Discovery and Characterisation of Novel Protein Interactions with Death Associated Protein Kinase

Death Associated Protein Kinase (DAPK) has a wide-ranging role in cell death signaling and growth control. Over the past decade the importance of DAPK as a tumour suppressor has been highlighted by numerous studies that show its expression is ablated in many cancer types by epigenetic silencing. However the mechanisms by which this multi-domain protein exerts death-inducing effects have not been well defined, given that very few substrates or interaction partners have been discovered. Many protein-protein interactions involving cell signaling processes are driven by linear interaction motifs. Therefore combinatorial peptide libraries displayed on Ml3 filamentous bacteriophage were used to identify peptide consensus binding sites for the kinase domain of DAPK. Peptides that bound to the DAPKcore kinase domain were then isolated and sequenced leading to the discovery of binding peptides with striking homology to the SHI-4 family of transcription factors, the Promyelocytic Leukemia protein (PML) and the microtubule associating protein MAP IB. Immunobinding assays, immunofluorescent cell staining studies and biochemical fractionations demonstrated that DAPK can interact with human MAP IB via an Nterminal interface in-vitro and in cells and so this interaction was subject to further study. DAPK has been shown to integrate death inducing signals through a number of pathways including the p53 tumour suppression pathway and apoptotic and autophagic cell death inducing pathways. Therefore a range of assays were developed to characterise the biological significance of DAPK interaction with MAP IB in the context of each pathway. Cell growth and viability assays demonstrated that MAP IB co-operates with DAPK to reduce cell proliferation. This co-operative cell growth inhibition was independent of the p53 pathway and apoptotic (Type I) cell death, but induced autophagic (Type II) cell death. MAP IB co-operation with DAPK was marked by a striking increase in the number of cells with membrane blebbing morphology, an effect previously shown to involve DAPK interaction with the actin cytoskeleton leading to actin-myosin contraction. This was in contrast to the known role of MAP IB that is primarily thought of as a tubulin associating protein that modifies microtubule dynamics. Therefore the role of the cytoskeleton in DAPK co-operation with MAP IB was studied in detail using immunofluorescent cytoskeleton staining and microtubule purification assays. During DAPK transfection induced membrane blebbing, a pool of DAPK and MAP IB co-localise and co-purify with tubulin whereas a separate pool is co-located to cortical actin. Thus DAPK and MAP IB cooperation-induced membrane blebbing involves a novel interaction with both microtubules and microfilaments. These studies highlight the utility of peptide combinatorial libraries to identify novel binding interfaces and highlight a positive role for MAP IB in DAPK dependent cytoskeletal rearrangement and the autophagic cell death program

Proclamation 305—Fort Berthold Reservation in the State of North Dakota

This 1904 reprint of President Benjamin Harrison’s 1891 proclamation was transcribed and published in vol. I of Charles Kappler’s Indian Affairs. Laws and Treaties. Originally signed on May 20, 1891, this proclamation announced that the “Law of 1891,” passed by US Congress on March 3, 1891, was accepted, ratified, and confirmed.https://commons.und.edu/indigenous-gov-docs/1018/thumbnail.jp

A trained perceptual bias that lasts for weeks

AbstractClassical (Pavlovian) conditioning procedures can be used to bias the appearance of physical stimuli. Under natural conditions this form of perceptual learning could cause perception to become more accurate by changing prior belief to be in accord with what is statistically likely. However, for learning to be of functional significance, it must last until similar stimuli are encountered again. Here, we used the apparent rotation direction of a revolving wire frame (Necker) cube to test whether a learned perceptual bias is long lasting. Apparent rotation direction was trained to have a different bias at two different retinal locations by interleaving the presentation of ambiguous cubes with presentation of cubes that were disambiguated by disparity and occlusion cues. Four groups of eight subjects were subsequently tested either 1, 7, 14, or 28days after initial training, respectively, using a counter-conditioning procedure. All four groups showed incomplete re-learning of the reversed contingency relationship during their second session. One group repeated the counter-conditioning and showed an increase in the reverse bias, showing that the first counter-conditioning session also had a long-lasting effect. The fact that the original learning was still evident four weeks after the initial training is consistent with the operation of a mechanism that ordinarily would improve the accuracy and efficiency of perception

Disambiguation of Necker cube rotation by monocular and binocular depth cues: Relative effectiveness for establishing long-term bias

AbstractThe apparent direction of rotation of perceptually bistable wire-frame (Necker) cubes can be conditioned to depend on retinal location by interleaving their presentation with cubes that are disambiguated by depth cues (Haijiang, Saunders, Stone, & Backus, 2006; Harrison & Backus, 2010a). The long-term nature of the learned bias is demonstrated by resistance to counter-conditioning on a consecutive day. In previous work, either binocular disparity and occlusion, or a combination of monocular depth cues that included occlusion, internal occlusion, haze, and depth-from-shading, were used to control the rotation direction of disambiguated cubes. Here, we test the relative effectiveness of these two sets of depth cues in establishing the retinal location bias. Both cue sets were highly effective in establishing a perceptual bias on Day 1 as measured by the perceived rotation direction of ambiguous cubes. The effect of counter-conditioning on Day 2, on perceptual outcome for ambiguous cubes, was independent of whether the cue set was the same or different as Day 1. This invariance suggests that a common neural population instantiates the bias for rotation direction, regardless of the cue set used. However, in a further experiment where only disambiguated cubes were presented on Day 1, perceptual outcome of ambiguous cubes during Day 2 counter-conditioning showed that the monocular-only cue set was in fact more effective than disparity-plus-occlusion for causing long-term learning of the bias. These results can be reconciled if the conditioning effect of Day 1 ambiguous trials in the first experiment is taken into account (Harrison & Backus, 2010b). We suggest that monocular disambiguation leads to stronger bias either because it more strongly activates a single neural population that is necessary for perceiving rotation, or because ambiguous stimuli engage cortical areas that are also engaged by monocularly disambiguated stimuli but not by disparity-disambiguated stimuli

Paleomagnetic evidence for dynamo activity driven by inward crystallisation of a metallic asteroid

The direction in which a planetary core solidifies has fundamental implications for the feasibility and nature of dynamo generation. Although Earth's core is outwardly solidifying, the cores of certain smaller planetary bodies have been proposed to inwardly solidify due to their lower central pressures. However, there have been no unambiguous observations of inwardly solidified cores or the relationship between this solidification regime and planetary magnetic activity. To address this gap, we present the results of complimentary paleomagnetic techniques applied to the matrix metal and silicate inclusions within the IVA iron meteorites. This family of meteorites has been suggested to originate from a planetary core that had its overlaying silicate mantle removed by collisions during the early solar system. This process is thought to have produced a molten ball of metal that cooled rapidly and has been proposed to have inwardly solidified. Recent thermal evolution models of such a body predict that it should have generated an intense, multipolar and time-varying dynamo field. This field could have been recorded as a remanent magnetisation in the outer, cool layers of a solid crust on the IVA parent core. We find that the different components in the IVA iron meteorites display a range of paleomagnetic fidelities, depending crucially on the cooling rate of the meteorite. In particular, silicate inclusions in the quickly cooled São João Nepomuceno meteorite are poor paleomagnetic recorders. On the other hand, the matrix metal and some silicate subsamples from the relatively slowly cooled Steinbach meteorite are far better paleomagnetic recorders and provide evidence of an intense (≳100 μT) and directionally varying (exhibiting significant changes on a timescale ≲200 kyr) magnetic field. This is the first demonstration that some iron meteorites record ancient planetary magnetic fields. Furthermore, the observed field intensity, temporal variability and dynamo lifetime are consistent with thermal evolution models of the IVA parent core. Because the acquisition of remanent magnetisation by some IVA iron meteorites require that they cooled below their Curie temperature during the period of dynamo activity, the magnetisation carried by Steinbach also provides strong evidence favouring the inward solidification of its parent core

Crystal structures of two one-dimensional coordination polymers constructed from Mn2+ ions, chelating hexafluoro-acetylacetonate anions, and flexible bipyridyl bridging ligands

Original Russian Text © 2016 M. J. Plater, B. M. De Silva, M. R. St. J. Foreman, W. T. A. Harrison. The text was submitted by the authors in English. Zhurnal Strukturnoi Khimii, Vol. 57, No. 6, pp. 1230-1235, July-August, 2016.Peer reviewedPostprin

Achieving Acetylcholine Receptor Clustering in Tissue-Engineered Skeletal Muscle Constructs In vitro through a Materials-Directed Agrin Delivery Approach

Volumetric muscle loss (VML) can result from trauma, infection, congenital anomalies, or surgery, and produce permanent functional and cosmetic deficits. There are no effective treatment options for VML injuries, and recent advances toward development of muscle constructs lack the ability to achieve innervation necessary for long-term function. We sought to develop a proof-of-concept biomaterial construct that could achieve acetylcholine receptor (AChR) clustering on muscle-derived cells (MDCs) in vitro. The approach consisted of the presentation of neural (Z+) agrin from the surface of microspheres embedded with a fibrin hydrogel to muscle cells (C2C12 cell line or primary rat MDCs). AChR clustering was spatially restricted to areas of cell (C2C12)-microsphere contact when the microspheres were delivered in suspension or when they were incorporated into a thin (2D) fibrin hydrogel. AChR clusters were observed from 16 to 72 h after treatment when Z+ agrin was adsorbed to the microspheres, and for greater than 120 h when agrin was covalently coupled to the microspheres. Little to no AChR clustering was observed when agrin-coated microspheres were delivered from specially designed 3D fibrin constructs. However, cyclic stretch in combination with agrin-presenting microspheres led to dramatic enhancement of AChR clustering in cells cultured on these 3D fibrin constructs, suggesting a synergistic effect between mechanical strain and agrin stimulation of AChR clustering in vitro. These studies highlight a strategy for maintaining a physiological phenotype characterized by motor endplates of muscle cells used in tissue engineering strategies for muscle regeneration. As such, these observations may provide an important first step toward improving function of tissue-engineered constructs for treatment of VML injuries

Distributions of putative aerobic methanotrophs in diverse pelagic marine environments

Aerobic methane oxidization in the pelagic ocean serves an important role in limiting methane release to the atmosphere, yet little is known about the identity and distribution of bacteria that mediate this process. The distribution of putative methane-oxidizing marine groups, OPU1, OPU3 and Group X, was assessed in different ocean provinces using a newly developed fingerprinting method (monooxygenase intergenic spacer analysis (MISA)) in combination with pmoA clone library analysis and quantitative PCR (qPCR). The distribution of these three distinct monooxygenase groups, previously reported from pelagic marine environments, was examined in 39 samples including active methane seeps in the Gulf of Mexico and Santa Monica Bay, submarine canyon heads along the California continental margin, an oligotrophic subtropical gyre and areas proximal to a hydrothermal vent in the North Fiji back-arc basin. OPU1 and OPU3 were widely and similarly distributed within the meso-and bathypelagic zone (110 to similar to 2000 m water depth) and showed a >50-fold greater abundance near methane seeps relative to non-seep sites. In contrast, Group X was predominantly recovered from samples along the California margin, at both seep and non-seep sites. All three phylotypes were below detection in the epipelagic zone to depths of 100 m. Several additional deeply branching monooxygenase sequences were also identified in this study, indicating the presence of uncharacterized groups of microorganisms potentially involved in the cycling of methane or ammonium