The impacts of water quality on the amphibian chytrid fungal pathogen: A systematic review.

The pathogenic fungus Batrachochytrium dendrobatidis has caused declines of amphibians worldwide. Yet our understanding of how water quality influences fungal pathogenicity is limited. Here, we reviewed experimental studies on the effect of water quality on this pathogen to determine which parameters impacted disease dynamics consistently. The strongest evidence for protective effects is salinity which shows strong antifungal properties in hosts at natural levels. Although many fungicides had detrimental effects on the fungal pathogen in vitro, their impact on the host is variable and they can worsen infection outcomes. However, one fungicide, epoxiconazole, reduced disease effects experimentally and likely in the field. While heavy metals are frequently studied, there is weak evidence that they influence infection outcomes. Nitrogen and phosphorous do not appear to impact pathogen growth or infection in the amphibian host. The effects of other chemicals, like pesticides and disinfectants on infection were mostly unclear with mixed results or lacking an in vivo component. Our study shows that water chemistry does impact disease dynamics, but the effects of specific parameters require more investigation. Improving our understanding of how water chemistry influences disease dynamics will help predict the impact of chytridiomycosis, especially in amphibian populations affected by land use changes.

Comparison of methods for determining Batrachochytrium dendrobatidis zoospore viability.

The emerging fungal pathogen Batrachochytrium dendrobatidis (Bd) threatens hundreds of amphibian species globally. During laboratory-based experiments it is often essential to quantify live Bd cells, but a comparison of the effectiveness of methods for counting and assessing the viability of the infectious zoospore life stage has not been done. A direct comparison of staining methods that assess viability will ensure that the most accurate and efficient method is used. Here, we compared the use of 2 relatively cheap common stains, trypan blue and methylene blue, and assessed their accuracy and precision for estimating the viability of Bd zoospores during both manual counting and colorimetric assays. We stained known proportions of killed Bd zoospores (0, 0.25, 0.50, 0.75, and 1.00) with each stain and estimated the proportion of stained (dead) and unstained (viable) cells in each sample using both manual counting and colorimetric assays. Trypan blue was found to be a much more effective stain than methylene blue for both microscopy and colorimetric assays. Additionally, counting zoospores via microscopy was both a more accurate and precise technique. We recommend using manual counts via microscopy using the trypan blue stain for assessing Bd zoospore viability.

Systematic evaluation of multiple qPCR platforms, NanoString and miRNA-Seq for microRNA biomarker discovery in human biofluids.

Aberrant miRNA expression has been associated with many diseases, and extracellular miRNAs that circulate in the bloodstream are remarkably stable. Recently, there has been growing interest in identifying cell-free circulating miRNAs that can serve as non-invasive biomarkers for early detection of disease or selection of treatment options. However, quantifying miRNA levels in biofluids is technically challenging due to their low abundance. Using reference samples, we performed a cross-platform evaluation in which miRNA profiling was performed on four different qPCR platforms (MiRXES, Qiagen, Applied Biosystems, Exiqon), nCounter technology (NanoString), and miRNA-Seq. Overall, our results suggest that using miRNA-Seq for discovery and targeted qPCR for validation is a rational strategy for miRNA biomarker development in clinical samples that involve limited amounts of biofluids.

Identification and characterization of enhancers controlling the inflammatory gene expression program in macrophages.

Enhancers determine tissue-specific gene expression programs. Enhancers are marked by high histone H3 lysine 4 mono-methylation (H3K4me1) and by the acetyl-transferase p300, which has allowed genome-wide enhancer identification. However, the regulatory principles by which subsets of enhancers become active in specific developmental and/or environmental contexts are unknown. We exploited inducible p300 binding to chromatin to identify, and then mechanistically dissect, enhancers controlling endotoxin-stimulated gene expression in macrophages. In these enhancers, binding sites for the lineage-restricted and constitutive Ets protein PU.1 coexisted with those for ubiquitous stress-inducible transcription factors such as NF-kappaB, IRF, and AP-1. PU.1 was required for maintaining H3K4me1 at macrophage-specific enhancers. Reciprocally, ectopic expression of PU.1 reactivated these enhancers in fibroblasts. Thus, the combinatorial assembly of tissue- and signal-specific transcription factors determines the activity of a distinct group of enhancers. We suggest that this may represent a general paradigm in tissue-restricted and stimulus-responsive gene regulation.