Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.     

Semaphorin directs axon traffic in the fly olfactory system

The convergence of olfactory sensory axons that express the same receptor onto specific glomeruli is a common organizing principle in animal olfactory systems. In this issue of Neuron, two beautiful studies in Drosophila by Lattemann et al. and Sweeney et al. show that Semaphorin repulsion regulates interactions between olfactory receptor neurons to help axons find their correct targets.     

Impact categories for life cycle assessment research of seafood production systems: Review and prospectus

Goal, Scope and Background In face of continued declines in global fisheries landings and concurrent rapid aquaculture development, the sustainability of seafood production is of increasing concern. Life Cycle Assessment (LCA) offers a convenient means of quantifying the impacts associated with many of the energetic and material inputs and outputs in these industries. However, the relevant but limited suite of impact categories currently used in most LCA research fails to capture a number of important environmental and social burdens unique to fisheries and aquaculture. This article reviews the impact categories used in published LCA research of seafood production to date, reports on a number of methodological innovations, and discusses the challenges to and opportunities for further impact category developments. Main Features The range of environmental and socio-economic impacts associated with fisheries and aquaculture production are introduced, and both the commonly used and innovative impact categories employed in published LCA research of seafood production are discussed. Methodological innovations reported in agricultural LCAs are also reviewed for possible applications to seafood LCA research. Challenges and options for including additional environmental and socioeconomic impact categories are explored. Results A review of published LCA research in fisheries and aquaculture indicates the frequent use of traditional environmental impact categories as well as a number of interesting departures from the standard suite of categories employed in LCA studies in other sectors. Notable examples include the modeling of benthic impacts, by-catch, emissions from anti-fouling paints, and the use of Net Primary Productivity appropriation to characterize biotic resource use. Socio-economic impacts have not been quantified, nor does a generally accepted methodology for their consideration exist. However, a number of potential frameworks for the integration of such impacts into LCA have been proposed. Discussion LCA analyses of fisheries and aquaculture call attention to an important range of environmental interactions that are usually not considered in discussions of sustainability in the seafood sector. These include energy use, biotic resource use, and the toxicity of anti-fouling paints. However, certain important impacts are also currently overlooked in such research. While prospects clearly exist for improving and expanding on recent additions to environmental impact categories, the nature of the LCA framework may preclude treatment of some of these impacts. Socio-economic impact categories have only been described in a qualitative manner. Despite a number of challenges, significant opportunities exist to quantify several important socio-economic impacts. Conclusion The limited but increasing volume of LCA research of industrial fisheries and aquaculture indicates a growing interest in the use of LCA methodology to understand and improve the sustainability performance of seafood production systems. Recent impact category innovations, and the potential for further impact category developments that account for several of the unique interactions characteristic of fisheries and aquaculture will significantly improve the usefulness of LCA in this context, although quantitative analysis of certain types of impacts may remain beyond the scope of the LCA framework. The desirability of incorporating socio-economic impacts is clear, but such integration will require considerable methodological development. Recommendations and Perspectives While the quantity of published LCA research for seafood production systems is clearly increasing, the influence this research will have on the ground remains to be seen. In part, this will depend on the ability of LCA researchers to advance methodological innovations that enable consideration of a broader range of impacts specific to seafood production. It will also depend on the ability of researchers to communicate with a broader audience than the currently narrow LCA community.     

A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control

The heterodimeric Na,K-ATPase has been implicated in vertebrate and invertebrate epithelial cell junctions, morphogenesis and oncogenesis, but the mechanisms involved are unclear. We previously showed that the Drosophila Na,K-ATPase is required for septate junction (SJ) formation and that of the three beta-subunit loci, only Nrv2 isoforms support epithelial SJ barrier function and tracheal tube-size control. Here we show that Nrv1 is endogenously co-expressed with Nrv2 in the epidermis and tracheal system, but Nrv1 has a basolateral localization and appears to be excluded from the Nrv2-containing SJs. When the normally neuronal Nrv3 is expressed in epithelial cells, it does not associate with SJs. Thus, the beta-subunit is a key determinant of Na,K-ATPase subcellular localization as well as function. However, localization of the Na,K-ATPase to SJs is not sufficient for junctional activity because although several Nrv2/Nrv3 chimeric beta-subunits localize to SJs, only those containing the extracellular domain of Nrv2 have junctional activity. Junctional activity is also specific to different alpha-subunit isoforms, with only some isoforms from the major alpha-subunit locus being able to provide full barrier function and produce normal tracheal tubes. Importantly, mutations predicted to inactivate ATPalpha catalytic function do not compromise junctional activity, demonstrating that the Drosophila Na,K-ATPase has an ion-pump-independent role in junction formation and tracheal morphogenesis. These results define new functions for the intensively studied Na,K-ATPase. Strikingly, the rat alpha1 isoform has full junctional activity and can rescue Atpalpha-null mutants to viability, suggesting that the Na,K-ATPase has an evolutionarily conserved role in junction formation and function.     

Amateur boxing and risk of chronic traumatic brain injury: systematic review of observational studies

Objective To evaluate the risk of chronic traumatic brain injury from amateur boxing.Setting Secondary research performed by combination of sport physicians and clinical academics.Design, data sources, and methods Systematic review of observational studies in which chronic traumatic brain injury was defined as any abnormality on clinical neurological examination, psychometric testing, neuroimaging studies, and electroencephalography. Studies were identified through database (1950 to date) and bibliographic searches without language restrictions. Two reviewers extracted study characteristics, quality, and data, with adherence to a protocol developed from a widely recommended method for systematic review of observational studies (MOOSE).Results 36 papers had relevant extractable data (from a detailed evaluation of 93 studies of 943 identified from the initial search). Quality of evidence was generally poor. The best quality studies were those with a cohort design and those that used psychometric tests. These yielded the most negative results: only four of 17 (24%) better quality studies found any indication of chronic traumatic brain injury in a minority of boxers studied.Conclusion There is no strong evidence to associate chronic traumatic brain injury with amateur boxing.     

Phase studies of model biomembranes: Complex behavior of DSPC/DOPC/Cholesterol

We have undertaken a series of experiments to examine the behavior of individual components of cell membranes. Here we report an initial stage of these experiments, in which the properties of a chemically simple lipid mixture are carefully mapped onto a phase diagram. Four different experimental methods were used to establish the phase behavior of the 3-component mixture DSPC/DOPC/chol: (1) confocal fluorescence microscopy observation of giant unilamellar vesicles, GUVs; (2) FRET from perylene to C20:0-DiI; (3) fluorescence of dilute dyes C18:2-DiO and C20:0-DiI; and (4) wide angle X-ray diffraction. This particular 3-component mixture was chosen, in part, for a high level of immiscibility of the components in order to facilitate solving the phase behavior at all compositions. At 23 °C, a large fraction of the possible compositions for this mixture give rise to a solid phase. A region of 3-phase coexistence of {Lα + Lβ + Lo} was detected and defined based on a combination of fluorescence microscopy of GUVs, FRET, and dilute C20:0-DiI fluorescence. At very low cholesterol concentrations, the solid phase is the tilted-chain phase Lβ′. Most of the phase boundaries have been determined to be within a few percent of the composition. Measurements of the perturbations of the boundaries of this accurate phase diagram could serve as a means to understand the behaviors of a range of added lipids and proteins.     

An acetal-based PEGylation reagent for pH-sensitive shielding of DNA polyplexes

p-Piperazinobenzaldehyde methoxy poly(ethylene glycol) (mPEG, 5 kDa) acetal was synthesized by the Buchwald-Hartwig coupling reaction from piperazine and p-bromobenzaldehyde mPEG acetal. Introduction of a maleimide moiety yielded a novel acetal-based PEGylation reagent (PEG-acetal-MAL) for pH-sensitive conjugation of PEG to thiol-functionalized biomolecules. For reversible shielding of polyplexes, PEG-acetal-MAL was conjugated to polyethylenimine (PEI). At 37 degrees C, the PEG-acetal-PEI conjugate had a half-life of 3 min at endosomal pH 5.5 and 2 h at physiological pH 7.4, respectively. PEI polyplexes containing PEG-acetal-PEI had a zeta potential of +3 mV and were stable to salt-induced aggregation for 2 h at pH 7.4. In contrast, at endosomal pH, the particles were deshielded and aggregated within 0.5 h. Epidermal growth factor or transferrin receptor-targeted polyplexes shielded with the pH-sensitive PEG-acetal mediated enhanced luciferase gene expression in receptor-expressing target cells (Renca-EGFR or K562) as compared to stably shielded control polyplexes. Thus, the novel PEG-acetal-MAL reagent may present a versatile tool for drug and gene delivery formulations when pH-sensitive PEGylation is preferred.     

Structural analysis of flexible proteins in solution by small angle X-ray scattering combined with crystallography

In the last few years, SAXS of biological materials has been rapidly evolving and promises to move structural analysis to a new level. Recent innovations in SAXS data analysis allow ab initio shape predictions of proteins in solution. Furthermore, experimental scattering data can be compared to calculated scattering curves from the growing data base of solved structures and also identify aggregation and unfolded proteins. Combining SAXS results with atomic resolution structures enables detailed characterizations in solution of mass, radius, conformations, assembly, and shape changes associated with protein folding and functions. SAXS can efficiently reveal the spatial organization of protein domains, including domains missing from or disordered in known crystal structures, and establish cofactor or substrate-induced conformational changes. For flexible domains or unstructured regions that are not amenable for study by many other structural techniques, SAXS provides a unique technology. Here, we present SAXS shape predictions for PCNA that accurately predict a trimeric ring assembly and for a full-length DNA repair glycosylase with a large unstructured region. These new results in combination with illustrative published data show how SAXS combined with high resolution crystal structures efficiently establishes architectures, assemblies, conformations, and unstructured regions for proteins and protein complexes in solution.     

Spinal loading during manual materials handling in a kneeling posture.

Stooped, restricted, kneeling, and other awkward postures adopted during manual materials handling have frequently been associated with LBP onset. However, lift assessment tools have focused on materials handling performed in an upright, or nearly upright standing posture. Unfortunately, many of the tools designed to analyze standing postures are not easily adapted to jobs requiring restricted postures. Therefore, the objective of this study was to evaluate spinal loading during manual materials handing in kneeling postures and determine if those loads can be predicted using simple regression. An EMG-driven biomechanical model, previously validated for upright lifting, was adapted for use in kneeling tasks. Subjects knelt under a 1.07m ceiling and lifted luggage of six weights (6.8, 10.9, 15.0, 19.1, 23.1 and, 27.2kgf) to one of four destination heights (0, 25.4, 53.3, 78.7cm). Spine loading was significantly affected by both destination height and load weight. Destination height increased compression, AP shear and lateral shear by an average of 14.5, 3.7 and 6.6N respectively per cm height increase. Load weight increased compression, AP shear and lateral shear by an average of 83.8, 27.0 and 13.1N respectively per kgf lifted. Regression equations were developed to predict peak spine loading using subject height, load weight and destination height with R(2) values of 0.62, 0.51 and 0.57 for compression, AP and lateral shear respectively.