Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.     

Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase

The signaling pathways of mammalian Toll-like receptors (TLR) are well characterized, but the initial molecular mechanisms activated following ligand interactions with the receptors remain poorly defined. Here, we show a membrane controlling mechanism that is initiated by ligand binding to TLR-2, -3 and-4 to induce Neu1 sialidase activity within minutes in live primary bone marrow (BM) macrophage cells and macrophage and dendritic cell lines. Central to this process is that Neu1 and not Neu2,-3 and-4 forms a complex with TLR-2,-3 and-4 on the cell surface of naïve macrophage cells. Neuraminidase inhibitors BCX1827, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir and oseltamivir carboxylate have a limited significant inhibition of the LPS-induced sialidase activity in live BMC-2 macrophage cells but Tamiflu (oseltamivir phosphate) completely blocks this activity. Tamiflu inhibits LPS-induced sialidase activity in live BMC-2 cells with an IC₅₀ of 1.2?μM compared to an IC₅₀ of 1015?μM for its hydrolytic metabolite oseltamivir carboxylate. Tamiflu blockage of LPS-induced Neu1 sialidase activity is not affected in BMC-2 cells pretreated with anticarboxylesterase agent clopidogrel. Endotoxin LPS binding to TLR4 induces Neu1 with subsequent activation of NFκB and the production of nitric oxide and pro-inflammatory IL-6 and TNFα cytokines in primary and macrophage cell lines. Hypomorphic cathepsin A mice with a secondary Neu1 deficiency respond poorly to LPS-induced pro-inflammatory cytokines compared to the wild-type or hypomorphic cathepsin A with normal Neu1 mice. Our findings establish an unprecedented mechanism for pathogen molecule-induced TLR activation and cell function, which is critically dependent on Neu1 sialidase activity associated with TLR ligand treated live primary macrophage cells and macrophage and dendritic cell lines.     

A View From Both Ends: Shifts in Herbivore Assemblages Impact Top-Down and Bottom-Up Processes on Coral Reefs

Ecosystems - A fundamental goal in ecology is to understand the role of consumers in top-down (TD) and bottom-up (BU) processes that affect the functioning of ecosystems. Consumers ingest organic...     

Wetlands serve as natural sources for improvement of stream ecosystem health in regions affected by acid deposition

For over 40 years, acid deposition has been recognized as a serious international environmental problem, but efforts to restore acidified streams and biota have had limited success. The need to better understand the effects of different sources of acidity on streams has become more pressing with the recent increases in surface water organic acids, or ‘brownification,’ associated with climate change and decreased inorganic acid deposition. Here, we carried out a large scale multi‐seasonal investigation in the Adirondacks, one of the most acid‐impacted regions in the United States, to assess how acid stream producers respond to local and watershed influences and whether these influences can be used in acidification remediation. We explored the pathways of wetland control on aluminum chemistry and diatom taxonomic and functional composition. We demonstrate that streams with larger watershed wetlands have higher organic content, lower concentrations of acidic anions, and lower ratios of inorganic to organic monomeric aluminum, all beneficial for diatom biodiversity and guilds producing high biomass. Although brownification has been viewed as a form of pollution, our results indicate that it may be a stimulating force for biofilm producers with potentially positive consequences for higher trophic levels. Our research also reveals that the mechanism of watershed control of local stream diatom biodiversity through wetland export of organic matter is universal in running waters, operating not only in hard streams, as previously reported, but also in acid streams. Our findings that the negative impacts of acid deposition on Adirondack stream chemistry and biota can be mitigated by wetlands have important implications for biodiversity conservation and stream ecosystem management. Future acidification research should focus on the potential for wetlands to improve stream ecosystem health in acid‐impacted regions and their direct use in stream restoration, for example, through stream rechanneling or wetland construction in appropriate hydrologic settings.     

Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer

Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development.     

A Field Evaluation of an External and Neutrally Buoyant Acoustic Transmitter for Juvenile Salmon: Implications for Estimating Hydroturbine Passage Survival

Turbine-passed fish are exposed to rapid decreases in pressure which can cause barotrauma. The presence of an implanted telemetry tag increases the likelihood of injury or death from exposure to pressure changes, thus potentially biasing studies evaluating survival of turbine-passed fish. Therefore, a neutrally buoyant externally attached tag was developed to eliminate this bias in turbine passage studies. This new tag was designed not to add excess mass in water or take up space in the coelom, having an effective tag burden of zero with the goal of reducing pressure related biases to turbine survival studies. To determine if this new tag affects fish performance or susceptibility to predation, it was evaluated in the field relative to internally implanted acoustic transmitters (JSATS; Juvenile Salmon Acoustic Telemetry System) used widely for survival studies of juvenile salmonids. Survival and travel time through the study reach was compared between fish with either tag type in an area of high predation in the Snake and Columbia rivers, Washington. An additional group of fish affixed with neutrally-buoyant dummy external tags were implanted with passive integrated transponder (PIT) tags and recovered further downstream to assess external tag retention and injury. There were no significant differences in survival to the first detection site, 12 river kilometers (rkm) downstream of release. Travel times were also similar between groups. Conversely, externally-tagged fish had reduced survival (or elevated tag loss) to the second detection site, 65 rkm downstream. In addition, the retention study revealed that tag loss was first observed in fish recaptured approximately 9 days after release. Results suggest that this new tag may be viable for short term (<8 days) single-dam turbine-passage studies and under these situations, may alleviate the turbine passage-related bias encountered when using internal tags, however further research is needed to confirm this.     

Mutually dependent degradation of Ama1p and Cdc20p terminates APC/C ubiquitin ligase activity at the completion of meiotic development in yeast

Background

The execution of meiotic nuclear divisions in S. cerevisiae is regulated by protein degradation mediated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The correct timing of APC/C activity is essential for normal chromosome segregation. During meiosis, the APC/C is activated by the association of either Cdc20p or the meiosis-specific factor Ama1p. Both Ama1p and Cdc20p are targeted for degradation as cells exit meiosis II with Cdc20p being destroyed by APC/C^Ama1. In this study we investigated how Ama1p is down regulated at the completion of meiosis.

Findings

Here we show that Ama1p is a substrate of APC/C^Cdc20 but not APC/C^Cdh1 in meiotic cells. Cdc20p binds Ama1p in vivo and APC/C^Cdc20 ubiquitylates Ama1p in vitro. Ama1p ubiquitylation requires one of two degradation motifs, a D-box and a “KEN-box” like motif called GxEN. Finally, Ama1p degradation does not require its association with the APC/C via its conserved APC/C binding motifs (C-box and IR) and occurs simultaneously with APC/C^Ama1-mediated Cdc20p degradation.

Conclusions

Unlike the cyclical nature of mitotic cell division, meiosis is a linear pathway leading to the production of quiescent spores. This raises the question of how the APC/C is reset prior to spore germination. This and a previous study revealed that Cdc20p and Ama1p direct each others degradation via APC/C-dependent degradation. These findings suggest a model that the APC/C is inactivated by mutual degradation of the activators. In addition, these results support a model in which Ama1p and Cdc20p relocate to the substrate address within the APC/C cavity prior to degradation.