in semi-field trials conducted in Trinidad, West Indies, as well as in Bangkok, Thailand, in which the consumption of the yeast resulted in adult female mosquito death within 48 h, faster than what was observed in laboratory trials. These findings support the pursuit of large-scale field trials to further evaluate the Sh.463_56.10R insecticide, a member of a promising new class of species-specific RNAi insecticides that could help combat insecticide resistance and support effective mosquito control programs worldwide.

Yeast cells as microcapsules. Analytical tools and process variables in the encapsulation of hydrophobes in S. cerevisiae.

Yeast cells can be used as biocompatible and biodegradable containers for the microencapsulation of a variety of actives. Despite the wide application of this process, e.g. in the food industry, mechanism and controlling factors are yet poorly known. In this study we have studied kinetics and mechanistic aspects of the spontaneous internalization of terpenes (as model hydrophobic compounds) in Saccharomyces cerevisiae, quantifying their encapsulation through HPLC analysis and fluorescent staining of lipidic bodies with Nile Red, while in parallel monitoring cell viability. Our results showed that this encapsulation process is essentially a phenomenon of passive diffusion with negligible relevance of active transport. Further, our evidence shows that the major determinant of the encapsulation kinetics is the solubility of the hydrophobe in the cell wall, which is inversely related to partition coefficient (log P).

Probing (macro)molecular transport through cell walls.

We here report a study on the passive permeability of hydrophobic probes through the cell wall of Saccharomyces cerevisiae. In this study we have prepared a series of fluorescent probes with similar chemical composition and molecular weight ranging from a few hundreds to a few thousands of g mol(-1). Their permeation into the cell body exhibits a clear MW cut-off and the underlying mechanism is governed by the permeation of individual molecules rather than aggregates. We also show that it is possible to reversibly alter the cell wall permeation properties without compromising the essence of its structure, by modifying the polarity/dielectric constant of the wall through solvent exchange.

Emulsion macromonomer cross-linking. A preparative method for oxidation-responsive nanoparticles with a controlled network structure.

We here report on a preparative, template-based method for oxidation-responsive, poly(propylene sulfide) cross-linked nanoparticles. In this study we demonstrate that preformed, narrow polydispersity, and end-functional polysulfides can be dispersed to yield stable emulsions, which can then be converted into stable nanoparticles through photochemically initiated cross-linking. The nanoparticle size is substantially templated on that of the precursor emulsions; the nanoparticles bulk is an elastomeric material with a homogeneous cross-linking density. The nanoparticles show a peculiar, two-stage behavior upon exposure to oxidants, which is likely composed of a first agglomeration phase followed by swelling.

Disruption of epithelial tight junctions by yeast enhances the paracellular delivery of a model protein.

PURPOSE: The aim of this study was to investigate the effect of heat-killed yeast cells on the integrity of epithelial tight junctions in vitro. METHODS: Changes in barrier potential of Caco-2 cell monolayers were assessed by transepithelial electrical resistance (TEER) measurements and by an increasing permeability to a marker protein, horse-radish peroxidase (HRP). Visualisation of tight junction disruption was carried out directly through electron microscopy and indirectly through fluorescence confocal microscopy and immunoblotting of the tight junction-associated proteins zonula occludens ZO-1, occludin and actin. RESULTS: Yeast cells opened tight junctions in a reversible dose- and time-dependent manner, as shown by a decrease in TEER and an increase in HRP permeability. These changes to barrier potential were shown not to be due to cytotoxic effects but due to modulation of the tight junctions. ZO-1, actin and occludin proteins were demonstrated to be involved in yeast-induced tight junction opening through the use of confocal microscopy and western blotting. Electron microscopy confirmed a direct opening of tight junctions after application of yeast. CONCLUSION: Yeast modulated epithelial tight junctions in a reversible manner by contraction of the actin cytoskeleton and shift of ZO-1 and occludin tight junction proteins from the membrane to cytoskeletal areas of the cell.