Field test of an automated radio‐telemetry system: tracking local space use of aerial insectivores

Documenting local space use of birds that move rapidly, but are too small to carry GPS tags, such as swallows and swifts, can be challenging. For these species, tracking methods such as manual radio‐telemetry and visual observation are either inadequate or labor‐ and time‐intensive. Another option is use of an automated telemetry system, but equipment for such systems can be costly when many receivers are used. Our objective, therefore, was to determine if an automated radio‐telemetry system, consisting of just two receivers, could provide an alternative to manual tracking for gathering data on local space use of six individuals of three species of aerial insectivores, including one Cliff Swallow (Petrochelidon pyrrhonota), one Eastern Phoebe (Sayornis phoebe), and four Barn Swallows (Hirundo rustica). We established automated radio‐telemetry systems at three sites near the city of Peterborough in eastern Ontario, Canada, from May to August 2015. We evaluated the location error of our two‐receiver system using data from moving and stationary test transmitters at known locations, and used telemetry data from the aerial insectivores as a test of the system's ability to track rapidly moving birds under field conditions. Median location error was ~250 m for automated telemetry test locations after filtering. More than 90% of estimated locations had large location errors and were removed from analysis, including all locations > 1 km from receiver stations. Our automated telemetry receivers recorded 17,634 detections of the six radio‐tagged birds. However, filtering removed an average of 89% of bird location estimates, leaving only the Cliff Swallow with enough locations for analysis of space use. Our results demonstrate that a minimal automated radio‐telemetry system can be used to assess local space use by small, highly mobile birds, but the resolution of the data collected using only two receiver stations was coarse and had a limited range. To improve both location accuracy and increase the percentage of usable location estimates collected, we suggest that, in future studies, investigators use receivers that simultaneously record signals detected by all antennas, and use of a minimum of three receiver stations with more antennas at each station.     

Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase

The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.     

A second Warburg-like effect in cancer metabolism: The metabolic shift of glutamine-derived nitrogen

Carbon and nitrogen are essential elements for life. Glucose as a carbon source and glutamine as a nitrogen source are important nutrients for cell proliferation. About 100 years ago, it was discovered that cancer cells that have acquired unlimited proliferative capacity and undergone malignant evolution in their host manifest a cancer-specific remodeling of glucose metabolism (the Warburg effect). Only recently, however, was it shown that the metabolism of glutamine-derived nitrogen is substantially shifted from glutaminolysis to nucleotide biosynthesis during malignant progression of cancer—which might be referred to as a “second” Warburg effect. In this review, address the mechanism and relevance of this metabolic shift of glutamine-derived nitrogen in human cancer. We also examine the clinical potential of anticancer therapies that modulate the metabolic pathways of glutamine-derived nitrogen. This shift may be as important as the shift in carbon metabolism, which has long been known as the Warburg effect.     

An Optimized Proteomics Approach Reveals Novel Alternative Proteins in Mouse Liver Development

Alternative ORFs (AltORFs) are unannotated sequences in genome that encode novel peptides or proteins named alternative proteins (AltProts). Although ribosome profiling and bioinformatics predict a large number of AltProts, mass spectrometry as the only direct way of identification is hampered by the short lengths and relative low abundance of AltProts. There is an urgent need for improvement of mass spectrometry methodologies for AltProt identification. Here, we report an approach based on size-exclusion chromatography for simultaneous enrichment and fractionation of AltProts from complex proteome. This method greatly simplifies the variance of AltProts discovery by enriching small proteins smaller than 40 kDa. In a systematic comparison between 10 methods, the approach we reported enabled the discovery of more AltProts with overall higher intensities, with less cost of time and effort compared to other workflows. We applied this approach to identify 89 novel AltProts from mouse liver, 39 of which were differentially expressed between embryonic and adult mice. During embryonic development, the upregulated AltProts were mainly involved in biological pathways on RNA splicing and processing, whereas the AltProts involved in metabolisms were more active in adult livers. Our study not only provides an effective approach for identifying AltProts but also novel AltProts that are potentially important in developmental biology.     

Ecology of Hantaan virus at Twin Bridges Training Area,Gyeonggi Province,Republic of Korea, 2005–2007

The Twin Bridges Training Area (TBTA) in the Republic of Korea consists of dirt roads, barren training areas, and forested hillsides adjacent to linear and broad expanses of tall grasses, herbaceous, and scrub vegetation. Of the six species of small mammals, the striped field mouse, Apodemus agrarius, was the most frequently captured (96.1%). Apodemus agrarius capture rates varied from 17.7 to 33.2% during three trapping periods. Gravid females were observed during November‐December 2006 (8.4%) and March 2007 (5.1%). In 2005, the overall seroprevalence of Hantaan virus (HTNV) was high (34.4%) and lower during surveys in 2006 (14.2%) and 2007 (13.8%). Seroprevalence was directly correlated with weight increase of A. agrarius.     

Small Molecular Inhibitors Block TRPM4 Currents in Prostate Cancer Cells,with Limited Impact on Cancer Hallmark Functions

Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca²⁺ activated monovalent cation channel that contributes to the pathophysiology of several diseases.For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells.Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays.To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na⁺. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype.In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells.     

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.     

Enteric and serological distribution of serotonin and its precursor tryptophan in perinatal low and normal weight piglets

Perinatal mortality is high among small-for-gestational age (SGA) piglets and continues to be an economic burden and threat to animal welfare. As the physiological role of serotonin (5-hydroxytryptamine, 5-HT) in perinatal development and gastrointestinal function in the pig remains unknown, the aim of this study was to assess the enteric distribution of 5-HT cells and to determine 5-HT together with its precursor tryptophan in the serum of perinatal normal and SGA piglets. For this purpose, proximal and distal parts of the small intestine (SI) were processed for immunohistochemical analysis to assess the presence of 5-HT endocrine cells. Serum 5-HT was measured with ELISA, whereas its precursor, that is, the free fraction of tryptophan (FFT) together with albumin-bound tryptophan and total tryptophan, were analysed with HPLC in postnatal piglets. In addition, the morphological growth patterns of the different intestinal tissue layers of both normal and SGA piglets were stereologically analysed. The stereological volume density of 5-HT enteroendocrine cells showed a significant interaction effect between age and region. Indeed, the amount of 5-HT cells in both the proximal and distal part of the SI tended to decrease according to age, with the lowest values detected at day 3 postpartum. No differences could be observed related to BW. Interestingly, the serum concentration of 5-HT was higher in normal piglets compared with SGA piglets. Moreover, the ratio of FFT to total tryptophan was significantly affected by age and BW. Normal piglets had, on average, a lower FFT/total tryptophan ratio compared with SGA piglets. An approximate linear decrease was observed with increasing age. Finally, the immaturity of the intestinal system of the SGA piglets was not reflected in altered volume densities of the different intestinal layers. To conclude, although no BW effect could be detected in the distribution of enteric 5-HT cells, serum 5-HT and the ratio of FFT to total tryptophan ratio showed significant differences between normal piglets and their SGA littermates.     

Skp-cullin-F box E3 ligase component FBXL2 ubiquitinates Aurora B to inhibit tumorigenesis

Aurora B kinase is an integral regulator of cytokinesis, as it stabilizes the intercellular canal within the midbody to ensure proper chromosomal segregation during cell division. Here we identified that the ubiquitin E3 ligase complex SCF^FBXL2 mediates Aurora B ubiquitination and degradation within the midbody, which is sufficient to induce mitotic arrest and apoptosis. Three molecular acceptor sites (K¹⁰², K¹⁰³ and K²⁰⁷) within Aurora B protein were identified as important sites for its ubiquitination. A triple Lys mutant of Aurora B (K¹⁰²/¹⁰³/^207R) exhibited optimal resistance to SCF^FBXL2-directed polyubiquitination, and overexpression of this variant resulted in a significant delay in anaphase onset, resulting in apoptosis. A unique small molecule F-box/LRR-repeat protein 2 (FBXL2) activator, BC-1258, stabilized and increased levels of FBXL2 protein that promoted Aurora B degradation, resulting in tetraploidy, mitotic arrest and apoptosis of tumorigenic cells, and profoundly inhibiting tumor formation in athymic nude mice. These findings uncover a new proteolytic mechanism targeting a key regulator of cell replication that may serve as a basis for chemotherapeutic intervention in neoplasia.