Formaldehyde-based whole-mount in situ hybridization method for planarians.

Whole-mount in situ hybridization (WISH) is a powerful tool for visualizing gene expression patterns in specific cell and tissue types. Each model organism presents its own unique set of challenges for achieving robust and reproducible staining with cellular resolution. Here, we describe a formaldehyde-based WISH method for the freshwater planarian Schmidtea mediterranea developed by systematically comparing and optimizing techniques for fixation, permeabilization, hybridization, and postprocessing. The new method gives robust, high-resolution labeling in fine anatomical detail, allows co-labeling with fluorescent probes, and is sufficiently sensitive to resolve the expression pattern of a microRNA in planarians. Our WISH methodology not only provides significant advancements over current protocols that make it a valuable asset for the planarian community, but should also find wide applicability in WISH methods used in other systems.

Soil acidification and foliar nutrient status of Ontario's deciduous forest in 1986 and 2005.

To assess the impacts of the decline in sulphur (S) deposition over the past 20 years in Ontario, soil chemistry and sugar maple (Acer saccharum Marsh) foliar chemistry were measured at 17 sites in south and central Ontario in 2005 and compared with archived samples collected in 1986. Foliar S concentrations were lower in 2005, reflecting the decline in S deposition whereas foliar N remained unchanged, reflecting the lack of change in N deposition in Ontario. Mineral soil pH, exchangeable base cations were lower in 2005 whereas total S, N and cation exchange capacity (CEC) were unchanged. Foliar concentrations of Ca were positively related to soil Ca levels in the A-horizon and were lower in 2005. Despite evidence of increasing soil acidity and losses of calcium, foliar base cation concentrations at most sites were adequate for sugar maple and forest health in terms of canopy appearance (Decline Index) improved.

Molecular characterization of FRAXB and comparative common fragile site instability in cancer cells.

The common fragile site, FRA3B, has been shown to be a site of frequent homozygous deletions in some cancers, resulting in loss of expression of the associated FHIT gene. It has been proposed that FHIT is a tumor suppressor gene that is inactivated as a result of the instability of FRA3B in tumorigenesis. More recently, deletions at other common fragile sites, FRA7G and FRA16D, have been identified in a small number of cancer cell lines. Here, we have mapped and molecularly characterized the frequently observed common fragile site FRAXB, located at Xp22.3. Like other common fragile sites, it spans a large genomic region of approximately 500 kb. Three known genes, including the microsomal steroid sulfatase locus (STS), map within the fragile site region. We examined FRAXB and four other fragile sites (FRA3B, FRA7G, FRA7H, FRA16D), and several associated genes, for deletions and aberrant transcripts in a panel of cancer cell lines and primary tumors. Deletions within FRAXB were seen in 4/27 (14.8%) of the primary tumors and cell lines examined. Three of the 21 (14.3%) cell lines examined were characterized by loss of expression of one or more FRAXB-associated genes. Moreover, all of the fragile sites examined were characterized by genomic deletions within the fragile site regions in one or more tumors or cell lines, including FRAXB, which is not associated with any known tumor suppressor genes or activity. Our results further support the hypothesis that common fragile sites and their associated genes are, in general, unstable in some cancer cells.