Genetic data show that Carcharhinus tilstoni is not confined to the tropics, highlighting the importance of a multifaceted approach to species identification

This study shows a range extension for the Australian blacktip shark Carcharhinus tilstoni, which was believed to be restricted to Australia's tropical waters, of >1000 km into temperate waters, revealing its vulnerability to a wider commercial fishery.     

Multi Year Observations Reveal Variability in Residence of a Tropical Demersal Fish,Lethrinus nebulosus: Implications for Spatial Management

Off the Ningaloo coast of North West Western Australia, Spangled Emperor Lethrinus nebulosus are among the most highly targeted recreational fish species. The Ningaloo Reef Marine Park comprises an area of 4,566 km² of which 34% is protected from fishing by 18 no-take sanctuary zones ranging in size from 0.08–44.8 km². To better understand Spangled Emperor movements and the adequacy of sanctuary zones within the Ningaloo Reef Marine Park for this species, 84 Spangled Emperor of a broad spectrum of maturity and sex were tagged using internal acoustic tags in a range of lagoon and reef slope habitats both inside and adjacent to the Mangrove Bay Sanctuary zone. Kernel Utilisation Distribution (KUD) was calculated for 39 resident individuals that were detected for more than 30 days. There was no relationship with fish size and movement or site fidelity. Average home range (95% KUD) for residents was 8.5±0.5 km² compared to average sanctuary zone size of 30 km². Calculated home range was stable over time resulting in resident animals tagged inside the sanctuary zone spending ∼80% of time within the sanctuary boundaries. The number of fish remaining within the array of receivers declined steadily over time and after one year more than 60% of tagged fish had moved outside the sanctuary zone and also beyond the 28 km² array of receivers. Long term monitoring identified the importance of shifting home range and was essential for understanding overall residency within protected areas and also for identifying spawning related movements. This study indicates that despite exhibiting stable and small home ranges over periods of one to two years, more than half the population of spangled emperor move at scales greater than average sanctuary size within the Ningaloo Reef Marine Park.     

Conjugation to polyethylene glycol polymer promotes aptamer biodistribution to healthy and inflamed tissues

Here, we examine biodistribution of radiolabeled aptamers and assess the relative ability of different stabilized aptamer compositions (mixed 2'-F/2'-O-Me; fully 2'-O-Me modified) to access inflamed tissues in a murine inflammation model. Biodistribution of 3H-labeled aptamers, including pegylated and unpegylated compositions, was assessed 3 hours postadministration using quantitative whole body autoradiography (QWBA). Aptamer penetration of cells in kidney and liver was also examined at a qualitative level by microautoradiography. To evaluate aptamer distribution to diseased tissues, inflammation was induced locally in animal hind limbs by treatment with carrageenan just prior to aptamer dosing. Aptamer compositions examined exhibited significant variation in distribution levels among organs and tissues. Highest concentrations of radioactivity in whole body tissues for all animals were observed in the kidney and urinary bladder contents. Relatively little radioactivity was associated with brain, spinal cord, and adipose tissue. Overall, the total level of radioactivity in whole body tissues was significantly higher for a 20-kDa PEG conjugate than for other aptamers. Comparatively high levels of the 20-kDa conjugate were seen in well-perfused organs and tissues, including liver, lungs, spleen, bone marrow, and myocardium. A fully 2'-O-Me composition aptamer had the lowest level of radioactivity in whole body tissues but distributed to higher concentrations in the gastrointestinal tract contents relative to other aptamers. Interestingly, the 20-kDa PEG-conjugated aptamer showed significantly higher levels of distribution to inflamed paw tissues than did either unconjugated or fully 2'-O-Me-modified aptamers.     

Integrated modeling predicts shifts in waterbird population dynamics under climate change

Climate change has been identified as one of the most important drivers of wildlife population dynamics. The in‐depth knowledge of the complex relationships between climate and population sizes through density dependent demographic processes is important for understanding and predicting population shifts under climate change, which requires integrated population models (IPMs) that unify the analyses of demography and abundance data. In this study we developed an IPM based on Gaussian approximation to dynamic N‐mixture models for large scale population data. We then analyzed four decades (1972–2013) of mallard Anas platyrhynchos breeding population survey, band‐recovery and climate data covering a large spatial extent from North American prairies through boreal habitat to Alaska. We aimed to test the hypothesis that climate change will cause shifts in population dynamics if climatic effects on demographic parameters that have substantial contribution to population growth vary spatially. More specifically, we examined the spatial variation of climatic effects on density dependent population demography, identified the key demographic parameters that are influential to population growth, and forecasted population responses to climate change. Our results revealed that recruitment, which explained more variance of population growth than survival, was sensitive to the temporal variation of precipitation in the southern portion of the study area but not in the north. Survival, by contrast, was insensitive to climatic variation. We then forecasted a decrease in mallard breeding population density in the south and an increase in the northwestern portion of the study area, indicating potential shifts in population dynamics under future climate change. Our results implied that different strategies need to be considered across regions to conserve waterfowl populations in the face of climate change. Our modelling approach can be adapted for other species and thus has wide application to understanding and predicting population dynamics in the presence of global change.     

Targeted Knock-In Mice Expressing Mutations of CD28 Reveal an Essential Pathway for Costimulation

Despite extensive study, the role of phosphatidylinositol 3-kinase (PI3-kinase) activation in CD28 function has been highly contentious. To definitively address this question, we generated knock-in mice expressing mutations in two critical domains of the cytoplasmic tail of CD28. Mutation of the proximal tyrosine motif interrupted PI3-kinase binding and prevented CD28-dependent phosphorylation of protein kinase B (PKB)/Akt; however, there was no detectable effect on interleukin-2 (IL-2) secretion, expression of Bcl-X_L, or on T-cell function in vivo. Furthermore, we demonstrate that signaling initiated by the C-terminal proline motif is directly responsible for tyrosine phosphorylation of phosphoinosotide-dependent kinase 1, protein kinase Cθ, and glycogen synthase kinase 3β, as well as contributing to threonine phosphorylation of PKB. T cells mutated in this domain were profoundly impaired in IL-2 secretion, and the mice had marked impairment of humoral responses as well as less severe disease manifestations in experimental allergic encephalomyelitis. These data demonstrate that the distal proline motif initiates a critical nonredundant signaling pathway, whereas direct activation of PI3-kinase by the proximal tyrosine motif of CD28 is not required for normal T-cell function.CD28 and T-cell receptor (TCR)-derived signals act synergistically, leading to optimal T-cell proliferation, cytokine secretion, and cell survival (for a review, see reference 32). The importance of CD28 in vivo is evidenced by impaired responses of CD28-deficient mice in a number of model systems, including allergic airway inflammation and experimental allergic encephalomyelitis (EAE) (13, 34). In addition, the recent development of inhibitors of CD28 as effective therapeutics for autoimmune disease and transplant immunosuppression further emphasizes the critical role of this receptor in human disease (21, 57).Despite extensive study, the biochemical mechanism(s) that mediates CD28 function remains incompletely understood. Specific motifs within the cytoplasmic tail of CD28 have been identified that trigger distinct signaling pathways. Binding and activation of Src family kinases to the distal proline motif (sequence PYAP) initiates signaling, whereas the proximal tyrosine motif (sequence YMNM) binds and activates the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) as well as other adaptor proteins, including Grb2 and GADS (12, 27, 28, 33, 42, 48, 51). Studies have suggested that both motifs contribute to CD28-dependent interleukin-2 (IL-2) secretion and proliferation but that the upregulation of Bcl-X_L is uniquely dependent on PI3-kinase activation by the proximal tyrosine at position 170 (11, 25, 43). The potential for extensive overlap between pathways initiated by each motif exists, as well as overlap between CD28 and TCR-derived signals, making it unclear as to whether CD28 initiates any critical, nonredundant signaling pathway.We generated gene-targeted knock-in mice expressing either wild-type CD28 or mutations in the proximal tyrosine-based motif or the distal proline-based motif to determine the importance of these both biochemically and functionally. Our data demonstrate that the distal proline motif initiates an essential signaling pathway required for normal regulation of IL-2 secretion and CD28-dependent responses in vivo. Mutation of this motif resulted in the failure to normally phosphorylate phosphoinosotide-dependent kinase 1 (PDK1), glycogen synthase kinase 3β (GSK3β), and protein kinase Cθ (PKCθ), implicating these kinases as critical downstream mediators of CD28 function. In contrast, mutation of the proximal tyrosine motif resulted in no detectable defect in T-cell function, despite impaired binding of the p85 subunit of PI3-kinase and a lack of CD28-dependent serine phosphorylation of protein kinase B (PKB/Akt). These data strongly suggest that loss of the proximal tyrosine motif can be overcome by compensatory pathways, whereas no such redundancy exists for signaling initiated by the distal proline motif.     

Attenuation of T Cell Receptor Signaling by Serine Phosphorylation-mediated Lysine 30 Ubiquitination of SLP-76 Protein

SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) is an adaptor protein that is essential for T cell development and T cell receptor (TCR) signaling activation. Previous studies have identified an important negative feedback regulation of SLP-76 by HPK1 (hematopoietic progenitor kinase 1; MAP4K1)-induced Ser-376 phosphorylation. Ser-376 phosphorylation of SLP-76 mediates 14-3-3 binding, resulting in the attenuation of SLP-76 activation and downstream signaling; however, the underlying mechanism of this action remains unknown. Here, we report that phosphorylated SLP-76 is ubiquitinated and targeted for proteasomal degradation during TCR signaling. SLP-76 ubiquitination is mediated by Ser-376 phosphorylation. Furthermore, Lys-30 is identified as a ubiquitination site of SLP-76. Loss of Lys-30 ubiquitination of SLP-76 results in enhanced anti-CD3 antibody-induced ERK and JNK activation. These results reveal a novel regulation mechanism of SLP-76 by ubiquitination and proteasomal degradation of activated SLP-76, which is mediated by Ser-376 phosphorylation, leading to down-regulation of TCR signaling.     

Protein phosphatase 4 is a positive regulator of hematopoietic progenitor kinase 1

Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic specific mammalian Ste20-like protein kinase and has been implicated in many cellular signaling pathways including T cell receptor (TCR) signaling. However, little is known about the in vivo regulation of HPK1. We present evidence that HPK1 is positively regulated by protein phosphatase 4 (PP4; also called PPX and PPP4), a serine/threonine phosphatase. We found that PP4 interacted with HPK1 and that the proline-rich region of HPK1 was necessary and sufficient for this interaction. We also found that PP4 had phosphatase activity toward HPK1 in vivo and that co-transfection of PP4 with HPK1 resulted in specific kinase activation of HPK1. Moreover, we found that the PP4-induced HPK1 kinase activation was accompanied by an increase in protein expression of HPK1. Pulse-chase analysis showed that PP4 increased the half-life of HPK1. Further studies showed that HPK1 was subject to regulation by ubiquitination and ubiquitin-targeted degradation and that PP4 inhibited HPK1 ubiquitination. In addition, we found that TCR stimulation enhanced the PP4-HPK1 interaction and that wild-type PP4 enhanced, whereas a phosphatase-dead PP4 mutant inhibited, TCR-induced activation of HPK1 in Jurkat T cells. Combined with the observation that PP4 enhanced HPK1-induced JNK activation, our studies identify PP4 as a positive regulator for HPK1 and the HPK1-JNK signaling pathway.     

A novel strategy for selection of allosteric ribozymes yields RiboReporter sensors for caffeine and aspartame

We have utilized in vitro selection technology to develop allosteric ribozyme sensors that are specific for the small molecule analytes caffeine or aspartame. Caffeine- or aspartame-responsive ribozymes were converted into fluorescence-based RiboReporter™ sensor systems that were able to detect caffeine or aspartame in solution over a concentration range from 0.5 to 5 mM. With read-times as short as 5 min, these caffeine- or aspartame-dependent ribozymes function as highly specific and facile molecular sensors. Interestingly, successful isolation of allosteric ribozymes for the analytes described here was enabled by a novel selection strategy that incorporated elements of both modular design and activity-based selection methods typically used for generation of catalytic nucleic acids.