Busting Myths in Compound Handling Practices for Assay Developers.

Since the advent of modern-day screening collections in the early 2000s, various aspects of our knowledge of good handling practices have continued to evolve. Some early practices, however, continue to prevail due to the absence of defining data that would bust the myths of tradition. The lack of defining data leads to a gap between plate-based screeners, on the one hand, and compound sample handling groups, on the other, with the latter being the default party to blame when an assay goes awry.In this paper, we highlight recommended practices that ensure sample integrity and present myth busting data that can help determine the root cause of an assay gone bad. We show how a strong and collaborative relationship between screening and sample handling groups is the better state that leads to the accomplishment of the common goal of finding breakthrough medicines.

Oxygenase coordination is required for morphological transition and the host-fungus interaction of Aspergillus flavus.

Oxylipins, a class of oxygenase-derived unsaturated fatty acids, are important signal molecules in many biological systems. Recent characterization of an Aspergillus flavus lipoxygenase gene, lox, revealed its importance in maintaining a density-dependent morphology switch from sclerotia to conidia as population density increased. Here, we present evidence for the involvement of four more oxylipin-generating dioxygenases (PpoA, PpoB, PpoC, and PpoD) in A. flavus density-dependent phenomena and the effects of loss of these genes on aflatoxin production and seed colonization. Although several single mutants showed alterations in the sclerotia-to-conidia switch, the major effect was observed in a strain downregulated for all five oxygenases (invert repeat transgene [IRT] strain IRT4 = ppoA, ppoB, ppoC, ppoD, and lox). In strain IRT4, sclerotia production was increased up to 500-fold whereas conidiation was decreased down to 100-fold and the strain was unable to switch into conidial production. Aflatoxin (AF) production for all mutant strains and the wild type was greatest at low population densities and absent in high populations except for strain IRT4, which consistently produced high levels of the mycotoxin. Growth on host seed by both IRT4 and IRT2 (downregulated in ppoA, ppoB, and ppoD) was marked by decreased conidial but increased AF production. We propose that A. flavus oxygenases and the oxylipins they produce act in a highly interdependent network with some redundancy of biological function. These studies provide substantial evidence for oxylipin-based mechanisms in governing fungus-seed interactions and in regulating a coordinated quorum-sensing mechanism in A. flavus.

Requirement of LaeA for secondary metabolism and sclerotial production in Aspergillus flavus.

The nuclear regulator LaeA has been shown to govern production of multiple secondary metabolites in Aspergillus nidulans and Aspergillus fumigatus. Herein we examine the role of this protein in Aspergillus flavus. Similarly as in other Aspergilli, LaeA had a major effect on A. flavus secondary metabolism where DeltalaeA and over-expression laeA (OE::laeA) strains yielded opposite phenotypes resulting in decreased (increased) secondary metabolite production. The two mutant strains also exhibited striking morphological phenotypes in the loss (increase) of sclerotial production in comparison to wildtype. Growth on seed was marked by decreased (increased) conidial and aflatoxin production of the respective mutants; this was accompanied by decreased lipase activity in DeltalaeA, an enzymatic process correlated with seed maceration. Transcriptional examination of the mutants showed LaeA negatively regulates expression of its recently identified nuclear partner VeA, another global regulator of A. flavus secondary metabolites and sclerotia.