Cross-ecosystem effects of terrestrial predators link treefrogs, zooplankton, and aquatic primary production

Predators can directly or indirectly shape food webs through a combination of consumptive and non-consumptive effects. Yet, how these effects vary across natural populations and their consequences for adjacent ecosystems remains poorly resolved. We examined links between terrestrial predators and aquatic ecosystems through their effects on a locally abundant amphibian, the red-eyed treefrog (Agalychnis callidryas), which has arboreal eggs (heavily predated by snakes and wasps) and aquatic larvae; embryos can escape terrestrial threats by hatching at an earlier age and smaller size. Our multi-site field survey indicates that in natural populations, the relative contributions of these consumptive and non-consumptive effects of predators can be substantial and remarkably similar. However, in mesocosms where we experimentally mimicked these predator effects, changes in the density and initial hatching age of tadpoles carried distinct consequences for aquatic food webs. Density-dependent growth resulted in peak tadpole biomass at intermediate densities (reflecting intermediate predation), and early-hatched tadpoles grew 16% faster and produced 26% more biomass than their late-hatched counterparts. These changes in tadpole growth and size differentially affected zooplankton communities, and the production and stability of phytoplankton. Together, these results illustrate multiple pathways through which predators in one ecosystem can modulate the structure of adjacent food webs

A small molecule (pluripotin) as a tool for studying cancer stem cell biology: proof of concept.

BACKGROUND: Cancer stem cells (CSC) are thought to be responsible for tumor maintenance and heterogeneity. Bona fide CSC purified from tumor biopsies are limited in supply and this hampers study of CSC biology. Furthermore, purified stem-like CSC subpopulations from existing tumor lines are unstable in culture. Finding a means to overcome these technical challenges would be a useful goal. In a first effort towards this, we examined whether a chemical probe that promotes survival of murine embryonic stem cells without added exogenous factors can alter functional characteristics in extant tumor lines in a fashion consistent with a CSC phenotype. METHODOLOGY/PRINCIPAL FINDINGS: The seven tumor lines of the NCI60 colon subpanel were exposed to SC-1 (pluripotin), a dual kinase and GTPase inhibitor that promotes self-renewal, and then examined for tumorigenicity under limiting dilution conditions and clonogenic activity in soft agar. A statistically significant increase in tumor formation following SC-1 treatment was observed (p<0.04). Cloning efficiencies and expression of putative CSC surface antigens (CD133 and CD44) were also increased. SC-1 treatment led to sphere formation in some colon tumor lines. Finally, SC-1 inhibited in vitro kinase activity of RSK2, and another RSK2 inhibitor increased colony formation implicating a role for this kinase in eliciting a CSC phenotype. CONCLUSIONS/SIGNIFICANCE: These findings validate a proof of concept study exposure of extant tumor lines to a small molecule may provide a tractable in vitro model for understanding CSC biology

Tumor Lines Treated with SC-1 Possess Characteristics Consistent with the CSC Phenotype.

<p>The colon tumor lines were treated for five days with 0.1 µM SC-1, harvested and then analyzed for their ability to form colonies in soft agar, to alter expression of putative CSC surface markers, and to form spheres. A. The cloning efficiency of the colon tumor lines was determined following treatment with SC-1. In 5/7 tumor lines, SC-1 treated cells had statistically significant increases in cumulative colony forming unit (CFU) mass compared to control treated cells (***p<0.001, **p<0.1 *p<0.05, n = 3). In one instance (#p<0.05), SC-1 treatment reduced cloning efficiency. B. The CD133 positive subpopulation was increased 2.5-fold in SC-1 treated HT29 colon tumor line (p = 0.03, n = 3). The CD44+ CD24- subpopulation was increased after SC-1 treatment in the HCT-116 colon tumor line (*p<0.05, n = 3). The CD44+ subpopulation was increased significantly following SC-1 treatments (*p<0.05, n = 3) in SW-620 colon tumor line. C. Three tumor lines (COLO 205, HCT-116, HT-29) formed nonadherent spheres in the presence of 0.1 µM SC-1 cultured in standard media containing 5% FBS. These observations were also apparent at day 5 in culture. All images were prepared at 400× magnification.</p

Effect of SC-1 on the Tumor Take Rate of the Seven Colon Tumor Lines of the NCI60 Panel^*.

*<p>Female NOD.SCID mice were inoculated s.c. with bulk population colon cell lines treated for 5 days as indicated with control media or media containing 0.1 µM SC-1.</p><p><a href="mailto:@Significant" target="_blank">@Significant</a> difference between control and SC-1 treated groups in the HT29 colon tumor line, 0.0375&</p><p>Significant difference between control (10/35) and SC-1 (19/35) treated group for 7 colon tumor lines, p = 0.04.</p

Effect of SC-1 on <i>In Vitro</i> Kinase Inhibition in Selected Kinases^*.

*<p>SC-1 was evaluated for <i>in vitro</i> kinase inhibition utilizing a miniaturized ³³P-based screening assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057099#pone.0057099-Horiuchi1" target="_blank">[41]</a>. The maximum concentration tested was 10 µM. IC₅₀ (inhibitory concentration of 50% of the control (DMSO) treatment) was calculated from a 3-fold serial dilution (n = 2). Dashed lines indicate no effect at or below the maximum concentration tested.</p

Effect of SC-1 on Protein Expression Levels of phospho-ERK1/2, and OCT4 in Colon Tumor Lines.

<p>Colon tumor lines were treated with SC-1 (0.1 µM), harvested, lysed, and probed for the proteins of interest following electrophoresis in SDS-PAGE gels at the indicated time points. A. In the SC-1 treated HT29 tumor line, phospho-ERK1/2/total ERK1/2 protein levels were decreased to 67±0.06% of control value at 5 min, 56±0.06% of control value at 30 min, and 46±0.06% of control value at 1 hr (n = 3, p<0.05). B. Increased OCT4 protein expression due to SC-1 was dependent on the tumor line. Representative experiments are shown for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057099#pone.0057099.s004" target="_blank">Figure S4A</a>–B (n = 2).</p