Candida albicans, Cryptococcus neoformans, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Staphylococcus aureus (methicillin-susceptible and -resistant strains) are some of the most common pathogens of immunocompromised individuals. Since multi-drug resistance of these microorganisms is a major medical problem, we propose a convenient microdilution method for screening of natural products in a search for new antimicrobial agents that would be active against these organisms. To enable the screening process under standard laboratory conditions, Cr. albidus and M. smegmatis were used as model-organisms in place of their pathogenic counterparts. Antibiotics were used as positive controls, and their MIC values were in agreement with the MIC ranges recommended by the National Committee for Clinical Laboratory Standards. Organic plant extracts from Lemna minor and Ilex cornuta were used for developing the microdilution assay. The method can be used as a reliable tool for discovering antimicrobial agents with novel chemistry. It is adopted for organic extracts at a microgram scale and is currently being used for screens of more than one thousand extracts from the National Cancer Institute Open Repository.

The lignan podophyllotoxin (1) is highly valued as the precursor to clinically useful anticancer drugs. Substantial drug development of this compound class continues, including potential new use for inflammatory disease. We have isolated two endophyte fungi, both strains of Phialocephala fortinii, from rhizomes of the plant Podophyllum peltatum. The fungi were identified through DNA sequencing and morphology. Both strains of fungi are slow-growing and produce 1 at low but measurable amounts in broth culture. The compound was confirmed through matching HPLC retention times, absorption spectra, and MS data to authentic 1. The yield of 1 has ranged from 0.5 to 189 microg/L in 4 weeks of culture. These fungi have implications for the sustained production of 1 independent of wild populations of the source plants.

Biliary atresia (BA) is a rapidly progressive and destructive fibrotic disorder of unknown etiology affecting the extrahepatic biliary tree of neonates. Epidemiological studies suggest that an environmental factor, such as a virus or toxin, is the cause of the disease, although none have been definitively established. Several naturally occurring outbreaks of BA in Australian livestock have been associated with the ingestion of unusual plants by pregnant animals during drought conditions. We used a biliary secretion assay in zebrafish to isolate a previously undescribed isoflavonoid, biliatresone, from Dysphania species implicated in a recent BA outbreak. This compound caused selective destruction of the extrahepatic, but not intrahepatic, biliary system of larval zebrafish. A mutation that enhanced biliatresone toxicity mapped to a region of the zebrafish genome that has conserved synteny with an established human BA susceptibility locus. The toxin also caused loss of cilia in neonatal mouse extrahepatic cholangiocytes in culture and disrupted cell polarity and monolayer integrity in cholangiocyte spheroids. Together, these findings provide direct evidence that BA could be initiated by perinatal exposure to an environmental toxin.

Biliary atresia, the most common indication for pediatric liver transplantation, is a fibrotic disease of unknown etiology affecting the extrahepatic bile ducts of newborns. The recently-described toxin biliatresone causes lumen obstruction in mouse cholangiocyte spheroids and represents a new model of biliary atresia. Our aim was to determine the cellular changes caused by biliatresone in mammalian cells that ultimately lead to biliary atresia and extra-hepatic fibrosis. We treated mouse cholangiocytes in 3D spheroid culture and neonatal extra-hepatic duct explants with biliatresone and compounds that regulate glutathione. We examined the effects of biliatresone on SOX17 levels, and determined the effects of Sox17 knockdown on cholangiocytes in 3D culture. We found that biliatresone caused disruption of cholangiocyte apical polarity and loss of monolayer integrity. Spheroids treated with biliatresone had increased permeability as shown by rhodamine efflux within 5 hours compared to untreated spheroids, which retained rhodamine for longer than 12 hours. Neonatal bile duct explants treated with the toxin showed lumen obstruction with increased subepithelial staining for α-smooth muscle actin and collagen, consistent with fibrosis. Biliatresone caused a rapid and transient decrease in glutathione, which was both necessary and sufficient to mediate its effects in cholangiocyte spheroid and bile duct explant systems. It also caused a significant decrease in in cholangiocyte levels of SOX17, and Sox17 knockdown in cholangiocyte spheroids mimicked the effects of biliatresone. Conclusion Biliatresone decreases glutathione and SOX17 in mouse cholangiocytes. In 3D cell systems, this leads to cholangiocyte monolayer damage and increased permeability and in extrahepatic bile duct explants it leads to disruption of the extra-hepatic biliary tree and subepithelial fibrosis. This mechanism may be important in understanding human biliary atresia.

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