PNAS

Clinical Genomic Database.

Publication Date: 2013 May 21 PMID: 23696674
Authors: Solomon, B. D. - Nguyen, A. D. - Bear, K. A. - Wolfsberg, T. G.
Journal: Proc Natl Acad Sci U S A

Technological advances have greatly increased the availability of human genomic sequencing. However, the capacity to analyze genomic data in a clinically meaningful way lags behind the ability to generate such data. To help address this obstacle, we reviewed all conditions with genetic causes and constructed the Clinical Genomic Database (CGD) (http://research.nhgri.nih.gov/CGD/), a searchable, freely Web-accessible database of conditions based on the clinical utility of genetic diagnosis and the availability of specific medical interventions. The CGD currently includes a total of 2,616 genes organized clinically by affected organ systems and interventions (including preventive measures, disease surveillance, and medical or surgical interventions) that could be reasonably warranted by the identification of pathogenic mutations. To aid independent analysis and optimize new data incorporation, the CGD also includes all genetic conditions for which genetic knowledge may affect the selection of supportive care, informed medical decision-making, prognostic considerations, reproductive decisions, and allow avoidance of unnecessary testing, but for which specific interventions are not otherwise currently available. For each entry, the CGD includes the gene symbol, conditions, allelic conditions, clinical categorization (for both manifestations and interventions), mode of inheritance, affected age group, description of interventions/rationale, links to other complementary databases, including databases of variants and presumed pathogenic mutations, and links to PubMed references (>20,000). The CGD will be regularly maintained and updated to keep pace with scientific discovery. Further content-based expert opinions are actively solicited. Eventually, the CGD may assist the rapid curation of individual genomes as part of active medical care.

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Multispot, label-free biodetection at a phantom plastic-water interface.

Publication Date: 2013 May 21 PMID: 23696673
Authors: Giavazzi, F. - Salina, M. - Cerbino, R. - Bassi, M. - Prosperi, D. - Ceccarello, E. - Damin, F. - Sola, L. - Rusnati, M. - Chiari, M. - Chini, B. - Bellini, T. - Buscaglia, M.
Journal: Proc Natl Acad Sci U S A

Recognizing and quantifying specific biomolecules in aqueous samples are constantly needed in research and diagnostic laboratories. As the typical detection procedures are rather lengthy and involve the use of labeled secondary antibodies or other agents to provide a signal, efforts have been made over the last 10 y to develop alternative label-free methods that enable direct detection. We propose and demonstrate an extremely simple, low-cost, label-free biodetector based on measuring the intensity of light reflected by the interface between a fluid sample and an amorphous fluoropolymer substrate having a refractive index very close to that of water and hosting various antibodies immobilized in spots. Under these index-matching conditions, the amount of light reflected by the interface allows straightforward quantification of the amount of antigen binding to each spot. Using antibodies targeting heterologous immunoglobulins and antigens commonly used as markers for diagnoses of hepatitis B and HIV, we demonstrate the limit of detection of a few picograms per square millimeter of surface-bound molecules. We also show that direct and real-time access to the amount of binding molecules allows the precise extrapolation of adhesion rates, from which the concentrations of antigens in solution can be estimated down to fractions of nanograms per milliliter.

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Mosaic genome structure of the barley powdery mildew pathogen and conservation of transcriptional programs in divergent hosts.

Publication Date: 2013 May 21 PMID: 23696672
Authors: Hacquard, S. - Kracher, B. - Maekawa, T. - Vernaldi, S. - Schulze-Lefert, P. - Ver Loren van Themaat, E.
Journal: Proc Natl Acad Sci U S A

Barley powdery mildew, Blumeria graminis f. sp. hordei (Bgh), is an obligate biotrophic ascomycete fungal pathogen that can grow and reproduce only on living cells of wild or domesticated barley (Hordeum sp.). Domestication and deployment of resistant barley cultivars by humans selected for amplification of Bgh isolates with different virulence combinations. We sequenced the genomes of two European Bgh isolates, A6 and K1, for comparative analysis with the reference genome of isolate DH14. This revealed a mosaic genome structure consisting of large isolate-specific DNA blocks with either high or low SNP densities. Some of the highly polymorphic blocks likely accumulated SNPs for over 10,000 years, well before the domestication of barley. These isolate-specific blocks of alternating monomorphic and polymorphic regions imply an exceptionally large standing genetic variation in the Bgh population and might be generated and maintained by rare outbreeding and frequent clonal reproduction. RNA-sequencing experiments with isolates A6 and K1 during four early stages of compatible and incompatible interactions on leaves of partially immunocompromised Arabidopsis mutants revealed a conserved Bgh transcriptional program during pathogenesis compared with the natural host barley despite approximately 200 million years of reproductive isolation of these hosts. Transcripts encoding candidate-secreted effector proteins are massively induced in successive waves. A specific decrease in candidate-secreted effector protein transcript abundance in the incompatible interaction follows extensive transcriptional reprogramming of the host transcriptome and coincides with the onset of localized host cell death, suggesting a host-inducible defense mechanism that targets fungal effector secretion or production.

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Blast resistance of CC-NB-LRR protein Pb1 is mediated by WRKY45 through protein-protein interaction.

Publication Date: 2013 May 21 PMID: 23696671
Authors: Inoue, H. - Hayashi, N. - Matsushita, A. - Xinqiong, L. - Nakayama, A. - Sugano, S. - Jiang, C. J. - Takatsuji, H.
Journal: Proc Natl Acad Sci U S A

Panicle blast 1 (Pb1) is a panicle blast resistance gene derived from the indica rice cultivar "Modan." Pb1 encodes a coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NB-LRR) protein and confers durable, broad-spectrum resistance to Magnaporthe oryzae races. Here, we investigated the molecular mechanisms underlying Pb1-mediated blast resistance. The Pb1 protein interacted with WRKY45, a transcription factor involved in induced resistance via the salicylic acid signaling pathway that is regulated by the ubiquitin proteasome system. Pb1-mediated panicle blast resistance was largely compromised when WRKY45 was knocked down in a Pb1-containing rice cultivar. Leaf-blast resistance by Pb1 overexpression (Pb1-ox) was also compromised in WRKY45 knockdown/Pb1-ox rice. Blast infection induced higher accumulation of WRKY45 in Pb1-ox than in control Nipponbare rice. Overexpression of Pb1-Quad, a coiled-coil domain mutant that had weak interaction with WRKY45, resulted in significantly weaker blast resistance than that of wild-type Pb1. Overexpression of Pb1 with a nuclear export sequence failed to confer blast resistance to rice. These results suggest that the blast resistance of Pb1 depends on its interaction with WRKY45 in the nucleus. In a transient system using rice protoplasts, coexpression of Pb1 enhanced WRKY45 accumulation and increased WRKY45-dependent transactivation activity, suggesting that protection of WRKY45 from ubiquitin proteasome system degradation is possibly involved in Pb1-dependent blast resistance.

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Correction for Blehm et al., In vivo optical trapping indicates kinesin's stall force is reduced by dynein during intracellular transport.

Publication Date: 2013 May 21 PMID: 23696670
Authors:
Journal: Proc Natl Acad Sci U S A

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Rise and fall of competitiveness in individualistic and collectivistic societies.

Publication Date: 2013 May 21 PMID: 23696669
Authors: Leibbrandt, A. - Gneezy, U. - List, J. A.
Journal: Proc Natl Acad Sci U S A

Competitiveness pervades life: plants compete for sunlight and water, animals for territory and food, and humans for mates and income. Herein we investigate human competitiveness with a natural experiment and a set of behavioral experiments. We compare competitiveness in traditional fishing societies where local natural forces determine whether fishermen work in isolation or in collectives. We find sharp evidence that fishermen from individualistic societies are far more competitive than fishermen from collectivistic societies, and that this difference emerges with work experience. These findings suggest that humans can evolve traits to specific needs, support the idea that socio-ecological factors play a decisive role for individual competitiveness, and provide evidence how individualistic and collectivistic societies shape economic behavior.

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The receptor for complement component C3a mediates protection from intestinal ischemia-reperfusion injuries by inhibiting neutrophil mobilization.

Publication Date: 2013 May 21 PMID: 23696668
Authors: Wu, M. C. - Brennan, F. H. - Lynch, J. P. - Mantovani, S. - Phipps, S. - Wetsel, R. A. - Ruitenberg, M. J. - Taylor, S. M. - Woodruff, T. M.
Journal: Proc Natl Acad Sci U S A

C3a is a key complement activation fragment, yet its neutrophil-expressed receptor (C3aR) still has no clearly defined role. In this study, we used a neutrophil-dependent mouse model of intestinal ischemia-reperfusion (IR) injury to explore the role of C3aR in acute tissue injuries. C3aR deficiency worsened intestinal injury, which corresponded with increased numbers of tissue-infiltrating neutrophils. Circulating neutrophils were significantly increased in C3aR-/- mice after intestinal ischemia, and C3aR-/- mice also mobilized more circulating neutrophils after granulocyte colony-stimulating factor infusion compared with WT mice, indicating a specific role for C3aR in constraining neutrophil mobilization in response to intestinal injury. In support of this role, C3aR-/- mice reconstituted with WT bone marrow reversed IR pathology back to WT levels. Complement C5a receptor (C5aR) antagonism in C3aR-/- mice also rectified the worsened pathology after intestinal IR injury but had no effect on circulating neutrophils, highlighting the opposing roles of C3a and C5a in disease pathogenesis. Finally, we found that using a potent C3a agonist to activate C3aR in vivo reduced neutrophil mobilization and ameliorated intestinal IR pathology in WT, but not C3aR-/-, mice. This study identifies a role for C3aR in regulating neutrophil mobilization after acute intestinal injury and highlights C3aR agonism as a potential treatment option for acute, neutrophil-driven pathologies.

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Single-membrane-bounded peroxisome division revealed by isolation of dynamin-based machinery.

Publication Date: 2013 May 21 PMID: 23696667
Authors: Imoto, Y. - Kuroiwa, H. - Yoshida, Y. - Ohnuma, M. - Fujiwara, T. - Yoshida, M. - Nishida, K. - Yagisawa, F. - Hirooka, S. - Miyagishima, S. Y. - Misumi, O. - Kawano, S. - Kuroiwa, T.
Journal: Proc Natl Acad Sci U S A

Peroxisomes (microbodies) are ubiquitous single-membrane-bounded organelles and fulfill essential roles in the cellular metabolism. They are found in virtually all eukaryotic cells and basically multiply by division. However, the mechanochemical machinery involved in peroxisome division remains elusive. Here, we first identified the peroxisome-dividing (POD) machinery. We isolated the POD machinery from Cyanidioschyzon merolae, a unicellular red alga containing a single peroxisome. Peroxisomal division in C. merolae can be highly synchronized by light/dark cycles and the microtubule-disrupting agent oryzalin. By proteomic analysis based on the complete genome sequence of C. merolae, we identified a dynamin-related protein 3 (DRP3) ortholog, CmDnm1 (Dnm1), that predominantly accumulated with catalase in the dividing-peroxisome fraction. Immunofluorescence microscopy demonstrated that Dnm1 formed a ring at the division site of the peroxisome. The outlines of the isolated dynamin rings were dimly observed by phase-contrast microscopy and clearly stained for Dnm1. Electron microscopy revealed that the POD machinery was formed at the cytoplasmic side of the equator. Immunoelectron microscopy showed that the POD machinery consisted of an outer dynamin-based ring and an inner filamentous ring. Down-regulation of Dnm1 impaired peroxisomal division. Surprisingly, the same Dnm1 serially controlled peroxisomal division after mitochondrial division. Because genetic deficiencies of Dnm1 orthologs in multiperoxisomal organisms inhibited both mitochondrial and peroxisomal proliferation, it is thought that peroxisomal division by contraction of a dynamin-based machinery is universal among eukaryotes. These findings are useful for understanding the fundamental systems in eukaryotic cells.

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Spatiotemporal dynamics of neuronal population response in the primary visual cortex.

Publication Date: 2013 May 21 PMID: 23696666
Authors: Zhou, D. - Rangan, A. V. - McLaughlin, D. W. - Cai, D.
Journal: Proc Natl Acad Sci U S A

One of the fundamental questions in system neuroscience is how the brain encodes external stimuli in the early sensory cortex. It has been found in experiments that even some simple sensory stimuli can activate large populations of neurons. It is believed that information can be encoded in the spatiotemporal profile of these collective neuronal responses. Here, we use a large-scale computational model of the primary visual cortex (V1) to study the population responses in V1 as observed in experiments in which monkeys performed visual detection tasks. We show that our model can capture very well spatiotemporal activities measured by voltage-sensitive-dye-based optical imaging in V1 of the awake state. In our model, the properties of horizontal long-range connections with NMDA conductance play an important role in the correlated population responses and have strong implications for spatiotemporal coding of neuronal populations. Our computational modeling approach allows us to reveal intrinsic cortical dynamics, separating them from those statistical effects arising from averaging procedures in experiment. For example, in experiments, it was shown that there was a spatially antagonistic center-surround structure in optimal weights in signal detection theory, which was believed to underlie the efficiency of population coding. However, our study shows that this feature is an artifact of data processing.

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Development of cortical microstructure in the preterm human brain.

Publication Date: 2013 May 21 PMID: 23696665
Authors: Ball, G. - Srinivasan, L. - Aljabar, P. - Counsell, S. J. - Durighel, G. - Hajnal, J. V. - Rutherford, M. A. - Edwards, A. D.
Journal: Proc Natl Acad Sci U S A

Cortical maturation was studied in 65 infants between 27 and 46 wk postconception using structural and diffusion magnetic resonance imaging. Alterations in neural structure and complexity were inferred from changes in mean diffusivity and fractional anisotropy, analyzed by sampling regions of interest and also by a unique whole-cortex mapping approach. Mean diffusivity was higher in gyri than sulci and in frontal compared with occipital lobes, decreasing consistently throughout the study period. Fractional anisotropy declined until 38 wk, with initial values and rates of change higher in gyri, frontal and temporal poles, and parietal cortex; and lower in sulcal, perirolandic, and medial occipital cortex. Neuroanatomical studies and experimental diffusion-anatomic correlations strongly suggested the interpretation that cellular and synaptic complexity and density increased steadily throughout the period, whereas elongation and branching of dendrites orthogonal to cortical columns was later and faster in higher-order association cortex, proceeding rapidly before becoming undetectable after 38 wk. The rate of microstructural maturation correlated locally with cortical growth, and predicted higher neurodevelopmental test scores at 2 y of age. Cortical microstructural development was reduced in a dose-dependent fashion by longer premature exposure to the extrauterine environment, and preterm infants at term-corrected age possessed less mature cortex than term-born infants. The results are compatible with predictions of the tension theory of cortical growth and show that rapidly developing cortical microstructure is vulnerable to the effects of premature birth, suggesting a mechanism for the adverse effects of preterm delivery on cognitive function.

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Glutathione and tryptophan metabolism are required for Arabidopsis immunity during the hypersensitive response to hemibiotrophs.

Publication Date: 2013 May 21 PMID: 23696664
Authors: Hiruma, K. - Fukunaga, S. - Bednarek, P. - Pislewska-Bednarek, M. - Watanabe, S. - Narusaka, Y. - Shirasu, K. - Takano, Y.
Journal: Proc Natl Acad Sci U S A

The hypersensitive response (HR) is a type of strong immune response found in plants that is accompanied by localized cell death. However, it is unclear how HR can block a broad range of pathogens with different infective modes. In this study, we report that gamma-glutamylcysteine synthetase GSH1, which is critical for glutathione biosynthesis, and tryptophan (Trp) metabolism contribute to HR and block development of fungal pathogens with hemibiotrophic infective modes. We found that GSH1 is involved in the penetration2 (PEN2)-based entry control of the nonadapted hemibiotroph Colletotrichum gloeosporioides. However, Arabidopsis mutants specifically defective in entry control terminated further growth of the pathogen in the presence of HR cell death, whereas gsh1 mutants supported pathogen invasive growth in planta, demonstrating the requirement of GSH1 for postinvasive nonhost resistance. Remarkably, on the basis of the phenotypic and metabolic analysis of Arabidopsis mutants defective in Trp metabolism, we showed that biosynthesis of Trp-derived phytochemicals is also essential for resistance to C. gloeosporioides during postinvasive HR. By contrast, GSH1 and these metabolites are likely to be dispensable for the induction of cell death during postinvasive HR. Furthermore, the resistance to Ralstonia solanacearum 1/resistance to Pseudomonas syringae 4 dual Resistance gene-dependent immunity of Arabidopsis to the adapted hemibiotroph shared GSH1 and cytochromes P450 CYP79B2/CYP79B3 with postinvasive nonhost resistance, whereas resistance to P. syringae pv. maculicola 1 and resistance to P. syringae 2-based Resistance gene resistance against bacterial pathogens did not. These data suggest that the synthesis of glutathione and Trp-derived metabolites during HR play crucial roles in terminating the invasive growth of both nonadapted and adapted hemibiotrophs.

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Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells.

Publication Date: 2013 May 21 PMID: 23696663
Authors: Pennarossa, G. - Maffei, S. - Campagnol, M. - Tarantini, L. - Gandolfi, F. - Brevini, T. A.
Journal: Proc Natl Acad Sci U S A

The differentiated state of mature cells of adult organisms is achieved and maintained through the epigenetic regulation of gene expression, which consists of several mechanisms including DNA methylation. The advent of induced pluripotent stem cell technology enabled the conversion of adult cells into any other cell type passing through a stable pluripotency state. However, indefinite pluripotency is unphysiological, inherently labile, and makes cells prone to culture-induced alterations. The direct conversion of one cell type to another without an intermediate pluripotent stage is also possible but, at present, requires the viral transfection of appropriate transcription factors, limiting its therapeutic potential. The aim of this study was to investigate whether it is possible to achieve the direct conversion of an adult cell by exposing it to a demethylating agent immediately followed by differentiating culture conditions. Adult human skin fibroblasts were exposed for 18 h to the DNA methyltransferase inhibitor 5-azacytidine, followed by a three-step protocol for the induction of endocrine pancreatic differentiation that lasted 36 d. At the end of this treatment, 35 +/- 8.9% fibroblasts became pancreatic converted cells that acquired an epithelial morphology, produced insulin, and then released the hormone in response to a physiological glucose challenge in vitro. Furthermore, pancreatic converted cells were able to protect recipient mice against streptozotocin-induced diabetes, restoring a physiological response to glucose tolerance tests. This work shows that it is possible to convert adult fibroblasts into insulin-secreting cells, avoiding both a stable pluripotent stage and any transgenic modification.

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HIV-1 exploits CCR5 conformational heterogeneity to escape inhibition by chemokines.

Publication Date: 2013 May 21 PMID: 23696662
Authors: Colin, P. - Benureau, Y. - Staropoli, I. - Wang, Y. - Gonzalez, N. - Alcami, J. - Hartley, O. - Brelot, A. - Arenzana-Seisdedos, F. - Lagane, B.
Journal: Proc Natl Acad Sci U S A

CC chemokine receptor 5 (CCR5) is a receptor for chemokines and the coreceptor for R5 HIV-1 entry into CD4+ T lymphocytes. Chemokines exert anti-HIV-1 activity in vitro, both by displacing the viral envelope glycoprotein gp120 from binding to CCR5 and by promoting CCR5 endocytosis, suggesting that they play a protective role in HIV infection. However, we showed here that different CCR5 conformations at the cell surface are differentially engaged by chemokines and gp120, making chemokines weaker inhibitors of HIV infection than would be expected from their binding affinity constants for CCR5. These distinct CCR5 conformations rely on CCR5 coupling to nucleotide-free G proteins (NFG proteins). Whereas native CCR5 chemokines bind with subnanomolar affinity to NFG protein-coupled CCR5, gp120/HIV-1 does not discriminate between NFG protein-coupled and uncoupled CCR5. Interestingly, the antiviral activity of chemokines is G protein independent, suggesting that "low-chemokine affinity" NFG protein-uncoupled conformations of CCR5 represent a portal for viral entry. Furthermore, chemokines are weak inducers of CCR5 endocytosis, as is revealed by EC50 values for chemokine-mediated endocytosis reflecting their low-affinity constant value for NFG protein-uncoupled CCR5. Abolishing CCR5 interaction with NFG proteins eliminates high-affinity binding of CCR5 chemokines but preserves receptor endocytosis, indicating that chemokines preferentially endocytose low-affinity receptors. Finally, we evidenced that chemokine analogs achieve highly potent HIV-1 inhibition due to high-affinity interactions with internalizing and/or gp120-binding receptors. These data are consistent with HIV-1 evading chemokine inhibition by exploiting CCR5 conformational heterogeneity, shed light into the inhibitory mechanisms of anti-HIV-1 chemokine analogs, and provide insights for the development of unique anti-HIV molecules.

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Founder effects initiated rapid species radiation in Hawaiian cave planthoppers.

Publication Date: 2013 May 21 PMID: 23696661
Authors: Wessel, A. - Hoch, H. - Asche, M. - von Rintelen, T. - Stelbrink, B. - Heck, V. - Stone, F. D. - Howarth, F. G.
Journal: Proc Natl Acad Sci U S A

The Hawaiian Islands provide the venue of one of nature's grand experiments in evolution. Here, we present morphological, behavioral, genetic, and geologic data from a young subterranean insect lineage in lava tube caves on Hawai'i Island. The Oliarus polyphemus species complex has the potential to become a model for studying rapid speciation by stochastic events. All species in this lineage live in extremely similar environments but show strong differentiation in behavioral and morphometric characters, which are random with respect to cave age and geographic distribution. Our observation that phenotypic variability within populations decreases with increasing cave age challenges traditional views on founder effects. Furthermore, these cave populations are natural replicates that can be used to test the contradictory hypotheses. Moreover, Hawaiian cave planthoppers exhibit one of the highest speciation rates among animals and, thus, radically shift our perception on the evolutionary potential of obligate cavernicoles.

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Microbiota-induced activation of epithelial IL-6 signaling links inflammasome-driven inflammation with transmissible cancer.

Publication Date: 2013 May 21 PMID: 23696660
Authors: Hu, B. - Elinav, E. - Huber, S. - Strowig, T. - Hao, L. - Hafemann, A. - Jin, C. - Eisenbarth, S. C. - Flavell, R. A.
Journal: Proc Natl Acad Sci U S A

The microbiota is pivotal in the pathogenesis of inflammatory bowel disease (IBD)-associated inflammation-induced colorectal cancer (CRC), yet mechanisms for these effects remain poorly characterized. Here, we demonstrate that aberrant inflammasome-induced microbiota plays a critical role in CRC development, where mice deficient in the NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammasome feature enhanced inflammation-induced CRC formation. Intriguingly, WT mice cohoused either with inflammasome-deficient mice or with mice lacking IL-18 feature exacerbated inflammation-induced CRC compared with singly housed WT mice. Enhanced tumorigenesis is dependent on microbiota-induced chemokine (C-C motif) ligand 5 (CCL5)-driven inflammation, which in turn promotes epithelial cell proliferation through local activation of the IL-6 pathway, leading to cancer formation. Altogether, our results mechanistically link the altered microbiota with the pathogenesis of inflammation-induced CRC and suggest that in some conditions, microbiota components may transfer CRC susceptibility between individuals.

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Genome encapsidation by orthobunyavirus nucleoproteins.

Publication Date: 2013 May 21 PMID: 23696659
Authors: Zheng, W. - Tao, Y. J.
Journal: Proc Natl Acad Sci U S A

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Country-level operational implementation of the Global Plan for Insecticide Resistance Management.

Publication Date: 2013 May 21 PMID: 23696658
Authors: Hemingway, J. - Vontas, J. - Poupardin, R. - Raman, J. - Lines, J. - Schwabe, C. - Matias, A. - Kleinschmidt, I.
Journal: Proc Natl Acad Sci U S A

Malaria control is reliant on the use of long-lasting pyrethroid-impregnated nets and/or indoor residual spraying (IRS) of insecticide. The rapid selection and spread of operationally significant pyrethroid resistance in African malaria vectors threatens our ability to sustain malaria control. Establishing whether resistance is operationally significant is technically challenging. Routine monitoring by bioassay is inadequate, and there are limited data linking resistance selection with changes in disease transmission. The default is to switch insecticides when resistance is detected, but limited insecticide options and resistance to multiple insecticides in numerous locations make this approach unsustainable. Detailed analysis of the resistance situation in Anopheles gambiae on Bioko Island after pyrethroid resistance was detected in this species in 2004, and the IRS program switched to carbamate bendiocarb, has now been undertaken. The pyrethroid resistance selected is a target-site knock-down resistance kdr-form, on a background of generally elevated metabolic activity, compared with insecticide-susceptible A. gambiae, but the major cytochrome P450-based metabolic pyrethroid resistance mechanisms are not present. The available evidence from bioassays and infection data suggests that the pyrethroid resistance mechanisms in Bioko malaria vectors are not operationally significant, and on this basis, a different, long-lasting pyrethroid formulation is now being reintroduced for IRS in a rotational insecticide resistance management program. This will allow control efforts to be sustained in a cost-effective manner while reducing the selection pressure for resistance to nonpyrethroid insecticides. The methods used provide a template for evidence-based insecticide resistance management by malaria control programs.

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Heat transport in bubbling turbulent convection.

Publication Date: 2013 May 21 PMID: 23696657
Authors: Lakkaraju, R. - Stevens, R. J. - Oresta, P. - Verzicco, R. - Lohse, D. - Prosperetti, A.
Journal: Proc Natl Acad Sci U S A

Boiling is an extremely effective way to promote heat transfer from a hot surface to a liquid due to numerous mechanisms, many of which are not understood in quantitative detail. An important component of the overall process is that the buoyancy of the bubble compounds with that of the liquid to give rise to a much-enhanced natural convection. In this article, we focus specifically on this enhancement and present a numerical study of the resulting two-phase Rayleigh-Benard convection process in a cylindrical cell with a diameter equal to its height. We make no attempt to model other aspects of the boiling process such as bubble nucleation and detachment. The cell base and top are held at temperatures above and below the boiling point of the liquid, respectively. By keeping this difference constant, we study the effect of the liquid superheat in a Rayleigh number range that, in the absence of boiling, would be between 2 x 106 and 5 x 109. We find a considerable enhancement of the heat transfer and study its dependence on the number of bubbles, the degree of superheat of the hot cell bottom, and the Rayleigh number. The increased buoyancy provided by the bubbles leads to more energetic hot plumes detaching from the cell bottom, and the strength of the circulation in the cell is significantly increased. Our results are in general agreement with recent experiments on boiling Rayleigh-Benard convection.

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PNAS Plus Significance Statements.

Publication Date: 2013 May 21 PMID: 23696656
Authors:
Journal: Proc Natl Acad Sci U S A

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Metamaterials.

Publication Date: 2013 May 21 PMID: 23696655
Authors: Ornes, S.
Journal: Proc Natl Acad Sci U S A

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Three-dimensional viewing in the deep sea.

Publication Date: 2013 May 21 PMID: 23696654
Authors: Schrope, M.
Journal: Proc Natl Acad Sci U S A

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Radiated relics.

Publication Date: 2013 May 21 PMID: 23696653
Authors: Horne, R.
Journal: Proc Natl Acad Sci U S A

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Fratricide of natural killer cells dressed with tumor-derived NKG2D ligand.

Publication Date: 2013 May 20 PMID: 23690625
Authors: Nakamura, K. - Nakayama, M. - Kawano, M. - Amagai, R. - Ishii, T. - Harigae, H. - Ogasawara, K.
Journal: Proc Natl Acad Sci U S A

The natural killer group 2 membrane D (NKG2D) activating receptor plays crucial roles not only in host defense against tumors and viral infections, but also in autoimmune diseases. After NKG2D-mediated activation, Natural killer (NK) cells must be regulated to avoid potentially harmful reactivity. However, the negative regulation of these activated NK cells is poorly understood. Here, we reveal that the engagement of NKG2D by its ligand elicits not only target cell lysis, but also NK cell fratricide. Conventional mouse NK cells underwent cell death when cocultured with RMA cells expressing the NKG2D ligand retinoic acid early-inducible protein 1 (Rae-1), but not with RMA cells lacking MHC class I. NK cells from mice deficient for DAP10 and DAP12 or perforin did not undergo death, highlighting the importance of the NKG2D pathway for NK cell death. However, NKG2D does not transmit direct death signals in NK cells. Rather, the interaction between NKG2D and Rae-1 allowed NK cells to acquire tumor-derived Rae-1 by a membrane transfer process known as "trogocytosis," which was associated with clathrin-dependent NKG2D endocytosis. NK cells dressed with Rae-1 were lysed by neighboring NK cells through the NKG2D-induced perforin pathway in vitro and in vivo. These results provide the unique NKG2D function in negative regulation of activated NK cells.

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Macrophages are required for adult salamander limb regeneration.

Publication Date: 2013 May 20 PMID: 23690624
Authors: Godwin, J. W. - Pinto, A. R. - Rosenthal, N. A.
Journal: Proc Natl Acad Sci U S A

The failure to replace damaged body parts in adult mammals results from a muted growth response and fibrotic scarring. Although infiltrating immune cells play a major role in determining the variable outcome of mammalian wound repair, little is known about the modulation of immune cell signaling in efficiently regenerating species such as the salamander, which can regrow complete body structures as adults. Here we present a comprehensive analysis of immune signaling during limb regeneration in axolotl, an aquatic salamander, and reveal a temporally defined requirement for macrophage infiltration in the regenerative process. Although many features of mammalian cytokine/chemokine signaling are retained in the axolotl, they are more dynamically deployed, with simultaneous induction of inflammatory and anti-inflammatory markers within the first 24 h after limb amputation. Systemic macrophage depletion during this period resulted in wound closure but permanent failure of limb regeneration, associated with extensive fibrosis and disregulation of extracellular matrix component gene expression. Full limb regenerative capacity of failed stumps was restored by reamputation once endogenous macrophage populations had been replenished. Promotion of a regeneration-permissive environment by identification of macrophage-derived therapeutic molecules may therefore aid in the regeneration of damaged body parts in adult mammals.

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Regulation of T cell function by the ubiquitin-specific protease USP9X via modulating the Carma1-Bcl10-Malt1 complex.

Publication Date: 2013 May 20 PMID: 23690623
Authors: Park, Y. - Jin, H. S. - Liu, Y. C.
Journal: Proc Natl Acad Sci U S A

The ubiquitin conjugation system plays an important role in immune regulation; however, the ubiquitin-specific proteases (USPs) that carry out deubiquitination of cellular substrates are poorly understood. Here we show that in vivo knockdown of the deubiquitinating enzyme USP9X attenuates T-cell proliferation. In addition, naive CD4+ T cells from USP9X knockdown chimeric mice display decreased cytokine production and T helper cell differentiation in vitro, which we confirmed in vivo by performing adoptive transfer of transgenic T cells and subsequent immunization. USP9X silencing in both a human T-cell line and mouse primary T cells reduced T-cell receptor (TCR) signaling-induced NF-kappaB activation. Mechanistically, USP9X interacts with Bcl10 of the Carma1-Bcl10-Malt1 (CBM) complex and removes the TCR-induced ubiquitin chain from Bcl10, which facilitates the association of Carma1 with Bcl0-Malt1. These results demonstrate that USP9X is a crucial positive regulator of the TCR signaling pathway and is required for T-cell function through the modulation of CBM complex formation.

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Methionine oxidation activates a transcription factor in response to oxidative stress.

Publication Date: 2013 May 20 PMID: 23690622
Authors: Drazic, A. - Miura, H. - Peschek, J. - Le, Y. - Bach, N. C. - Kriehuber, T. - Winter, J.
Journal: Proc Natl Acad Sci U S A

Oxidant-mediated antibacterial response systems are broadly used to control bacterial proliferation. Hypochlorite (HOCl) is an important component of the innate immune system produced in neutrophils and specific epithelia. Its antimicrobial activity is due to damaging cellular macromolecules. Little is known about how bacteria escape HOCl-inflicted damage. Recently, the transcription factor YjiE was identified that specifically protects Escherichia coli from HOCl killing. According to its function, YjiE is now renamed HypT (hypochlorite-responsive transcription factor). Here we unravel that HypT is activated by methionine oxidation to methionine sulfoxide. Interestingly, so far only inactivation of cellular proteins by methionine oxidation has been reported. Mutational analysis revealed three methionines that are essential to confer HOCl resistance. Their simultaneous substitution by glutamine, mimicking the methionine sulfoxide state, increased the viability of E. coli cells upon HOCl stress. Triple glutamine substitution generates a constitutively active HypT that regulates target genes independently of HOCl stress and permanently down-regulates intracellular iron levels. Inactivation of HypT depends on the methionine sulfoxide reductases A/B. Thus, microbial protection mechanisms have evolved along the evolution of antimicrobial control systems, allowing bacteria to survive within the host environment.

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Interplay of physics and evolution in the likely origin of protein biochemical function.

Publication Date: 2013 May 20 PMID: 23690621
Authors: Skolnick, J. - Gao, M.
Journal: Proc Natl Acad Sci U S A

The intrinsic ability of protein structures to exhibit the geometric and sequence properties required for ligand binding without evolutionary selection is shown by the coincidence of the properties of pockets in native, single domain proteins with those in computationally generated, compact homopolypeptide, artificial (ART) structures. The library of native pockets is covered by a remarkably small number of representative pockets ( approximately 400), with virtually every native pocket having a statistically significant match in the ART library, suggesting that the library is complete. When sequences are selected for ART structures based on fold stability, pocket sequence conservation is coincident to native. The fact that structurally and sequentially similar pockets occur across fold classes combined with the small number of representative pockets in native proteins implies that promiscuous interactions are inherent to proteins. Based on comparison of PDB (real, single domain protein structures found in the Protein Data Bank) and ART structures and pockets, the widespread assumption that the co-occurrence of global structure, pocket similarity, and amino acid conservation demands an evolutionary relationship between proteins is shown to significantly underestimate the random background probability. Indeed, many features of biochemical function arise from the physical properties of proteins that evolution likely fine-tunes to achieve specificity. Finally, our study suggests that a repertoire of thermodynamically (marginally) stable proteins could engage in many of the biochemical reactions needed for living systems without selection for function, a conclusion with significant implications for the origin of life.

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Ninjurin1, a target of p53, regulates p53 expression and p53-dependent cell survival, senescence, and radiation-induced mortality.

Publication Date: 2013 May 20 PMID: 23690620
Authors: Cho, S. J. - Rossi, A. - Jung, Y. S. - Yan, W. - Liu, G. - Zhang, J. - Zhang, M. - Chen, X.
Journal: Proc Natl Acad Sci U S A

The tumor suppressor protein p53 plays a crucial role in coordinating cellular processes, such as cell cycle arrest, apoptosis, and senescence. The nerve injury-induced protein 1 (Ninjurin1, Ninj1) is a homophilic adhesion molecule and involved in nerve regeneration. Interestingly, Ninj1 is found to be overexpressed in human cancer, but its role in tumorigenesis is not clear. Here, we found that Ninj1 is transcriptionally regulated by p53 and can be induced by DNA damage in a p53-dependent manner. We also found that knockout or knockdown of Ninj1 increases p53 expression potentially through enhanced p53 mRNA translation. In addition, we found that Ninj1 deficiency suppresses cell proliferation but enhances apoptosis and premature senescence in a p53-dependent manner. Consistent with this, we found that mice heterozygous in ninj1 are hypersensitive to ionizing radiation-induced lethality, along with increased expression of p53 in thymus. Taken together, we provided evidence that Ninj1 is a p53 target and modulates p53 mRNA translation and p53-dependent premature senescence, cell proliferation, apoptosis, and radiation-induced mortality in vitro and in vivo. Thus, we postulate that as a membrane adhesion molecule, Ninj1 is an ideal target to regulate p53 activity via the p53-Ninj1 loop.

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Brain homogenates from human tauopathies induce tau inclusions in mouse brain.

Publication Date: 2013 May 20 PMID: 23690619
Authors: Clavaguera, F. - Akatsu, H. - Fraser, G. - Crowther, R. A. - Frank, S. - Hench, J. - Probst, A. - Winkler, D. T. - Reichwald, J. - Staufenbiel, M. - Ghetti, B. - Goedert, M. - Tolnay, M.
Journal: Proc Natl Acad Sci U S A

Filamentous inclusions made of hyperphosphorylated tau are characteristic of numerous human neurodegenerative diseases, including Alzheimer's disease, tangle-only dementia, Pick disease, argyrophilic grain disease (AGD), progressive supranuclear palsy, and corticobasal degeneration. In Alzheimer's disease and AGD, it has been shown that filamentous tau appears to spread in a stereotypic manner as the disease progresses. We previously demonstrated that the injection of brain extracts from human mutant P301S tau-expressing transgenic mice into the brains of mice transgenic for wild-type human tau (line ALZ17) resulted in the assembly of wild-type human tau into filaments and the spreading of tau inclusions from the injection sites to anatomically connected brain regions. Here we injected brain extracts from humans who had died with various tauopathies into the hippocampus and cerebral cortex of ALZ17 mice. Argyrophilic tau inclusions formed in all cases and following the injection of the corresponding brain extracts, we recapitulated the hallmark lesions of AGD, PSP and CBD. Similar inclusions also formed after intracerebral injection of brain homogenates from human tauopathies into nontransgenic mice. Moreover, the induced formation of tau aggregates could be propagated between mouse brains. These findings suggest that once tau aggregates have formed in discrete brain areas, they become self-propagating and spread in a prion-like manner.

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Recruitment and remodeling of an ancient gene regulatory network during land plant evolution.

Publication Date: 2013 May 20 PMID: 23690618
Authors: Pires, N. D. - Yi, K. - Breuninger, H. - Catarino, B. - Menand, B. - Dolan, L.
Journal: Proc Natl Acad Sci U S A

The evolution of multicellular organisms was made possible by the evolution of underlying gene regulatory networks. In animals, the core of gene regulatory networks consists of kernels, stable subnetworks of transcription factors that are highly conserved in distantly related species. However, in plants it is not clear when and how kernels evolved. We show here that RSL (ROOT HAIR DEFECTIVE SIX-LIKE) transcription factors form an ancient land plant kernel controlling caulonema differentiation in the moss Physcomitrella patens and root hair development in the flowering plant Arabidopsis thaliana. Phylogenetic analyses suggest that RSL proteins evolved in aquatic charophyte algae or in early land plants, and have been conserved throughout land plant radiation. Genetic and transcriptional analyses in loss of function A. thaliana and P. patens mutants suggest that the transcriptional interactions in the RSL kernel were remodeled and became more hierarchical during the evolution of vascular plants. We predict that other gene regulatory networks that control development in derived groups of plants may have originated in the earliest land plants or in their ancestors, the Charophycean algae.

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Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump.

Publication Date: 2013 May 20 PMID: 23690617
Authors: Marcoux, J. - Wang, S. C. - Politis, A. - Reading, E. - Ma, J. - Biggin, P. C. - Zhou, M. - Tao, H. - Zhang, Q. - Chang, G. - Morgner, N. - Robinson, C. V.
Journal: Proc Natl Acad Sci U S A

Multidrug resistance is a serious barrier to successful treatment of many human diseases, including cancer, wherein chemotherapeutics are exported from target cells by membrane-embedded pumps. The most prevalent of these pumps, the ATP-Binding Cassette transporter P-glycoprotein (P-gp), consists of two homologous halves each comprising one nucleotide-binding domain and six transmembrane helices. The transmembrane region encapsulates a hydrophobic cavity, accessed by portals in the membrane, that binds cytotoxic compounds as well as lipids and peptides. Here we use mass spectrometry (MS) to probe the intact P-gp small molecule-bound complex in a detergent micelle. Activation in the gas phase leads to formation of ions, largely devoid of detergent, yet retaining drug molecules as well as charged or zwitterionic lipids. Measuring the rates of lipid binding and calculating apparent KD values shows that up to six negatively charged diacylglycerides bind more favorably than zwitterionic lipids. Similar experiments confirm binding of cardiolipins and show that prior binding of the immunosuppressant and antifungal antibiotic cyclosporin A enhances subsequent binding of cardiolipin. Ion mobility MS reveals that P-gp exists in an equilibrium between different states, readily interconverted by ligand binding. Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.

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Simple computation of reaction-diffusion processes on point clouds.

Publication Date: 2013 May 20 PMID: 23690616
Authors: Macdonald, C. B. - Merriman, B. - Ruuth, S. J.
Journal: Proc Natl Acad Sci U S A

The study of reaction-diffusion processes is much more complicated on general curved surfaces than on standard Cartesian coordinate spaces. Here we show how to formulate and solve systems of reaction-diffusion equations on surfaces in an extremely simple way, using only the standard Cartesian form of differential operators, and a discrete unorganized point set to represent the surface. Our method decouples surface geometry from the underlying differential operators. As a consequence, it becomes possible to formulate and solve rather general reaction-diffusion equations on general surfaces without having to consider the complexities of differential geometry or sophisticated numerical analysis. To illustrate the generality of the method, computations for surface diffusion, pattern formation, excitable media, and bulk-surface coupling are provided for a variety of complex point cloud surfaces.

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Analysis of core circadian feedback loop in suprachiasmatic nucleus of mCry1-luc transgenic reporter mouse.

Publication Date: 2013 May 20 PMID: 23690615
Authors: Maywood, E. S. - Drynan, L. - Chesham, J. E. - Edwards, M. D. - Dardente, H. - Fustin, J. M. - Hazlerigg, D. G. - O'Neill, J. S. - Codner, G. F. - Smyllie, N. J. - Brancaccio, M. - Hastings, M. H.
Journal: Proc Natl Acad Sci U S A

The suprachiasmatic nucleus (SCN) coordinates circadian rhythms that adapt the individual to solar time. SCN pacemaking revolves around feedback loops in which expression of Period (Per) and Cryptochrome (Cry) genes is periodically suppressed by their protein products. Specifically, PER/CRY complexes act at E-box sequences in Per and Cry to inhibit their transactivation by CLOCK/BMAL1 heterodimers. To function effectively, these closed intracellular loops need to be synchronized between SCN cells and to the light/dark cycle. For Per expression, this is mediated by neuropeptidergic and glutamatergic extracellular cues acting via cAMP/calcium-responsive elements (CREs) in Per genes. Cry genes, however, carry no CREs, and how CRY-dependent SCN pacemaking is synchronized remains unclear. Furthermore, whereas reporter lines are available to explore Per circadian expression in real time, no Cry equivalent exists. We therefore created a mouse, B6.Cg-Tg(Cry1-luc)01Ld, carrying a transgene (mCry1-luc) consisting of mCry1 elements containing an E-box and E'-box driving firefly luciferase. mCry1-luc organotypic SCN slices exhibited stable circadian bioluminescence rhythms with appropriate phase, period, profile, and spatial organization. In SCN lacking vasoactive intestinal peptide or its receptor, mCry1 expression was damped and desynchronized between cells. Despite the absence of CREs, mCry1-luc expression was nevertheless (indirectly) sensitive to manipulation of cAMP-dependent signaling. In mPer1/2-null SCN, mCry1-luc bioluminescence was arrhythmic and no longer suppressed by elevation of cAMP. Finally, an SCN graft procedure showed that PER-independent as well as PER-dependent mechanisms could sustain circadian expression of mCry1. The mCry1-luc mouse therefore reports circadian mCry1 expression and its interactions with vasoactive intestinal peptide, cAMP, and PER at the heart of the SCN pacemaker.

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High flight costs, but low dive costs, in auks support the biomechanical hypothesis for flightlessness in penguins.

Publication Date: 2013 May 20 PMID: 23690614
Authors: Elliott, K. H. - Ricklefs, R. E. - Gaston, A. J. - Hatch, S. A. - Speakman, J. R. - Davoren, G. K.
Journal: Proc Natl Acad Sci U S A

Flight is a key adaptive trait. Despite its advantages, flight has been lost in several groups of birds, notably among seabirds, where flightlessness has evolved independently in at least five lineages. One hypothesis for the loss of flight among seabirds is that animals moving between different media face tradeoffs between maximizing function in one medium relative to the other. In particular, biomechanical models of energy costs during flying and diving suggest that a wing designed for optimal diving performance should lead to enormous energy costs when flying in air. Costs of flying and diving have been measured in free-living animals that use their wings to fly or to propel their dives, but not both. Animals that both fly and dive might approach the functional boundary between flight and nonflight. We show that flight costs for thick-billed murres (Uria lomvia), which are wing-propelled divers, and pelagic cormorants (Phalacrocorax pelagicus) (foot-propelled divers), are the highest recorded for vertebrates. Dive costs are high for cormorants and low for murres, but the latter are still higher than for flightless wing-propelled diving birds (penguins). For murres, flight costs were higher than predicted from biomechanical modeling, and the oxygen consumption rate during dives decreased with depth at a faster rate than estimated biomechanical costs. These results strongly support the hypothesis that function constrains form in diving birds, and that optimizing wing shape and form for wing-propelled diving leads to such high flight costs that flying ceases to be an option in larger wing-propelled diving seabirds, including penguins.

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Entropy-driven crystal formation on highly strained substrates.

Publication Date: 2013 May 20 PMID: 23690613
Authors: Savage, J. R. - Hopp, S. F. - Ganapathy, R. - Gerbode, S. J. - Heuer, A. - Cohen, I.
Journal: Proc Natl Acad Sci U S A

In heteroepitaxy, lattice mismatch between the deposited material and the underlying surface strongly affects nucleation and growth processes. The effect of mismatch is well studied in atoms with growth kinetics typically dominated by bond formation with interaction lengths on the order of one lattice spacing. In contrast, less is understood about how mismatch affects crystallization of larger particles, such as globular proteins and nanoparticles, where interparticle interaction energies are often comparable to thermal fluctuations and are short ranged, extending only a fraction of the particle size. Here, using colloidal experiments and simulations, we find particles with short-range attractive interactions form crystals on isotropically strained lattices with spacings significantly larger than the interaction length scale. By measuring the free-energy cost of dimer formation on monolayers of increasing uniaxial strain, we show the underlying mismatched substrate mediates an entropy-driven attractive interaction extending well beyond the interaction length scale. Remarkably, because this interaction arises from thermal fluctuations, lowering temperature causes such substrate-mediated attractive crystals to dissolve. Such counterintuitive results underscore the crucial role of entropy in heteroepitaxy in this technologically important regime. Ultimately, this entropic component of lattice mismatched crystal growth could be used to develop unique methods for heterogeneous nucleation and growth of single crystals for applications ranging from protein crystallization to controlling the assembly of nanoparticles into ordered, functional superstructures. In particular, the construction of substrates with spatially modulated strain profiles would exploit this effect to direct self-assembly, whereby nucleation sites and resulting crystal morphology can be controlled directly through modifications of the substrate.

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Parasitoid wasp venom SERCA regulates Drosophila calcium levels and inhibits cellular immunity.

Publication Date: 2013 May 20 PMID: 23690612
Authors: Mortimer, N. T. - Goecks, J. - Kacsoh, B. Z. - Mobley, J. A. - Bowersock, G. J. - Taylor, J. - Schlenke, T. A.
Journal: Proc Natl Acad Sci U S A

Because parasite virulence factors target host immune responses, identification and functional characterization of these factors can provide insight into poorly understood host immune mechanisms. The fruit fly Drosophila melanogaster is a model system for understanding humoral innate immunity, but Drosophila cellular innate immune responses remain incompletely characterized. Fruit flies are regularly infected by parasitoid wasps in nature and, following infection, flies mount a cellular immune response culminating in the cellular encapsulation of the wasp egg. The mechanistic basis of this response is largely unknown, but wasps use a mixture of virulence proteins derived from the venom gland to suppress cellular encapsulation. To gain insight into the mechanisms underlying wasp virulence and fly cellular immunity, we used a joint transcriptomic/proteomic approach to identify venom genes from Ganaspis sp.1 (G1), a previously uncharacterized Drosophila parasitoid species, and found that G1 venom contains a highly abundant sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. Accordingly, we found that fly immune cells termed plasmatocytes normally undergo a cytoplasmic calcium burst following infection, and that this calcium burst is required for activation of the cellular immune response. We further found that the plasmatocyte calcium burst is suppressed by G1 venom in a SERCA-dependent manner, leading to the failure of plasmatocytes to become activated and migrate toward G1 eggs. Finally, by genetically manipulating plasmatocyte calcium levels, we were able to alter fly immune success against G1 and other parasitoid species. Our characterization of parasitoid wasp venom proteins led us to identify plasmatocyte cytoplasmic calcium bursts as an important aspect of fly cellular immunity.

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Evidence for deposition of 10 million tonnes of impact spherules across four continents 12,800 y ago.

Publication Date: 2013 May 20 PMID: 23690611
Authors: Wittke, J. H. - Weaver, J. C. - Bunch, T. E. - Kennett, J. P. - Kennett, D. J. - Moore, A. M. - Hillman, G. C. - Tankersley, K. B. - Goodyear, A. C. - Moore, C. R. - Daniel, I. R. Jr - Ray, J. H. - Lopinot, N. H. - Ferraro, D. - Israde-Alcantara, I. - Bischoff, J. L. - Decarli, P. S. - Hermes, R. E. - Kloosterman, J. B. - Revay, Z. - Howard, G. A. - Kimbel, D. R. - Kletetschka, G. - Nabelek, L. - Lipo, C. P. - Sakai, S. - West, A. - Firestone, R. B.
Journal: Proc Natl Acad Sci U S A

Airbursts/impacts by a fragmented comet or asteroid have been proposed at the Younger Dryas onset (12.80 +/- 0.15 ka) based on identification of an assemblage of impact-related proxies, including microspherules, nanodiamonds, and iridium. Distributed across four continents at the Younger Dryas boundary (YDB), spherule peaks have been independently confirmed in eight studies, but unconfirmed in two others, resulting in continued dispute about their occurrence, distribution, and origin. To further address this dispute and better identify YDB spherules, we present results from one of the largest spherule investigations ever undertaken regarding spherule geochemistry, morphologies, origins, and processes of formation. We investigated 18 sites across North America, Europe, and the Middle East, performing nearly 700 analyses on spherules using energy dispersive X-ray spectroscopy for geochemical analyses and scanning electron microscopy for surface microstructural characterization. Twelve locations rank among the world's premier end-Pleistocene archaeological sites, where the YDB marks a hiatus in human occupation or major changes in site use. Our results are consistent with melting of sediments to temperatures >2,200 degrees C by the thermal radiation and air shocks produced by passage of an extraterrestrial object through the atmosphere; they are inconsistent with volcanic, cosmic, anthropogenic, lightning, or authigenic sources. We also produced spherules from wood in the laboratory at >1,730 degrees C, indicating that impact-related incineration of biomass may have contributed to spherule production. At 12.8 ka, an estimated 10 million tonnes of spherules were distributed across approximately 50 million square kilometers, similar to well-known impact strewnfields and consistent with a major cosmic impact event.

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Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response.

Publication Date: 2013 May 20 PMID: 23690610
Authors: Tseng, D. - Volkmer, J. P. - Willingham, S. B. - Contreras-Trujillo, H. - Fathman, J. W. - Fernhoff, N. B. - Seita, J. - Inlay, M. A. - Weiskopf, K. - Miyanishi, M. - Weissman, I. L.
Journal: Proc Natl Acad Sci U S A

Mobilization of the T-cell response against cancer has the potential to achieve long-lasting cures. However, it is not known how to harness antigen-presenting cells optimally to achieve an effective antitumor T-cell response. In this study, we show that anti-CD47 antibody-mediated phagocytosis of cancer by macrophages can initiate an antitumor T-cell immune response. Using the ovalbumin model antigen system, anti-CD47 antibody-mediated phagocytosis of cancer cells by macrophages resulted in increased priming of OT-I T cells [cluster of differentiation 8-positive (CD8+)] but decreased priming of OT-II T cells (CD4+). The CD4+ T-cell response was characterized by a reduction in forkhead box P3-positive (Foxp3+) regulatory T cells. Macrophages following anti-CD47-mediated phagocytosis primed CD8+ T cells to exhibit cytotoxic function in vivo. This response protected animals from tumor challenge. We conclude that anti-CD47 antibody treatment not only enables macrophage phagocytosis of cancer but also can initiate an antitumor cytotoxic T-cell immune response.

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Ultrastructural analysis of hepatitis C virus particles.

Publication Date: 2013 May 20 PMID: 23690609
Authors: Catanese, M. T. - Uryu, K. - Kopp, M. - Edwards, T. J. - Andrus, L. - Rice, W. J. - Silvestry, M. - Kuhn, R. J. - Rice, C. M.
Journal: Proc Natl Acad Sci U S A

Hepatitis C virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people infected worldwide. Low yields, poor stability, and inefficient binding to conventional EM grids have posed significant challenges to the purification and structural analysis of HCV. In this report, we generated an infectious HCV genome with an affinity tag fused to the E2 envelope glycoprotein. Using affinity grids, previously described to isolate proteins and macromolecular complexes for single-particle EM, we were able to purify enveloped particles directly from cell culture media. This approach allowed for rapid in situ purification of virions and increased particle density that were instrumental for cryo-EM and cryoelectron tomography (cryo-ET). Moreover, it enabled ultrastructural analysis of virions produced by primary human hepatocytes. HCV appears to be the most structurally irregular member of the Flaviviridae family. Particles are spherical, with spike-like projections, and heterogeneous in size ranging from 40 to 100 nm in diameter. Exosomes, although isolated from unfractionated culture media, were absent in highly infectious, purified virus preparations. Cryo-ET studies provided low-resolution 3D structural information of highly infectious virions. In addition to apolipoprotein (apo)E, HCV particles also incorporate apoB and apoA-I. In general, host apolipoproteins were more readily accessible to antibody labeling than HCV glycoproteins, suggesting either lower abundance or masking by host proteins.

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Genetic screens to identify pathogenic gene variants in the common cancer predisposition Lynch syndrome.

Publication Date: 2013 May 20 PMID: 23690608
Authors: Drost, M. - Lutzen, A. - van Hees, S. - Ferreira, D. - Calleja, F. - Zonneveld, J. B. - Nielsen, F. C. - Rasmussen, L. J. - de Wind, N.
Journal: Proc Natl Acad Sci U S A

In many individuals suspected of the common cancer predisposition Lynch syndrome, variants of unclear significance (VUS), rather than an obviously pathogenic mutations, are identified in one of the DNA mismatch repair (MMR) genes. The uncertainty of whether such VUS inactivate MMR, and therefore are pathogenic, precludes targeted healthcare for both carriers and their relatives. To facilitate the identification of pathogenic VUS, we have developed an in cellulo genetic screen-based procedure for the large-scale mutagenization, identification, and cataloging of residues of MMR genes critical for MMR gene function. When a residue identified as mutated in an individual suspected of Lynch syndrome is listed as critical in such a reverse diagnosis catalog, there is a high probability that the corresponding human VUS is pathogenic. To investigate the applicability of this approach, we have generated and validated a prototypic reverse diagnosis catalog for the MMR gene MutS Homolog 2 (Msh2) by mutagenizing, identifying, and cataloging 26 deleterious mutations in 23 amino acids. Extensive in vivo and in vitro analysis of mutants listed in the catalog revealed both recessive and dominant-negative phenotypes. Nearly half of these critical residues match with VUS previously identified in individuals suspected of Lynch syndrome. This aids in the assignment of pathogenicity to these human VUS and validates the approach described here as a diagnostic tool. In a wider perspective, this work provides a model for the translation of personalized genomics into targeted healthcare.

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NF-kappaB inhibits osteogenic differentiation of mesenchymal stem cells by promoting beta-catenin degradation.

Publication Date: 2013 May 20 PMID: 23690607
Authors: Chang, J. - Liu, F. - Lee, M. - Wu, B. - Ting, K. - Zara, J. N. - Soo, C. - Al Hezaimi, K. - Zou, W. - Chen, X. - Mooney, D. J. - Wang, C. Y.
Journal: Proc Natl Acad Sci U S A

Mesenchymal stem cell (MSC)-based transplantation is a promising therapeutic approach for bone regeneration and repair. In the realm of therapeutic bone regeneration, the defect or injured tissues are frequently inflamed with an abnormal expression of inflammatory mediators. Growing evidence suggests that proinflammatory cytokines inhibit osteogenic differentiation and bone formation. Thus, for successful MSC-mediated repair, it is important to overcome the inflammation-mediated inhibition of tissue regeneration. In this study, using genetic and chemical approaches, we found that proinflammatory cytokines TNF and IL-17 stimulated IkappaB kinase (IKK)-NF-kappaB and impaired osteogenic differentiation of MSCs. In contrast, the inhibition of IKK-NF-kappaB significantly enhanced MSC-mediated bone formation. Mechanistically, we found that IKK-NF-kappaB activation promoted beta-catenin ubiquitination and degradation through induction of Smurf1 and Smurf2. To translate our basic findings to potential clinic applications, we showed that the IKK small molecule inhibitor, IKKVI, enhanced osteogenic differentiation of MSCs. More importantly, the delivery of IKKVI promoted MSC-mediated craniofacial bone regeneration and repair in vivo. Considering the well established role of NF-kappaB in inflammation and infection, our results suggest that targeting IKK-NF-kappaB may have dual benefits in enhancing bone regeneration and repair and inhibiting inflammation, and this concept may also have applicability in many other tissue regeneration situations.

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Visual arrestin interaction with clathrin adaptor AP-2 regulates photoreceptor survival in the vertebrate retina.

Publication Date: 2013 May 20 PMID: 23690606
Authors: Moaven, H. - Koike, Y. - Jao, C. C. - Gurevich, V. V. - Langen, R. - Chen, J.
Journal: Proc Natl Acad Sci U S A

Arrestins bind ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) and terminate the activation of G proteins. Additionally, nonvisual arrestin/GPCR complex can initiate G protein-independent intracellular signals through their ability to act as scaffolds that bring other signaling molecules to the internalized GPCR. Like nonvisual arrestins, vertebrate visual arrestin (ARR1) terminates G protein signaling from light-activated, phosphorylated GPCR, rhodopsin. Unlike nonvisual arrestins, its role as a transducer of signaling from internalized rhodopsin has not been reported in the vertebrate retina. Formation of signaling complexes with arrestins often requires recruitment of the endocytic adaptor protein, AP-2. We have previously shown that Lys296 --> Glu (K296E), which is a naturally occurring rhodopsin mutation in certain humans diagnosed with autosomal dominant retinitis pigmentosa, causes toxicity through forming a stable complex with ARR1. Here we investigated whether recruitment of AP-2 by the K296E/ARR1 complex plays a role in generating the cell death signal in a transgenic mouse model of retinal degeneration. We measured the binding affinity of ARR1 for AP-2 and found that, although the affinity is much lower than that of the other arrestins, the unusually high concentration of ARR1 in rods would favor this interaction. We further demonstrate that p44, a splice variant of ARR1 that binds light-activated, phosphorylated rhodopsin but lacks the AP-2 binding motif, prevents retinal degeneration and rescues visual function in K296E mice. These results reveal a unique role of ARR1 in a G protein-independent signaling cascade in the vertebrate retina.

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Nematic liquid crystal boojums with handles on colloidal handlebodies.

Publication Date: 2013 May 20 PMID: 23690605
Authors: Liu, Q. - Senyuk, B. - Tasinkevych, M. - Smalyukh, I. I.
Journal: Proc Natl Acad Sci U S A

Topological defects that form on surfaces of ordered media, dubbed boojums, are ubiquitous in superfluids, liquid crystals (LCs), Langmuir monolayers, and Bose-Einstein condensates. They determine supercurrents in superfluids, impinge on electrooptical switching in polymer-dispersed LCs, and mediate chemical response at nematic-isotropic fluid interfaces, but the role of surface topology in the appearance, stability, and core structure of these defects remains poorly understood. Here, we demonstrate robust generation of boojums by controlling surface topology of colloidal particles that impose tangential boundary conditions for the alignment of LC molecules. To do this, we design handlebody-shaped polymer particles with different genus g. When introduced into a nematic LC, these particles distort the nematic molecular alignment field while obeying topological constraints and induce at least 2g - 2 boojums that allow for topological charge conservation. We characterize 3D textures of boojums using polarized nonlinear optical imaging of molecular alignment and explain our findings by invoking symmetry considerations and numerical modeling of experiment-matching director fields, order parameter variations, and nontrivial handle-shaped core structure of defects. Finally, we discuss how this interplay between the topologies of colloidal surfaces and boojums may lead to controlled self-assembly of colloidal particles in nematic and paranematic hosts, which, in turn, may enable reconfigurable topological composites.

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Sustained accurate recording of intracellular acidification in living tissues with a photo-controllable bioluminescent protein.

Publication Date: 2013 May 20 PMID: 23690604
Authors: Hattori, M. - Haga, S. - Takakura, H. - Ozaki, M. - Ozawa, T.
Journal: Proc Natl Acad Sci U S A

Regulation of an intracellular acidic environment plays a pivotal role in biological processes and functions. However, spatiotemporal analysis of the acidification in complex tissues of living subjects persists as an important challenge. We developed a photo-inactivatable bioluminescent indicator, based on a combination of luciferase-fragment complementation and a photoreaction of a light, oxygen, and voltage domain from Avena sativa Phototropin1 (LOV2), to visualize temporally dynamic acidification in living tissue samples. Bioluminescence of the indicator diminished upon light irradiation and it recovered gradually in the dark state thereafter. The recovery rate was remarkably sensitive to pH changes but unsusceptible to fluctuation of luciferin or ATP concentrations. Bioluminescence imaging, taken as an index of the recovery rates, enabled long-time recording of acidification in apoptotic and autophagous processes in a cell population and an ischemic condition in living mice. This technology using the indicator is widely applicable to sense organelle-specific acidic changes in target biological tissues.

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Deliberate reduction of hemagglutinin and neuraminidase expression of influenza virus leads to an ultraprotective live vaccine in mice.

Publication Date: 2013 May 20 PMID: 23690603
Authors: Yang, C. - Skiena, S. - Futcher, B. - Mueller, S. - Wimmer, E.
Journal: Proc Natl Acad Sci U S A

A long-held dogma posits that strong presentation to the immune system of the dominant influenza virus glycoprotein antigens neuraminidase (NA) and hemagglutinin (HA) is paramount for inducing protective immunity against influenza virus infection. We have deliberately violated this dogma by constructing a recombinant influenza virus strain of A/PR8/34 (H1N1) in which expression of NA and HA genes was suppressed. We down-regulated NA and HA expression by recoding the respective genes with suboptimal codon pair bias, thereby introducing hundreds of nucleotide changes while preserving their codon use and protein sequence. The variants PR8-NAMin, PR8-HAMin, and PR8-(NA+HA)Min (Min, minimal expression) were used to assess the contribution of reduced glycoprotein expression to growth in tissue culture and pathogenesis in BALB/c mice. All three variants proliferated in Madin-Darby canine kidney cells to nearly the degree as WT PR8. In mice, however, they expressed explicit attenuation phenotypes, as revealed by their LD50 values: PR8, 32 plaque-forming units (PFU); HAMin, 1.7 x 103 PFU; NAMin, 2.4 x 105 PFU; (NA+HA)Min, >/=3.16 x 106 PFU. Remarkably, (NA+HA)Min was attenuated >100,000-fold, with NAMin the major contributor to attenuation. In vaccinated mice (NA+HA)Min was highly effective in providing long-lasting protective immunity against lethal WT challenge at a median protective dose (PD50) of 2.4 PFU. Moreover, at a PD50 of only 147 or 237, (NA+HA)Min conferred protection against heterologous lethal challenges with two mouse-adapted H3N2 viruses. We conclude that the suppression of HA and NA is a unique strategy in live vaccine development.

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Dietary choice affects Shiga toxin-producing Escherichia coli (STEC) O157:H7 colonization and disease.

Publication Date: 2013 May 20 PMID: 23690602
Authors: Zumbrun, S. D. - Melton-Celsa, A. R. - Smith, M. A. - Gilbreath, J. J. - Merrell, D. S. - O'Brien, A. D.
Journal: Proc Natl Acad Sci U S A

The likelihood that a single individual infected with the Shiga toxin (Stx)-producing, food-borne pathogen Escherichia coli O157:H7 will develop a life-threatening sequela called the hemolytic uremic syndrome is unpredictable. We reasoned that conditions that enhance Stx binding and uptake within the gut after E. coli O157:H7 infection should result in greater disease severity. Because the receptor for Stx, globotriaosylceramide, is up-regulated in the presence of butyrate in vitro, we asked whether a high fiber diet (HFD) that reportedly enhances butyrate production by normal gut flora can influence the outcome of an E. coli O157 infection in mice. To address that question, groups of BALB/c mice were fed high (10%) or low (2%) fiber diets and infected with E. coli O157:H7 strain 86-24 (Stx2+). Mice fed an HFD exhibited a 10- to 100-fold increase in colonization, lost 15% more body weight, exhibited signs of morbidity, and had 25% greater mortality relative to the low fiber diet (LFD)-fed group. Additionally, sections of intestinal tissue from HFD-fed mice bound more Stx1 and expressed more globotriaosylceramide than did such sections from LFD-fed mice. Furthermore, the gut microbiota of HFD-fed mice compared with LFD-fed mice contained reduced levels of native Escherichia species, organisms that might protect the gut from colonization by incoming E. coli O157:H7. Taken together, these results suggest that susceptibility to infection and subsequent disease after ingestion of E. coli O157:H7 may depend, at least in part, on individual diet and/or the capacity of the commensal flora to produce butyrate.

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Interplay between superconductivity and magnetism in Fe1-xPdxTe.

Publication Date: 2013 May 20 PMID: 23690601
Authors: Karki, A. B. - Garlea, V. O. - Custelcean, R. - Stadler, S. - Plummer, E. W. - Jin, R.
Journal: Proc Natl Acad Sci U S A

The attractive/repulsive relationship between superconductivity and magnetic ordering has fascinated the condensed matter physics community for a century. In the early days, magnetic impurities doped into a superconductor were found to quickly suppress superconductivity. Later, a variety of systems, such as cuprates, heavy fermions, and Fe pnictides, showed superconductivity in a narrow region near the border to antiferromagnetism (AFM) as a function of pressure or doping. However, the coexistence of superconductivity and ferromagnetic (FM) or AFM ordering is found in a few compounds [RRh4B4 (R = Nd, Sm, Tm, Er), R'Mo6X8 (R' = Tb, Dy, Er, Ho, and X = S, Se), UMGe (M = Ge, Rh, Co), CeCoIn5, EuFe2(As1-xPx)2, etc.], providing evidence for their compatibility. Here, we present a third situation, where superconductivity coexists with FM and near the border of AFM in Fe1-xPdxTe. The doping of Pd for Fe gradually suppresses the first-order AFM ordering at temperature TN/S, and turns into short-range AFM correlation with a characteristic peak in magnetic susceptibility at T'N. Superconductivity sets in when T'N reaches zero. However, there is a gigantic ferromagnetic dome imposed in the superconducting-AFM (short-range) cross-over regime. Such a system is ideal for studying the interplay between superconductivity and two types of magnetic (FM and AFM) interactions.

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Structural insights into the N-terminal GIY-YIG endonuclease activity of Arabidopsis glutaredoxin AtGRXS16 in chloroplasts.

Publication Date: 2013 May 20 PMID: 23690600
Authors: Liu, X. - Liu, S. - Feng, Y. - Liu, J. Z. - Chen, Y. - Pham, K. - Deng, H. - Hirschi, K. D. - Wang, X. - Cheng, N.
Journal: Proc Natl Acad Sci U S A

Glutaredoxins (Grxs) have been identified across taxa as important mediators in various physiological functions. A chloroplastic monothiol glutaredoxin, AtGRXS16 from Arabidopsis thaliana, comprises two distinct functional domains, an N-terminal domain (NTD) with GlyIleTyr-TyrIleGly (GIY-YIG) endonuclease motif and a C-terminal Grx module, to coordinate redox regulation and DNA cleavage in chloroplasts. Structural determination of AtGRXS16-NTD showed that it possesses a GIY-YIG endonuclease fold, but the critical residues for the nuclease activity are different from typical GIY-YIG endonucleases. AtGRXS16-NTD was able to cleave lambdaDNA and chloroplast genomic DNA, and the nuclease activity was significantly reduced in AtGRXS16. Functional analysis indicated that AtGRXS16-NTD could inhibit the ability of AtGRXS16 to suppress the sensitivity of yeast grx5 cells to oxidative stress; however, the C-terminal Grx domain itself and AtGRXS16 with a Cys123Ser mutation were active in these cells and able to functionally complement a Grx5 deficiency in yeast. Furthermore, the two functional domains were shown to be negatively regulated through the formation of an intramolecular disulfide bond. These findings unravel a manner of regulation for Grxs and provide insights into the mechanistic link between redox regulation and DNA metabolism in chloroplasts.

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Mapping microbubble viscosity using fluorescence lifetime imaging of molecular rotors.

Publication Date: 2013 May 20 PMID: 23690599
Authors: Hosny, N. A. - Mohamedi, G. - Rademeyer, P. - Owen, J. - Wu, Y. - Tang, M. X. - Eckersley, R. J. - Stride, E. - Kuimova, M. K.
Journal: Proc Natl Acad Sci U S A

Encapsulated microbubbles are well established as highly effective contrast agents for ultrasound imaging. There remain, however, some significant challenges to fully realize the potential of microbubbles in advanced applications such as perfusion mapping, targeted drug delivery, and gene therapy. A key requirement is accurate characterization of the viscoelastic surface properties of the microbubbles, but methods for independent, nondestructive quantification and mapping of these properties are currently lacking. We present here a strategy for performing these measurements that uses a small fluorophore termed a "molecular rotor" embedded in the microbubble surface, whose fluorescence lifetime is directly related to the viscosity of its surroundings. We apply fluorescence lifetime imaging to show that shell viscosities vary widely across the population of the microbubbles and are influenced by the shell composition and the manufacturing process. We also demonstrate that heterogeneous viscosity distributions exist within individual microbubble shells even with a single surfactant component.

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Bundling ecosystem services in the Panama Canal watershed.

Publication Date: 2013 May 20 PMID: 23690598
Authors: Simonit, S. - Perrings, C.
Journal: Proc Natl Acad Sci U S A

Land cover change in watersheds affects the supply of a number of ecosystem services, including water supply, the production of timber and nontimber forest products, the provision of habitat for forest species, and climate regulation through carbon sequestration. The Panama Canal watershed is currently being reforested to protect the dry-season flows needed for Canal operations. Whether reforestation of the watershed is desirable depends on its impacts on all services. We develop a spatially explicit model to evaluate the implications of reforestation both for water flows and for other services. We find that reforestation does not necessarily increase water supply, but does increase carbon sequestration and timber production.

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Local circadian clock gates cell cycle progression of transient amplifying cells during regenerative hair cycling.

Publication Date: 2013 May 20 PMID: 23690597
Authors: Plikus, M. V. - Vollmers, C. - de la Cruz, D. - Chaix, A. - Ramos, R. - Panda, S. - Chuong, C. M.
Journal: Proc Natl Acad Sci U S A

Regenerative cycling of hair follicles offers an unique opportunity to explore the role of circadian clock in physiological tissue regeneration. We focused on the role of circadian clock in actively proliferating transient amplifying cells, as opposed to quiescent stem cells. We identified two key sites of peripheral circadian clock activity specific to regenerating anagen hair follicles, namely epithelial matrix and mesenchymal dermal papilla. We showed that peripheral circadian clock in epithelial matrix cells generates prominent daily mitotic rhythm. As a consequence of this mitotic rhythmicity, hairs grow faster in the morning than in the evening. Because cells are the most susceptible to DNA damage during mitosis, this cycle leads to a remarkable time-of-day-dependent sensitivity of growing hair follicles to genotoxic stress. Same doses of gamma-radiation caused dramatic hair loss in wild-type mice when administered in the morning, during mitotic peak, compared with the evening, when hair loss is minimal. This diurnal radioprotective effect becomes lost in circadian mutants, consistent with asynchronous mitoses in their hair follicles. Clock coordinates cell cycle progression with genotoxic stress responses by synchronizing Cdc2/Cyclin B-mediated G2/M checkpoint. Our results uncover diurnal mitotic gating as the essential protective mechanism in highly proliferative hair follicles and offer strategies for minimizing or maximizing cytotoxicity of radiation therapies.

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Orbital pacing and ocean circulation-induced collapses of the Mesoamerican monsoon over the past 22,000 y.

Publication Date: 2013 May 20 PMID: 23690596
Authors: Lachniet, M. S. - Asmerom, Y. - Bernal, J. P. - Polyak, V. J. - Vazquez-Selem, L.
Journal: Proc Natl Acad Sci U S A

The dominant controls on global paleomonsoon strength include summer insolation driven by precession cycles, ocean circulation through its influence on atmospheric circulation, and sea-surface temperatures. However, few records from the summer North American Monsoon system are available to test for a synchronous response with other global monsoons to shared forcings. In particular, the monsoon response to widespread atmospheric reorganizations associated with disruptions of the Atlantic Meridional Overturning Circulation (AMOC) during the deglacial period remains unconstrained. Here, we present a high-resolution and radiometrically dated monsoon rainfall reconstruction over the past 22,000 y from speleothems of tropical southwestern Mexico. The data document an active Last Glacial Maximum (18-24 cal ka B.P.) monsoon with similar delta18O values to the modern, and that the monsoon collapsed during periods of weakened AMOC during Heinrich stadial 1 (ca. 17 ka) and the Younger Dryas (12.9-11.5 ka). The Holocene was marked by a trend to a weaker monsoon that was paced by orbital insolation. We conclude that the Mesoamerican monsoon responded in concert with other global monsoon regions, and that monsoon strength was driven by variations in the strength and latitudinal position of the Intertropical Convergence Zone, which was forced by AMOC variations in the North Atlantic Ocean. The surprising observation of an active Last Glacial Maximum monsoon is attributed to an active but shallow AMOC and proximity to the Intertropical Convergence Zone. The emergence of agriculture in southwestern Mexico was likely only possible after monsoon strengthening in the Early Holocene at ca. 11 ka.

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In vitro reconstitution of lipid-dependent dual topology and postassembly topological switching of a membrane protein.

Publication Date: 2013 May 20 PMID: 23690595
Authors: Vitrac, H. - Bogdanov, M. - Dowhan, W.
Journal: Proc Natl Acad Sci U S A

Phospholipids could exert their effect on membrane protein topology either directly by interacting with topogenic signals of newly inserted proteins or indirectly by influencing the protein assembly machinery. In vivo lactose permease (LacY) of Escherichia coli displays a mixture of topological conformations ranging from complete inversion of the N-terminal helical bundle to mixed topology and then to completely native topology as phosphatidylethanolamine (PE) is increased from 0% to 70% of membrane phospholipids. These topological conformers are interconvertible by postassembly synthesis or dilution of PE in vivo. To investigate whether coexistence of multiple topological conformers is dependent solely on the membrane lipid composition, we determined the topological organization of LacY in an in vitro proteoliposome system in which lipid composition can be systematically controlled before (liposomes) and after (fliposomes) reconstitution using a lipid exchange technique. Purified LacY reconstituted into preformed liposomes of increasing PE content displayed inverted topology at low PE and then a mixture of inverted and proper topologies with the latter increasing with increasing PE until all LacY adopted its native topology. Interconversion between topological conformers of LacY was observed in a PE dose-dependent manner by either increasing or decreasing PE levels in proteoliposomes postreconstitution of LacY, clearly demonstrating that membrane protein topology can be changed simply by changing membrane lipid composition independent of other cellular factors. The results provide a thermodynamic-based lipid-dependent model for shifting the equilibrium between different conformational states of a membrane protein.

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GPR37 and GPR37L1 are receptors for the neuroprotective and glioprotective factors prosaptide and prosaposin.

Publication Date: 2013 May 20 PMID: 23690594
Authors: Meyer, R. C. - Giddens, M. M. - Schaefer, S. A. - Hall, R. A.
Journal: Proc Natl Acad Sci U S A

GPR37 (also known as Pael-R) and GPR37L1 are orphan G protein-coupled receptors that are almost exclusively expressed in the nervous system. We screened these receptors for potential activation by various orphan neuropeptides, and these screens yielded a single positive hit: prosaptide, which promoted the endocytosis of GPR37 and GPR37L1, bound to both receptors and activated signaling in a GPR37- and GPR37L1-dependent manner. Prosaptide stimulation of cells transfected with GPR37 or GPR37L1 induced the phosphorylation of ERK in a pertussis toxin-sensitive manner, stimulated 35S-GTPgammaS binding, and promoted the inhibition of forskolin-stimulated cAMP production. Because prosaptide is the active fragment of the secreted neuroprotective and glioprotective factor prosaposin (also known as sulfated glycoprotein-1), we purified full-length prosaposin and found that it also stimulated GPR37 and GPR37L1 signaling. Moreover, both prosaptide and prosaposin were found to protect primary astrocytes against oxidative stress, with these protective effects being attenuated by siRNA-mediated knockdown of endogenous astrocytic GPR37 or GPR37L1. These data reveal that GPR37 and GPR37L1 are receptors for the neuroprotective and glioprotective factors prosaptide and prosaposin.

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Surviving rapid climate change in the deep sea during the Paleogene hyperthermals.

Publication Date: 2013 May 20 PMID: 23690593
Authors: Foster, L. C. - Schmidt, D. N. - Thomas, E. - Arndt, S. - Ridgwell, A.
Journal: Proc Natl Acad Sci U S A

Predicting the impact of ongoing anthropogenic CO2 emissions on calcifying marine organisms is complex, owing to the synergy between direct changes (acidification) and indirect changes through climate change (e.g., warming, changes in ocean circulation, and deoxygenation). Laboratory experiments, particularly on longer-lived organisms, tend to be too short to reveal the potential of organisms to acclimatize, adapt, or evolve and usually do not incorporate multiple stressors. We studied two examples of rapid carbon release in the geological record, Eocene Thermal Maximum 2 ( approximately 53.2 Ma) and the Paleocene Eocene Thermal Maximum (PETM, approximately 55.5 Ma), the best analogs over the last 65 Ma for future ocean acidification related to high atmospheric CO2 levels. We use benthic foraminifers, which suffered severe extinction during the PETM, as a model group. Using synchrotron radiation X-ray tomographic microscopy, we reconstruct the calcification response of survivor species and find, contrary to expectations, that calcification significantly increased during the PETM. In contrast, there was no significant response to the smaller Eocene Thermal Maximum 2, which was associated with a minor change in diversity only. These observations suggest that there is a response threshold for extinction and calcification response, while highlighting the utility of the geological record in helping constrain the sensitivity of biotic response to environmental change.

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Adaptive governance and institutional strategies for climate-induced community relocations in Alaska.

Publication Date: 2013 May 20 PMID: 23690592
Authors: Bronen, R. - Chapin, F. S. 3rd
Journal: Proc Natl Acad Sci U S A

This article presents governance and institutional strategies for climate-induced community relocations. In Alaska, repeated extreme weather events coupled with climate change-induced coastal erosion impact the habitability of entire communities. Community residents and government agencies concur that relocation is the only adaptation strategy that can protect lives and infrastructure. Community relocation stretches the financial and institutional capacity of existing governance institutions. Based on a comparative analysis of three Alaskan communities, Kivalina, Newtok, and Shishmaref, which have chosen to relocate, we examine the institutional constraints to relocation in the United States. We identify policy changes and components of a toolkit that can facilitate community-based adaptation when environmental events threaten people's lives and protection in place is not possible. Policy changes include amendment of the Stafford Act to include gradual geophysical processes, such as erosion, in the statutory definition of disaster and the creation of an adaptive governance framework to allow communities a continuum of responses from protection in place to community relocation. Key components of the toolkit are local leadership and integration of social and ecological well-being into adaptation planning.

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Capillarity-induced ordering of spherical colloids on an interface with anisotropic curvature.

Publication Date: 2013 May 20 PMID: 23690591
Authors: Ershov, D. - Sprakel, J. - Appel, J. - Cohen Stuart, M. A. - van der Gucht, J.
Journal: Proc Natl Acad Sci U S A

Objects floating at a liquid interface, such as breakfast cereals floating in a bowl of milk or bubbles at the surface of a soft drink, clump together as a result of capillary attraction. This attraction arises from deformation of the liquid interface due to gravitational forces; these deformations cause excess surface area that can be reduced if the particles move closer together. For micrometer-sized colloids, however, the gravitational force is too small to produce significant interfacial deformations, so capillary forces between spherical colloids at a flat interface are negligible. Here, we show that this is different when the confining liquid interface has a finite curvature that is also anisotropic. In that case, the condition of constant contact angle along the three-phase contact line can only be satisfied when the interface is deformed. We present experiments and numerical calculations that demonstrate how this leads to quadrupolar capillary interactions between the particles, giving rise to organization into regular square lattices. We demonstrate that the strength of the governing anisotropic interactions can be rescaled with the deviatoric curvature alone, irrespective of the exact shape of the liquid interface. Our results suggest that anisotropic interactions can easily be induced between isotropic colloids through tailoring of the interfacial curvature.

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Bacteriophage adhering to mucus provide a non-host-derived immunity.

Publication Date: 2013 May 20 PMID: 23690590
Authors: Barr, J. J. - Auro, R. - Furlan, M. - Whiteson, K. L. - Erb, M. L. - Pogliano, J. - Stotland, A. - Wolkowicz, R. - Cutting, A. S. - Doran, K. S. - Salamon, P. - Youle, M. - Rohwer, F.
Journal: Proc Natl Acad Sci U S A

Mucosal surfaces are a main entry point for pathogens and the principal sites of defense against infection. Both bacteria and phage are associated with this mucus. Here we show that phage-to-bacteria ratios were increased, relative to the adjacent environment, on all mucosal surfaces sampled, ranging from cnidarians to humans. In vitro studies of tissue culture cells with and without surface mucus demonstrated that this increase in phage abundance is mucus dependent and protects the underlying epithelium from bacterial infection. Enrichment of phage in mucus occurs via binding interactions between mucin glycoproteins and Ig-like protein domains exposed on phage capsids. In particular, phage Ig-like domains bind variable glycan residues that coat the mucin glycoprotein component of mucus. Metagenomic analysis found these Ig-like proteins present in the phages sampled from many environments, particularly from locations adjacent to mucosal surfaces. Based on these observations, we present the bacteriophage adherence to mucus model that provides a ubiquitous, but non-host-derived, immunity applicable to mucosal surfaces. The model suggests that metazoan mucosal surfaces and phage coevolve to maintain phage adherence. This benefits the metazoan host by limiting mucosal bacteria, and benefits the phage through more frequent interactions with bacterial hosts. The relationships shown here suggest a symbiotic relationship between phage and metazoan hosts that provides a previously unrecognized antimicrobial defense that actively protects mucosal surfaces.

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Climbing, falling, and jamming during ant locomotion in confined environments.

Publication Date: 2013 May 20 PMID: 23690589
Authors: Gravish, N. - Monaenkova, D. - Goodisman, M. A. - Goldman, D. I.
Journal: Proc Natl Acad Sci U S A

Locomotion emerges from effective interactions of an individual with its environment. Principles of biological terrestrial locomotion have been discovered on unconfined vertical and horizontal substrates. However, a diversity of organisms construct, inhabit, and move within confined spaces. Such animals are faced with locomotor challenges including limited limb range of motion, crowding, and visual sensory deprivation. Little is known about how these organisms accomplish their locomotor tasks, and such environments challenge human-made devices. To gain insight into how animals move within confined spaces, we study the locomotion of the fire ant Solenopsis invicta, which constructs subterranean tunnel networks (nests). Laboratory experiments reveal that ants construct tunnels with diameter, D, comparable to body length, L = 3.5 +/- 0.5 mm. Ants can move rapidly (> 9 bodylengths per s) within these environments; their tunnels allow for effective limb, body, and antennae interaction with walls, which facilitate rapid slip-recovery during ascending and descending climbs. To examine the limits of slip-recovery in artificial tunnels, we perform perturbations consisting of rapid downward accelerations of the tunnels, which induce falls. Below a critical tunnel diameter, Ds = 1.31 +/- 0.02 L, falls are always arrested through rapid interaction of appendages and antennae with tunnel walls to jam the falls. Ds is comparable to the size of incipient nest tunnels (D = 1.06 +/- 0.23 L), supporting our hypothesis that fire ants construct environments that simplify their control task when moving through the nest, likely without need for rapid nervous system intervention.

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Direct observation of ground-state lactam-lactim tautomerization using temperature-jump transient 2D IR spectroscopy.

Publication Date: 2013 May 20 PMID: 23690588
Authors: Peng, C. S. - Baiz, C. R. - Tokmakoff, A.
Journal: Proc Natl Acad Sci U S A

We provide a systematic characterization of the nanosecond ground-state lactam-lactim tautomerization of pyridone derivatives in aqueous solution under ambient conditions using temperature-jump transient 2D IR spectroscopy. Although electronic excited-state tautomerization has been widely studied, experimental work on the ground electronic state, most relevant to chemistry and biology, is lacking. Using 2D IR spectroscopy, lactam and lactim tautomers of 6-chloro-2-pyridone and 2-chloro-4-pyridone are unambiguously identified by their unique cross-peak patterns. Monitoring the correlated exponential relaxation of these signals in response to a laser temperature jump provides a direct measurement of the nanosecond tautomerization kinetics. By studying the temperature, concentration, solvent, and pH dependence, we extract a thermodynamic and kinetic characterization and conclude that the tautomerization proceeds through a two-state concerted mechanism. We find that the intramolecular proton transfer is mediated by bridging water molecules and the reaction barrier is dictated by the release of a proton from pyridone, as would be expected for an efficient Grothuss-type proton transfer mechanism.

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Deciphering the impacts of vaccination and immunity on pertussis epidemiology in Thailand.

Publication Date: 2013 May 20 PMID: 23690587
Authors: Blackwood, J. C. - Cummings, D. A. - Broutin, H. - Iamsirithaworn, S. - Rohani, P.
Journal: Proc Natl Acad Sci U S A

Pertussis is a highly infectious respiratory disease that is currently responsible for nearly 300,000 annual deaths worldwide, primarily in infants in developing countries. Despite sustained high vaccine uptake, a resurgence in pertussis incidence has been reported in a number of countries. This resurgence has led to critical questions regarding the transmission impacts of vaccination and pertussis immunology. We analyzed pertussis incidence in Thailand-both age-stratified and longitudinal aggregate reports-over the past 30 y. To dissect the contributions of waning pertussis immunity and repeat infections to pertussis epidemiology in Thailand following a pronounced increase in vaccine uptake, we used likelihood-based statistical inference methods to evaluate the support for multiple competing transmission models. We found that, in contrast to other settings, there is no evidence for pertussis resurgence in Thailand, with each model examined pointing to a substantial rise in herd immunity over the past 30 y. Using a variety of empirical metrics, we verified our findings by documenting signatures of changing herd immunity over the study period. Importantly, this work leads to the conclusion that repeat infections have played little role in shaping pertussis epidemiology in Thailand. Our results are surprisingly emphatic in support of measurable impact of herd immunity given the uncertainty associated with pertussis epidemiology.

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Impairing existing declarative memory in humans by disrupting reconsolidation.

Publication Date: 2013 May 20 PMID: 23690586
Authors: Chan, J. C. - Lapaglia, J. A.
Journal: Proc Natl Acad Sci U S A

During the past decade, a large body of research has shown that memory traces can become labile upon retrieval and must be restabilized. Critically, interrupting this reconsolidation process can abolish a previously stable memory. Although a large number of studies have demonstrated this reconsolidation associated amnesia in nonhuman animals, the evidence for its occurrence in humans is far less compelling, especially with regard to declarative memory. In fact, reactivating a declarative memory often makes it more robust and less susceptible to subsequent disruptions. Here we show that existing declarative memories can be selectively impaired by using a noninvasive retrieval-relearning technique. In six experiments, we show that this reconsolidation-associated amnesia can be achieved 48 h after formation of the original memory, but only if relearning occurred soon after retrieval. Furthermore, the amnesic effect persists for at least 24 h, cannot be attributed solely to source confusion and is attainable only when relearning targets specific existing memories for impairment. These results demonstrate that human declarative memory can be selectively rewritten during reconsolidation.

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Identifying dynamic tuberculosis case-finding policies for HIV/TB coepidemics.

Publication Date: 2013 May 20 PMID: 23690585
Authors: Yaesoubi, R. - Cohen, T.
Journal: Proc Natl Acad Sci U S A

The global tuberculosis (TB) control plan has historically emphasized passive case finding (PCF) as the most practical approach for identifying TB suspects in high burden settings. The success of this approach in controlling TB depends on infectious individuals recognizing their symptoms and voluntarily seeking diagnosis rapidly enough to reduce onward transmission. It now appears, at least in some settings, that more intensified case-finding (ICF) approaches may be needed to control TB transmission; these more aggressive approaches for detecting as-yet undiagnosed cases obviously require additional resources to implement. Given that TB control programs are resource constrained and that the incremental yield of ICF is expected to wane over time as the pool of undiagnosed cases is depleted, a tool that can help policymakers to identify when to implement or suspend an ICF intervention would be valuable. In this article, we propose dynamic case-finding policies that allow policymakers to use existing observations about the epidemic and resource availability to determine when to switch between PCF and ICF to efficiently use resources to optimize population health. Using mathematical models of TB/HIV coepidemics, we show that dynamic policies strictly dominate static policies that prespecify a frequency and duration of rounds of ICF. We also find that the use of a diagnostic tool with better sensitivity for detecting smear-negative cases (e.g., Xpert MTB/RIF) further improves the incremental benefit of these dynamic case-finding policies.

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Experimentally calibrated population of models predicts and explains intersubject variability in cardiac cellular electrophysiology.

Publication Date: 2013 May 20 PMID: 23690584
Authors: Britton, O. J. - Bueno-Orovio, A. - Van Ammel, K. - Lu, H. R. - Towart, R. - Gallacher, D. J. - Rodriguez, B.
Journal: Proc Natl Acad Sci U S A

Cellular and ionic causes of variability in the electrophysiological activity of hearts from individuals of the same species are unknown. However, improved understanding of this variability is key to enable prediction of the response of specific hearts to disease and therapies. Limitations of current mathematical modeling and experimental techniques hamper our ability to provide insight into variability. Here, we describe a methodology to unravel the ionic determinants of intersubject variability exhibited in experimental recordings, based on the construction and calibration of populations of models. We illustrate the methodology through its application to rabbit Purkinje preparations, because of their importance in arrhythmias and safety pharmacology assessment. We consider a set of equations describing the biophysical processes underlying rabbit Purkinje electrophysiology, and we construct a population of over 10,000 models by randomly assigning specific parameter values corresponding to ionic current conductances and kinetics. We calibrate the model population by closely comparing simulation output and experimental recordings at three pacing frequencies. We show that 213 of the 10,000 candidate models are fully consistent with the experimental dataset. Ionic properties in the 213 models cover a wide range of values, including differences up to +/-100% in several conductances. Partial correlation analysis shows that particular combinations of ionic properties determine the precise shape, amplitude, and rate dependence of specific action potentials. Finally, we demonstrate that the population of models calibrated using data obtained under physiological conditions quantitatively predicts the action potential duration prolongation caused by exposure to four concentrations of the potassium channel blocker dofetilide.

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Functional diversity among sensory receptors in a Drosophila olfactory circuit.

Publication Date: 2013 May 20 PMID: 23690583
Authors: Mathew, D. - Martelli, C. - Kelley-Swift, E. - Brusalis, C. - Gershow, M. - Samuel, A. D. - Emonet, T. - Carlson, J. R.
Journal: Proc Natl Acad Sci U S A

The ability of an animal to detect, discriminate, and respond to odors depends on the function of its olfactory receptor neurons (ORNs), which in turn depends ultimately on odorant receptors. To understand the diverse mechanisms used by an animal in olfactory coding and computation, it is essential to understand the functional diversity of its odor receptors. The larval olfactory system of Drosophila melanogaster contains 21 ORNs and a comparable number of odorant receptors whose properties have been examined in only a limited way. We systematically screened them with a panel of approximately 500 odorants, yielding >10,000 receptor-odorant combinations. We identify for each of 19 receptors an odorant that excites it strongly. The responses elicited by each of these odorants are analyzed in detail. The odorants elicited little cross-activation of other receptors at the test concentration; thus, low concentrations of many of these odorants in nature may be signaled by a single ORN. The receptors differed dramatically in sensitivity to their cognate odorants. The responses showed diverse temporal dynamics, with some odorants eliciting supersustained responses. An intriguing question in the field concerns the roles of different ORNs and receptors in driving behavior. We found that the cognate odorants elicited behavioral responses that varied across a broad range. Some odorants elicited strong physiological responses but weak behavioral responses or weak physiological responses but strong behavioral responses.

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Preventing Alzheimer's disease-related gray matter atrophy by B-vitamin treatment.

Publication Date: 2013 May 20 PMID: 23690582
Authors: Douaud, G. - Refsum, H. - de Jager, C. A. - Jacoby, R. - E Nichols, T. - Smith, S. M. - Smith, A. D.
Journal: Proc Natl Acad Sci U S A

Is it possible to prevent atrophy of key brain regions related to cognitive decline and Alzheimer's disease (AD)? One approach is to modify nongenetic risk factors, for instance by lowering elevated plasma homocysteine using B vitamins. In an initial, randomized controlled study on elderly subjects with increased dementia risk (mild cognitive impairment according to 2004 Petersen criteria), we showed that high-dose B-vitamin treatment (folic acid 0.8 mg, vitamin B6 20 mg, vitamin B12 0.5 mg) slowed shrinkage of the whole brain volume over 2 y. Here, we go further by demonstrating that B-vitamin treatment reduces, by as much as seven fold, the cerebral atrophy in those gray matter (GM) regions specifically vulnerable to the AD process, including the medial temporal lobe. In the placebo group, higher homocysteine levels at baseline are associated with faster GM atrophy, but this deleterious effect is largely prevented by B-vitamin treatment. We additionally show that the beneficial effect of B vitamins is confined to participants with high homocysteine (above the median, 11 micromol/L) and that, in these participants, a causal Bayesian network analysis indicates the following chain of events: B vitamins lower homocysteine, which directly leads to a decrease in GM atrophy, thereby slowing cognitive decline. Our results show that B-vitamin supplementation can slow the atrophy of specific brain regions that are a key component of the AD process and that are associated with cognitive decline. Further B-vitamin supplementation trials focusing on elderly subjets with high homocysteine levels are warranted to see if progression to dementia can be prevented.

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Differential effects of cocaine on histone posttranslational modifications in identified populations of striatal neurons.

Publication Date: 2013 May 20 PMID: 23690581
Authors: Jordi, E. - Heiman, M. - Marion-Poll, L. - Guermonprez, P. - Cheng, S. K. - Nairn, A. C. - Greengard, P. - Girault, J. A.
Journal: Proc Natl Acad Sci U S A

Drugs of abuse, such as cocaine, induce changes in gene expression and epigenetic marks including alterations in histone posttranslational modifications in striatal neurons. These changes are thought to participate in physiological memory mechanisms and to be critical for long-term behavioral alterations. However, the striatum is composed of multiple cell types, including two distinct populations of medium-sized spiny neurons, and little is known concerning the cell-type specificity of epigenetic modifications. To address this question we used bacterial artificial chromosome transgenic mice, which express EGFP fused to the N-terminus of the large subunit ribosomal protein L10a driven by the D1 or D2 dopamine receptor (D1R, D2R) promoter, respectively. Fluorescence in nucleoli was used to sort nuclei from D1R- or D2R-expressing neurons and to quantify by flow cytometry the cocaine-induced changes in histone acetylation and methylation specifically in these two types of nuclei. The two populations of medium-sized spiny neurons displayed different patterns of histone modifications 15 min or 24 h after a single injection of cocaine or 24 h after seven daily injections. In particular, acetylation of histone 3 on Lys 14 and of histone 4 on Lys 5 and 12, and methylation of histone 3 on Lys 9 exhibited distinct and persistent changes in the two cell types. Our data provide insights into the differential epigenetic responses to cocaine in D1R- and D2R-positive neurons and their potential regulation, which may participate in the persistent effects of cocaine in these neurons. The method described should have general utility for studying nuclear modifications in different types of neuronal or nonneuronal cell types.

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Pressure-induced superconductivity in CaC2.

Publication Date: 2013 May 20 PMID: 23690580
Authors: Li, Y. L. - Luo, W. - Zeng, Z. - Lin, H. Q. - Mao, H. K. - Ahuja, R.
Journal: Proc Natl Acad Sci U S A

Carbon can exist as isolated dumbbell, 1D chain, 2D plane, and 3D network in carbon solids or carbon-based compounds, which attributes to its rich chemical binding way, including sp-, sp2-, and sp3-hybridized bonds. sp2-hybridizing carbon always captures special attention due to its unique physical and chemical property. Here, using an evolutionary algorithm in conjunction with ab initio method, we found that, under compression, dumbbell carbon in CaC2 can be polymerized first into 1D chain and then into ribbon and further into 2D graphite sheet at higher pressure. The C2/m structure transforms into an orthorhombic Cmcm phase at 0.5 GPa, followed by another orthorhombic Immm phase, which is stabilized in a wide pressure range of 15.2-105.8 GPa and then forced into MgB2-type phase with wide range stability up to at least 1 TPa. Strong electron-phonon coupling lambda in compressed CaC2 is found, in particular for Immm phase, which has the highest lambda value (0.562-0.564) among them, leading to its high superconducting critical temperature Tc (7.9 approximately 9.8 K), which is comparable with the 11.5 K value of CaC6. Our results show that calcium not only can stabilize carbon sp2 hybridization at a larger range of pressure but also can contribute in superconducting behavior, which would further ignite experimental and theoretical interest in alkaline-earth metal carbides to uncover their peculiar physical properties under extreme conditions.

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Evidence for multiple roles for grainyheadlike 2 in the establishment and maintenance of human mucociliary airway epithelium.

Publication Date: 2013 May 20 PMID: 23690579
Authors: Gao, X. - Vockley, C. M. - Pauli, F. - Newberry, K. M. - Xue, Y. - Randell, S. H. - Reddy, T. E. - Hogan, B. L.
Journal: Proc Natl Acad Sci U S A

Most of the airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated basal progenitor cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia, there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in coordinating multiple cellular processes required for epithelial morphogenesis, differentiation, remodeling, and repair. However, only a few target genes have been identified, and little is known about GRHL function in the adult lung. Here we focus on the role of GRHL2 in primary human bronchial epithelial cells, both as undifferentiated progenitors and as they differentiate in air-liquid interface culture into an organized mucociliary epithelium with transepithelial resistance. Using a dominant-negative protein or shRNA to inhibit GRHL2, we follow changes in epithelial phenotype and gene transcription using RNA sequencing or microarray analysis. We identify several hundreds of genes that are directly or indirectly regulated by GRHL2 in both undifferentiated cells and air-liquid interface cultures. Using ChIP sequencing to map sites of GRHL2 binding in the basal cells, we identify 7,687 potential primary targets and confirm that GRHL2 binding is strongly enriched near GRHL2-regulated genes. Taken together, the results support the hypothesis that GRHL2 plays a key role in regulating many physiological functions of human airway epithelium, including those involving cell morphogenesis, adhesion, and motility.

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The lipopolysaccharide modification regulator PmrA limits Salmonella virulence by repressing the type three-secretion system Spi/Ssa.

Publication Date: 2013 May 20 PMID: 23690578
Authors: Choi, J. - Groisman, E. A.
Journal: Proc Natl Acad Sci U S A

The regulatory protein PmrA controls expression of lipopolysaccharide (LPS) modification genes in Salmonella enterica serovar Typhimurium, the etiologic agent of human gastroenteritis and murine typhoid fever. PmrA-dependent LPS modifications confer resistance to serum, Fe3+, and several antimicrobial peptides, suggesting that the pmrA gene is required for Salmonella virulence. We now report that, surprisingly, a pmrA null mutant is actually hypervirulent when inoculated i.p. into C3H/HeN mice. We establish that the PmrA protein binds to the promoter and represses transcription of ssrB, a virulence regulatory gene required for expression of the Spi/Ssa type three-secretion system inside macrophages. The pmrA mutant displayed heightened expression of SsrB-dependent genes and faster Spi/Ssa-dependent macrophage killing than wild-type Salmonella. A mutation in the ssrB promoter that abolished repression by the PmrA protein rendered Salmonella as hypervirulent as the pmrA null mutant. The antivirulence function of the PmrA protein may limit the acute phase of Salmonella infection, thereby enhancing pathogen persistence in host tissues.

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Polysaccharide chemistry regulates kinetics of calcite nucleation through competition of interfacial energies.

Publication Date: 2013 May 20 PMID: 23690577
Authors: Giuffre, A. J. - Hamm, L. M. - Han, N. - De Yoreo, J. J. - Dove, P. M.
Journal: Proc Natl Acad Sci U S A

Calcified skeletons are produced within complex assemblages of proteins and polysaccharides whose roles in mineralization are not well understood. Here we quantify the kinetics of calcite nucleation onto a suite of high-purity polysaccharide (PS) substrates under controlled conditions. The energy barriers to nucleation are PS-specific by a systematic relationship to PS charge density and substrate structure that is rooted in minimization of the competing substrate-crystal and substrate-liquid interfacial energies. Chitosan presents a low-energy barrier to nucleation because its near-neutral charge favors formation of a substrate-crystal interface, thus reducing substrate interactions with water. Progressively higher barriers are measured for negatively charged alginates and heparin that favor contact with the solution over the formation of new substrate-crystal interfaces. The findings support a directing role for PS in biomineral formation and demonstrate that substrate-crystal interactions are one end-member in a larger continuum of competing forces that regulate heterogeneous crystal nucleation.

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Phosphatidylinositol-4,5-biphosphate-dependent rearrangement of TRPV4 cytosolic tails enables channel activation by physiological stimuli.

Publication Date: 2013 May 20 PMID: 23690576
Authors: Garcia-Elias, A. - Mrkonjic, S. - Pardo-Pastor, C. - Inada, H. - Hellmich, U. A. - Rubio-Moscardo, F. - Plata, C. - Gaudet, R. - Vicente, R. - Valverde, M. A.
Journal: Proc Natl Acad Sci U S A

Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP2), although the structural rearrangements occurring on PIP2 binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP2 binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence 121KRWRK125, which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4alpha-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP2 from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP2 facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5'-6'-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP2-binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP2 mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4-PIP2 interacting site and conditions that deplete PIP2 or restrict access of TRPV4 to PIP2-in the case of PACSIN3-change tail conformation and negatively affect channel activation by hypotonicity and heat.

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Construction of self-recognizing regulatory T cells from conventional T cells by controlling CTLA-4 and IL-2 expression.

Publication Date: 2013 May 20 PMID: 23690575
Authors: Yamaguchi, T. - Kishi, A. - Osaki, M. - Morikawa, H. - Prieto-Martin, P. - Wing, K. - Saito, T. - Sakaguchi, S.
Journal: Proc Natl Acad Sci U S A

Thymus-produced CD4+ regulatory T (Treg) cells, which specifically express the transcription factor forkhead box p3, are potently immunosuppressive and characteristically possess a self-reactive T-cell receptor (TCR) repertoire. To determine the molecular basis of Treg suppressive activity and their self-skewed TCR repertoire formation, we attempted to reconstruct these Treg-specific properties in conventional T (Tconv) cells by genetic manipulation. We show that Tconv cells rendered IL-2 deficient and constitutively expressing transgenic cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) were potently suppressive in vitro when they were preactivated by antigenic stimulation. They also suppressed in vivo inflammatory bowel disease and systemic autoimmunity/inflammation produced by Treg deficiency. In addition, in the thymus, transgenic CTLA-4 expression in developing Tconv cells skewed their TCR repertoire toward higher self-reactivity, whereas CTLA-4 deficiency specifically in developing thymic Treg cells cancelled their physiological TCR self-skewing. The extracellular portion of CTLA-4 was sufficient for the suppression and repertoire shifting. It interfered with CD28 signaling to responder Tconv cells via outcompeting CD28 for binding to CD80 and CD86,or modulating CD80/CD86 expression on antigen-presenting cells. Thus, a triad of IL-2 repression, CTLA-4 expression, and antigenic stimulation is a minimalistic requirement for conferring Treg-like suppressive activity on Tconv cells, in accordance with the function of forkhead box p3 to strongly repress IL-2 and maintain CTLA-4 expression in natural Treg cells. Moreover, CTLA-4 expression is a key element for the formation of a self-reactive TCR repertoire in natural Treg cells. These findings can be exploited to control immune responses by targeting IL-2 and CTLA-4 in Treg and Tconv cells.

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Miniature curved artificial compound eyes.

Publication Date: 2013 May 20 PMID: 23690574
Authors: Floreano, D. - Pericet-Camara, R. - Viollet, S. - Ruffier, F. - Bruckner, A. - Leitel, R. - Buss, W. - Menouni, M. - Expert, F. - Juston, R. - Dobrzynski, M. K. - L'eplattenier, G. - Recktenwald, F. - Mallot, H. A. - Franceschini, N.
Journal: Proc Natl Acad Sci U S A

In most animal species, vision is mediated by compound eyes, which offer lower resolution than vertebrate single-lens eyes, but significantly larger fields of view with negligible distortion and spherical aberration, as well as high temporal resolution in a tiny package. Compound eyes are ideally suited for fast panoramic motion perception. Engineering a miniature artificial compound eye is challenging because it requires accurate alignment of photoreceptive and optical components on a curved surface. Here, we describe a unique design method for biomimetic compound eyes featuring a panoramic, undistorted field of view in a very thin package. The design consists of three planar layers of separately produced arrays, namely, a microlens array, a neuromorphic photodetector array, and a flexible printed circuit board that are stacked, cut, and curved to produce a mechanically flexible imager. Following this method, we have prototyped and characterized an artificial compound eye bearing a hemispherical field of view with embedded and programmable low-power signal processing, high temporal resolution, and local adaptation to illumination. The prototyped artificial compound eye possesses several characteristics similar to the eye of the fruit fly Drosophila and other arthropod species. This design method opens up additional vistas for a broad range of applications in which wide field motion detection is at a premium, such as collision-free navigation of terrestrial and aerospace vehicles, and for the experimental testing of insect vision theories.

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Systematic functional regulatory assessment of disease-associated variants.

Publication Date: 2013 May 20 PMID: 23690573
Authors: Karczewski, K. J. - Dudley, J. T. - Kukurba, K. R. - Chen, R. - Butte, A. J. - Montgomery, S. B. - Snyder, M.
Journal: Proc Natl Acad Sci U S A

Genome-wide association studies have discovered many genetic loci associated with disease traits, but the functional molecular basis of these associations is often unresolved. Genome-wide regulatory and gene expression profiles measured across individuals and diseases reflect downstream effects of genetic variation and may allow for functional assessment of disease-associated loci. Here, we present a unique approach for systematic integration of genetic disease associations, transcription factor binding among individuals, and gene expression data to assess the functional consequences of variants associated with hundreds of human diseases. In an analysis of genome-wide binding profiles of NFkappaB, we find that disease-associated SNPs are enriched in NFkappaB binding regions overall, and specifically for inflammatory-mediated diseases, such as asthma, rheumatoid arthritis, and coronary artery disease. Using genome-wide variation in transcription factor-binding data, we find that NFkappaB binding is often correlated with disease-associated variants in a genotype-specific and allele-specific manner. Furthermore, we show that this binding variation is often related to expression of nearby genes, which are also found to have altered expression in independent profiling of the variant-associated disease condition. Thus, using this integrative approach, we provide a unique means to assign putative function to many disease-associated SNPs.

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Graphene-layered steps and their fields visualized by 4D electron microscopy.

Publication Date: 2013 May 20 PMID: 23690572
Authors: Park, S. T. - Yurtsever, A. - Baskin, J. S. - Zewail, A. H.
Journal: Proc Natl Acad Sci U S A

Enhanced image contrast has been seen at graphene-layered steps a few nanometers in height by means of photon-induced near-field electron microscopy (PINEM) using synchronous femtosecond pulses of light and electrons. The observed steps are formed by the edges of graphene strips lying on the surface of a graphene substrate, where the strips are hundreds of nanometers in width and many micrometers in length. PINEM measurements reflect the interaction of imaging electrons and induced (near) electric fields at the steps, and this leads to a much higher contrast than that achieved in bright-field transmission electron microscopy imaging of the same strips. Theory and numerical simulations support the experimental PINEM findings and elucidate the nature of the electric field at the steps formed by the graphene layers. These results extend the range of applications of the experimental PINEM methodology, which has previously been demonstrated for spherical, cylindrical, and triangular nanostructures, to shapes of high aspect ratio (rectangular strips), as well as into the regime of atomic layer thicknesses.

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Coherent Fano resonances in a plasmonic nanocluster enhance optical four-wave mixing.

Publication Date: 2013 May 20 PMID: 23690571
Authors: Zhang, Y. - Wen, F. - Zhen, Y. R. - Nordlander, P. - Halas, N. J.
Journal: Proc Natl Acad Sci U S A

Plasmonic nanoclusters, an ordered assembly of coupled metallic nanoparticles, support unique spectral features known as Fano resonances due to the coupling between their subradiant and superradiant plasmon modes. Within the Fano resonance, absorption is significantly enhanced, giving rise to highly localized, intense near fields with the potential to enhance nonlinear optical processes. Here, we report a structure supporting the coherent oscillation of two distinct Fano resonances within an individual plasmonic nanocluster. We show how this coherence enhances the optical four-wave mixing process in comparison with other double-resonant plasmonic clusters that lack this property. A model that explains the observed four-wave mixing features is proposed, which is generally applicable to any third-order process in plasmonic nanostructures. With a larger effective susceptibility chi(3) relative to existing nonlinear optical materials, this coherent double-resonant nanocluster offers a strategy for designing high-performance third-order nonlinear optical media.

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Stable nematic droplets with handles.

Publication Date: 2013 May 20 PMID: 23690570
Authors: Pairam, E. - Vallamkondu, J. - Koning, V. - van Zuiden, B. C. - Ellis, P. W. - Bates, M. A. - Vitelli, V. - Fernandez-Nieves, A.
Journal: Proc Natl Acad Sci U S A

We stabilize nematic droplets with handles against surface tension-driven instabilities, using a yield-stress material as outer fluid, and study the complex nematic textures and defect structures that result from the competition between topological constraints and the elasticity of the nematic liquid crystal. We uncover a surprisingly persistent twisted configuration of the nematic director inside the droplets when tangential anchoring is established at their boundaries, which we explain after considering the influence of saddle splay on the elastic free energy. For toroidal droplets, we find that the saddle-splay energy screens the twisting energy, resulting in a spontaneous breaking of mirror symmetry; the chiral twisted state persists for aspect ratios as large as approximately 20. For droplets with additional handles, we observe in experiments and computer simulations that there are two additional -1 surface defects per handle; these are located in regions with local saddle geometry to minimize the nematic distortions and hence the corresponding elastic free energy.

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Space can substitute for time in predicting climate-change effects on biodiversity.

Publication Date: 2013 May 20 PMID: 23690569
Authors: Blois, J. L. - Williams, J. W. - Fitzpatrick, M. C. - Jackson, S. T. - Ferrier, S.
Journal: Proc Natl Acad Sci U S A

"Space-for-time" substitution is widely used in biodiversity modeling to infer past or future trajectories of ecological systems from contemporary spatial patterns. However, the foundational assumption-that drivers of spatial gradients of species composition also drive temporal changes in diversity-rarely is tested. Here, we empirically test the space-for-time assumption by constructing orthogonal datasets of compositional turnover of plant taxa and climatic dissimilarity through time and across space from Late Quaternary pollen records in eastern North America, then modeling climate-driven compositional turnover. Predictions relying on space-for-time substitution were approximately 72% as accurate as "time-for-time" predictions. However, space-for-time substitution performed poorly during the Holocene when temporal variation in climate was small relative to spatial variation and required subsampling to match the extent of spatial and temporal climatic gradients. Despite this caution, our results generally support the judicious use of space-for-time substitution in modeling community responses to climate change.

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Broader perspective on ecosystem sustainability: Consequences for decision making.

Publication Date: 2013 May 17 PMID: 23686583
Authors: Sidle, R. C. - Benson, W. H. - Carriger, J. F. - Kamai, T.
Journal: Proc Natl Acad Sci U S A

Although the concept of ecosystem sustainability has a long-term focus, it is often viewed from a static system perspective. Because most ecosystems are dynamic, we explore sustainability assessments from three additional perspectives: resilient systems; systems where tipping points occur; and systems subject to episodic resetting. Whereas foundations of ecosystem resilience originated in ecology, recent discussions have focused on geophysical attributes, and it is recognized that dynamic system components may not return to their former state following perturbations. Tipping points emerge when chronic changes (typically anthropogenic, but sometimes natural) push ecosystems to thresholds that cause collapse of process and function and may become permanent. Ecosystem resetting occurs when episodic natural disasters breach thresholds with little or no warning, resulting in long-term changes to environmental attributes or ecosystem function. An example of sustainability assessment of ecosystem goods and services along the Gulf Coast (USA) demonstrates the need to include both the resilient and dynamic nature of biogeomorphic components. Mountain road development in northwest Yunnan, China, makes rivers and related habitat vulnerable to tipping points. Ecosystems reset by natural disasters are also presented, emphasizing the need to understand the magnitude frequency and interrelationships among major disturbances, as shown by (i) the 2011 Great East Japan Earthquake and resulting tsunami, including how unsustainable urban development exacerbates geodisaster propagation, and (ii) repeated major earthquakes and associated geomorphic and vegetation disturbances in Papua New Guinea. Although all of these ecosystem perturbations and shifts are individually recognized, they are not embraced in contemporary sustainable decision making.

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Hilary Koprowski (1916-2013): Vaccine pioneer, art lover, and scientific leader.

Publication Date: 2013 May 17 PMID: 23686582
Authors: Croce, C. M.
Journal: Proc Natl Acad Sci U S A

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Ten principles for a landscape approach to reconciling agriculture, conservation, and other competing land uses.

Publication Date: 2013 May 21 PMID: 23686581
Authors: Sayer, J. - Sunderland, T. - Ghazoul, J. - Pfund, J. L. - Sheil, D. - Meijaard, E. - Venter, M. - Boedhihartono, A. K. - Day, M. - Garcia, C. - van Oosten, C. - Buck, L. E.
Journal: Proc Natl Acad Sci U S A

"Landscape approaches" seek to provide tools and concepts for allocating and managing land to achieve social, economic, and environmental objectives in areas where agriculture, mining, and other productive land uses compete with environmental and biodiversity goals. Here we synthesize the current consensus on landscape approaches. This is based on published literature and a consensus-building process to define good practice and is validated by a survey of practitioners. We find the landscape approach has been refined in response to increasing societal concerns about environment and development tradeoffs. Notably, there has been a shift from conservation-orientated perspectives toward increasing integration of poverty alleviation goals. We provide 10 summary principles to support implementation of a landscape approach as it is currently interpreted. These principles emphasize adaptive management, stakeholder involvement, and multiple objectives. Various constraints are recognized, with institutional and governance concerns identified as the most severe obstacles to implementation. We discuss how these principles differ from more traditional sectoral and project-based approaches. Although no panacea, we see few alternatives that are likely to address landscape challenges more effectively than an approach circumscribed by the principles outlined here.

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Assessment of multidrug resistance on cell coculture patterns using scanning electrochemical microscopy.

Publication Date: 2013 May 17 PMID: 23686580
Authors: Kuss, S. - Polcari, D. - Geissler, M. - Brassard, D. - Mauzeroll, J.
Journal: Proc Natl Acad Sci U S A

The emergence of resistance to multiple unrelated chemotherapeutic drugs impedes the treatment of several cancers. Although the involvement of ATP-binding cassette transporters has long been known, there is no in situ method capable of tracking this transporter-related resistance at the single-cell level without interfering with the cell's environment or metabolism. Here, we demonstrate that scanning electrochemical microscopy (SECM) can quantitatively and noninvasively track multidrug resistance-related protein 1-dependent multidrug resistance in patterned adenocarcinoma cervical cancer cells. Nonresistant human cancer cells and their multidrug resistant variants are arranged in a side-by-side format using a stencil-based patterning scheme, allowing for precise positioning of target cells underneath the SECM sensor. SECM measurements of the patterned cells, performed with ferrocenemethanol and [Ru(NH3)6]3+ serving as electrochemical indicators, are used to establish a kinetic "map" of constant-height SECM scans, free of topography contributions. The concept underlying the work described herein may help evaluate the effectiveness of treatment administration strategies targeting reduced drug efflux.

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Warm temperatures induce transgenerational epigenetic release of RNA silencing by inhibiting siRNA biogenesis in Arabidopsis.

Publication Date: 2013 May 17 PMID: 23686579
Authors: Zhong, S. H. - Liu, J. Z. - Jin, H. - Lin, L. - Li, Q. - Chen, Y. - Yuan, Y. X. - Wang, Z. Y. - Huang, H. - Qi, Y. J. - Chen, X. Y. - Vaucheret, H. - Chory, J. - Li, J. - He, Z. H.
Journal: Proc Natl Acad Sci U S A

Owing to their sessile nature, plants have evolved sophisticated genetic and epigenetic regulatory systems to respond quickly and reversibly to daily and seasonal temperature changes. However, our knowledge of how plants sense and respond to warming ambient temperatures is rather limited. Here we show that an increase in growth temperature from 22 degrees C to 30 degrees C effectively inhibited transgene-induced posttranscriptional gene silencing (PTGS) in Arabidopsis. Interestingly, warmth-induced PTGS release exhibited transgenerational epigenetic inheritance. We discovered that the warmth-induced PTGS release occurred during a critical step that leads to the formation of double-stranded RNA (dsRNA) for producing small interfering RNAs (siRNAs). Deep sequencing of small RNAs and RNA blot analysis indicated that the 22-30 degrees C increase resulted in a significant reduction in the abundance of many trans-acting siRNAs that require dsRNA for biogenesis. We discovered that the temperature increase reduced the protein abundance of SUPPRESSOR OF GENE SILENCING 3, as a consequence, attenuating the formation of stable dsRNAs required for siRNA biogenesis. Importantly, SUPPRESSOR OF GENE SILENCING 3 overexpression released the warmth-triggered inhibition of siRNA biogenesis and reduced the transgenerational epigenetic memory. Thus, our study reveals a previously undescribed association between warming temperatures, an epigenetic system, and siRNA biogenesis.

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Original antigenic sin in dengue revisited.

Publication Date: 2013 May 16 PMID: 23686453
Authors: Zompi, S. - Harris, E.
Journal: Proc Natl Acad Sci U S A

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STED super-resolution microscopy reveals an array of MINOS clusters along human mitochondria.

Publication Date: 2013 May 15 PMID: 23676277
Authors: Jans, D. C. - Wurm, C. A. - Riedel, D. - Wenzel, D. - Stagge, F. - Deckers, M. - Rehling, P. - Jakobs, S.
Journal: Proc Natl Acad Sci U S A

The mitochondrial inner membrane organizing system (MINOS) is a conserved large hetero-oligomeric protein complex in the mitochondrial inner membrane, crucial for the maintenance of cristae morphology. MINOS has been suggested to represent the core of an extended protein network that controls mitochondrial function and structure, and has been linked to several human diseases. The spatial arrangement of MINOS within mitochondria is ill-defined, however. Using super-resolution stimulated emission depletion (STED) microscopy and immunogold electron microscopy, we determined the distribution of three known human MINOS subunits (mitofilin, MINOS1, and CHCHD3) in mammalian cells. Super-resolution microscopy revealed that all three subunits form similar clusters within mitochondria, and that MINOS is more abundant in mitochondria around the nucleus than in peripheral mitochondria. At the submitochondrial level, mitofilin, a core MINOS subunit, is preferentially localized at cristae junctions. In primary human fibroblasts, mitofilin labeling uncovered a regularly spaced pattern of clusters arranged in parallel to the cell growth surfaces. We suggest that this array of MINOS complexes might explain the observed phenomenon of largely horizontally arranged cristae junctions that connect the inner boundary membrane to lamellar cristae. The super-resolution images demonstrate an unexpectedly high level of regularity in the nanoscale distribution of the MINOS complex in human mitochondria, supporting an integrating role of MINOS in the structural organization of the organelle.

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A unified selection signal for attention and reward in primary visual cortex.

Publication Date: 2013 May 15 PMID: 23676276
Authors: Stanisor, L. - van der Togt, C. - Pennartz, C. M. - Roelfsema, P. R.
Journal: Proc Natl Acad Sci U S A

Stimuli associated with high rewards evoke stronger neuronal activity than stimuli associated with lower rewards in many brain regions. It is not well understood how these reward effects influence activity in sensory cortices that represent low-level stimulus features. Here, we investigated the effects of reward information in the primary visual cortex (area V1) of monkeys. We found that the reward value of a stimulus relative to the value of other stimuli is a good predictor of V1 activity. Relative value biases the competition between stimuli, just as has been shown for selective attention. The neuronal latency of this reward value effect in V1 was similar to the latency of attentional influences. Moreover, V1 neurons with a strong value effect also exhibited a strong attention effect, which implies that relative value and top-down attention engage overlapping, if not identical, neuronal selection mechanisms. Our findings demonstrate that the effects of reward value reach down to the earliest sensory processing levels of the cerebral cortex and imply that theories about the effects of reward coding and top-down attention on visual representations should be unified.

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Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol.

Publication Date: 2013 May 15 PMID: 23676275
Authors: Yadid, I. - Rudolph, J. - Hlouchova, K. - Copley, S. D.
Journal: Proc Natl Acad Sci U S A

Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolved pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly reactive intermediate; tetrachlorobenzoquinone (TCBQ) is a potent alkylating agent that reacts with cellular thiols at a diffusion-controlled rate. TCBQ reductase (PcpD), an FMN- and NADH-dependent reductase, catalyzes the reduction of TCBQ to tetrachlorohydroquinone. In the presence of PcpD, TCBQ formed by pentachlorophenol hydroxylase (PcpB) is sequestered until it is reduced to the less toxic tetrachlorohydroquinone, protecting the bacterium from the toxic effects of TCBQ and maintaining flux through the pathway. The toxicity of TCBQ may have exerted selective pressure to maintain slow turnover of PcpB (0.02 s-1) so that a transient interaction between PcpB and PcpD can occur before TCBQ is released from the active site of PcpB.

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Interaction of 14-3-3 proteins with the Estrogen Receptor Alpha F domain provides a drug target interface.

Publication Date: 2013 May 15 PMID: 23676274
Authors: De Vries-van Leeuwen, I. J. - da Costa Pereira, D. - Flach, K. D. - Piersma, S. R. - Haase, C. - Bier, D. - Yalcin, Z. - Michalides, R. - Feenstra, K. A. - Jimenez, C. R. - de Greef, T. F. - Brunsveld, L. - Ottmann, C. - Zwart, W. - de Boer, A. H.
Journal: Proc Natl Acad Sci U S A

Estrogen receptor alpha (ERalpha) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERalpha, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ERalpha dimerization is essential for its activity, interference with receptor dimerization offers a new opportunity to exploit in drug design. Here we describe a unique mechanism of how ERalpha dimerization is negatively controlled by interaction with 14-3-3 proteins at the extreme C terminus of the receptor. Moreover, the small-molecule fusicoccin (FC) stabilizes this ERalpha/14-3-3 interaction. Cocrystallization of the trimeric ERalpha/14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ERalpha-T594) for high-affinity interaction. We confirm that T594 is a distinct ERalpha phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T594-specific antibody and by mass spectrometry. In line with its ERalpha/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ERalpha dimerization, inhibits ERalpha/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ERalpha are provided, together with a small molecule that selectively targets this ERalpha/14-3-3 interface.

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Prefrontal microcircuit underlies contextual learning after hippocampal loss.

Publication Date: 2013 May 15 PMID: 23676273
Authors: Zelikowsky, M. - Bissiere, S. - Hast, T. A. - Bennett, R. Z. - Abdipranoto, A. - Vissel, B. - Fanselow, M. S.
Journal: Proc Natl Acad Sci U S A

Specific brain circuits have been classically linked to dedicated functions. However, compensation following brain damage suggests that these circuits are capable of dynamic adaptation. Such compensation is exemplified by Pavlovian fear conditioning following damage to the dorsal hippocampus (DH). Although the DH normally underlies contextual fear and fear renewal after extinction, both can be learned in the absence of the DH, although the mechanisms and nature of this compensation are currently unknown. Here, we report that recruitment of alternate structures, specifically the infralimbic and prelimbic prefrontal cortices, is required for compensation following damage to the hippocampus. Disconnection of these cortices in DH-compromised animals and immediate early gene induction profiles for amygdala-projecting prefrontal cells revealed that communication and dynamic rebalancing within this prefrontal microcircuit is critical. Additionally, the infralimbic cortex normally plays a role in limiting generalization of contextual fear. These discoveries reveal that plasticity through recruitment of alternate circuits allows the brain to compensate following damage, offering promise for targeted treatment of memory disorders.

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Arid5a controls IL-6 mRNA stability, which contributes to elevation of IL-6 level in vivo.

Publication Date: 2013 May 15 PMID: 23676272
Authors: Masuda, K. - Ripley, B. - Nishimura, R. - Mino, T. - Takeuchi, O. - Shioi, G. - Kiyonari, H. - Kishimoto, T.
Journal: Proc Natl Acad Sci U S A

Posttranscriptional regulation of IL-6 has been largely uncharacterized, with the exception of the ribonuclease Regnase-1, which prevents autoimmunity by destabilizing IL-6 mRNA. Here, we identified AT-rich interactive domain-containing protein 5A (Arid5a) as a unique RNA binding protein, which stabilizes IL-6 but not TNF-alpha mRNA through binding to the 3' untranslated region of IL-6 mRNA. Arid5a was enhanced in macrophages in response to LPS, IL-1beta, and IL-6. Arid5a deficiency inhibited elevation of IL-6 serum level in LPS-treated mice and suppressed IL-6 levels and the development of TH17 cells in experimental autoimmune encephalomyelitis. Importantly, Arid5a inhibited the destabilizing effect of Regnase-1 on IL-6 mRNA. These results indicate that Arid5a plays an important role in promotion of inflammatory processes and autoimmune diseases.

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Notch2 regulates BMP signaling and epithelial morphogenesis in the ciliary body of the mouse eye.

Publication Date: 2013 May 15 PMID: 23676271
Authors: Zhou, Y. - Tanzie, C. - Yan, Z. - Chen, S. - Duncan, M. - Gaudenz, K. - Li, H. - Seidel, C. - Lewis, B. - Moran, A. - Libby, R. T. - Kiernan, A. E. - Xie, T.
Journal: Proc Natl Acad Sci U S A

The ciliary body (CB) of the mammalian eye is responsible for secreting aqueous humor to maintain intraocular pressure, which is elevated in the eyes of glaucoma patients. It contains a folded two-layered epithelial structure comprising the nonpigmented inner ciliary epithelium (ICE), the pigmented outer ciliary epithelium (OCE), and the underlying stroma. Although the CB has an important function in the eye, its morphogenesis remains poorly studied. In this study, we show that conditional inactivation of the Jagged 1 (Jag1)-Notch2 signaling pathway in the developing CB abolishes its morphogenesis. Notch2 is expressed in the OCE of the CB, whereas Jag1 is expressed in the ICE. Conditional inactivation of Jag1 in the ICE or Notch2 in the OCE disrupts CB morphogenesis, but neither affects the specification of the CB region. Notch2 signaling in the OCE is required for promoting cell proliferation and maintaining bone morphogenetic protein (BMP) signaling, both of which have been suggested to be important for CB morphogenesis. Although Notch and BMP signaling pathways are known to cross-talk via the interaction between their downstream transcriptional factors, this study suggests that Notch2 maintains BMP signaling in the OCE possibly by repressing expression of secreted BMP inhibitors. Based on our findings, we propose that Jag1-Notch2 signaling controls CB morphogenesis at least in part by regulating cell proliferation and BMP signaling.

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Coupled Ca2+/H+ transport by cytoplasmic buffers regulates local Ca2+ and H+ ion signaling.

Publication Date: 2013 May 15 PMID: 23676270
Authors: Swietach, P. - Youm, J. B. - Saegusa, N. - Leem, C. H. - Spitzer, K. W. - Vaughan-Jones, R. D.
Journal: Proc Natl Acad Sci U S A

Ca2+ signaling regulates cell function. This is subject to modulation by H+ ions that are universal end-products of metabolism. Due to slow diffusion and common buffers, changes in cytoplasmic [Ca2+] ([Ca2+]i) or [H+] ([H+]i) can become compartmentalized, leading potentially to complex spatial Ca2+/H+ coupling. This was studied by fluorescence imaging of cardiac myocytes. An increase in [H+]i, produced by superfusion of acetate (salt of membrane-permeant weak acid), evoked a [Ca2+]i rise, independent of sarcolemmal Ca2+ influx or release from mitochondria, sarcoplasmic reticulum, or acidic stores. Photolytic H+ uncaging from 2-nitrobenzaldehyde also raised [Ca2+]i, and the yield was reduced following inhibition of glycolysis or mitochondrial respiration. H+ uncaging into buffer mixtures in vitro demonstrated that Ca2+ unloading from proteins, histidyl dipeptides (HDPs; e.g., carnosine), and ATP can underlie the H+-evoked [Ca2+]i rise. Raising [H+]i tonically at one end of a myocyte evoked a local [Ca2+]i rise in the acidic microdomain, which did not dissipate. The result is consistent with uphill Ca2+ transport into the acidic zone via Ca2+/H+ exchange on diffusible HDPs and ATP molecules, energized by the [H+]i gradient. Ca2+ recruitment to a localized acid microdomain was greatly reduced during intracellular Mg2+ overload or by ATP depletion, maneuvers that reduce the Ca2+-carrying capacity of HDPs. Cytoplasmic HDPs and ATP underlie spatial Ca2+/H+ coupling in the cardiac myocyte by providing ion exchange and transport on common buffer sites. Given the abundance of cellular HDPs and ATP, spatial Ca2+/H+ coupling is likely to be of general importance in cell signaling.

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ADAM-10 and -17 regulate endometriotic cell migration via concerted ligand and receptor shedding feedback on kinase signaling.

Publication Date: 2013 May 14 PMID: 23674691
Authors: Miller, M. A. - Meyer, A. S. - Beste, M. T. - Lasisi, Z. - Reddy, S. - Jeng, K. W. - Chen, C. H. - Han, J. - Isaacson, K. - Griffith, L. G. - Lauffenburger, D. A.
Journal: Proc Natl Acad Sci U S A

A Disintegrin and Metalloproteinases (ADAMs) are the principal enzymes for shedding receptor tyrosine kinase (RTK) ectodomains and ligands from the cell surface. Multiple layers of activity regulation, feedback, and catalytic promiscuity impede our understanding of context-dependent ADAM "sheddase" function and our ability to predictably target that function in disease. This study uses combined measurement and computational modeling to examine how various growth factor environments influence sheddase activity and cell migration in the invasive disease of endometriosis. We find that ADAM-10 and -17 dynamically integrate numerous signaling pathways to direct cell motility. Data-driven modeling reveals that induced cell migration is a quantitative function of positive feedback through EGF ligand release and negative feedback through RTK shedding. Although sheddase inhibition prevents autocrine ligand shedding and resultant EGF receptor transactivation, it also leads to an accumulation of phosphorylated receptors (HER2, HER4, and MET) on the cell surface, which subsequently enhances Jnk/p38 signaling. Jnk/p38 inhibition reduces cell migration by blocking sheddase activity while additionally preventing the compensatory signaling from accumulated RTKs. In contrast, Mek inhibition reduces ADAM-10 and -17 activities but fails to inhibit compensatory signaling from accumulated RTKs, which actually enhances cell motility in some contexts. Thus, here we present a sheddase-based mechanism of rapidly acquired resistance to Mek inhibition through reduced RTK shedding that can be overcome with rationally directed combination inhibitor treatment. We investigate the clinical relevance of these findings using targeted proteomics of peritoneal fluid from endometriosis patients and find growth-factor-driven ADAM-10 activity and MET shedding are jointly dysregulated with disease.

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End-binding proteins sensitize microtubules to the action of microtubule-targeting agents.

Publication Date: 2013 May 14 PMID: 23674690
Authors: Mohan, R. - Katrukha, E. A. - Doodhi, H. - Smal, I. - Meijering, E. - Kapitein, L. C. - Steinmetz, M. O. - Akhmanova, A.
Journal: Proc Natl Acad Sci U S A

Microtubule-targeting agents (MTAs) are widely used for treatment of cancer and other diseases, and a detailed understanding of the mechanism of their action is important for the development of improved microtubule-directed therapies. Although there is a large body of data on the interactions of different MTAs with purified tubulin and microtubules, much less is known about how the effects of MTAs are modulated by microtubule-associated proteins. Among the regulatory factors with a potential to have a strong impact on MTA activity are the microtubule plus end-tracking proteins, which control multiple aspects of microtubule dynamic instability. Here, we reconstituted microtubule dynamics in vitro to investigate the influence of end-binding proteins (EBs), the core components of the microtubule plus end-tracking protein machinery, on the effects that MTAs exert on microtubule plus-end growth. We found that EBs promote microtubule catastrophe induction in the presence of all MTAs tested. Analysis of microtubule growth times supported the view that catastrophes are microtubule age dependent. This analysis indicated that MTAs affect microtubule aging in multiple ways: destabilizing MTAs, such as colchicine and vinblastine, accelerate aging in an EB-dependent manner, whereas stabilizing MTAs, such as paclitaxel and peloruside A, induce not only catastrophes but also rescues and can reverse the aging process.

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Mutant presenilin 1 expression in excitatory neurons impairs enrichment-mediated phenotypes of adult hippocampal progenitor cells.

Publication Date: 2013 May 14 PMID: 23674689
Authors: Veeraraghavalu, K. - Sisodia, S. S.
Journal: Proc Natl Acad Sci U S A

Inheritance of mutant presenilin 1 genes (PSEN1) encoding presenilin 1 (PS1)variants causes autosomal dominant forms of familial Alzheimer's disease (FAD). We previously reported that ubiquitous expression of FAD-linked PS1 variants in mice impairs environmental enrichment (EE)-induced proliferation and neuronal commitment of adult hippocampal neural progenitor cells (AHNPCs). Notably, the self-renewal and differentiation properties of cultured AHNPCs expressing either human PS1 wild-type or PS1 variants were identical, suggesting that accessory cells within the hippocampal niche expressing PS1 variants may modulate AHNPC phenotypes in vivo. We now report that nontransgenic mouse AHNPCs transduced with retroviruses harboring cDNAs that encode either human PS1 wild-type or FAD-linked PS1 variants show no differences in EE-mediated proliferation and neuronal differentiation. Moreover, conditional inactivation of a mutant PS1 transgene in type-1 primary progenitor cells failed to rescue impairments of EE-induced proliferation, survival, or neurogenesis. In contrast, conditional inactivation of the mutant PS1 transgene in excitatory neurons of the mouse forebrain largely rescued the deficits in EE-induced proliferation and survival of AHNPCs, but not their differentiation into mature neuronal phenotypes. These results persuasively argue for a noncell autonomous effect of FAD-linked PS1 mutants on EE-mediated adult hippocampal neurogenesis.

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Tissue plasminogen activator regulates Purkinje neuron development and survival.

Publication Date: 2013 May 14 PMID: 23674688
Authors: Li, J. - Yu, L. - Gu, X. - Ma, Y. - Pasqualini, R. - Arap, W. - Snyder, E. Y. - Sidman, R. L.
Journal: Proc Natl Acad Sci U S A

The cerebellar cortex is centrally involved in motor coordination and learning, and its sole output is provided by Purkinje neurons (PNs). Growth of PN dendrites and their major synaptic input from granule cell parallel fiber axons takes place almost entirely in the first several postnatal weeks. PNs are more vulnerable to cell death than most other neurons, but the mechanisms remain unclear. We find that the homozygous nervous (nr) mutant mouse's 10-fold-increased cerebellar tissue plasminogen activator (tPA), a part of the tPA/plasmin proteolytic system, influences several different molecular mechanisms, each regulating a key aspect of postnatal PN development, followed by selective PN necrosis, as follows. (i) Excess endogenous or exogenous tPA inhibits dendritic growth in vivo and in vitro by activating protein kinase Cgamma and phosphorylation of microtubule-associated protein 2. (ii) tPA/plasmin proteolysis impairs parallel fiber-PN synaptogenesis by blocking brain-derived neurotrophic factor/tyrosine kinase receptor B signaling. (iii) Voltage-dependent anion channel 1 (a mitochondrial and plasma membrane protein) bound with kringle 5 (a peptide derived from the excess plasminogen) promotes pathological enlargement and rounding of PN mitochondria, reduces mitochondrial membrane potential, and damages plasma membranes. These abnormalities culminate in young nr PN necrosis that can be mimicked in wild-type PNs by exogenous tPA injection into cerebellum or prevented by endogenous tPA deletion in nr:tPA-knockout double mutants. In sum, excess tPA/plasmin, through separate downstream molecular mechanisms, regulates postnatal PN dendritogenesis, synaptogenesis, mitochondrial structure and function, and selective PN viability.

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LYM2-dependent chitin perception limits molecular flux via plasmodesmata.

Publication Date: 2013 May 14 PMID: 23674687
Authors: Faulkner, C. - Petutschnig, E. - Benitez-Alfonso, Y. - Beck, M. - Robatzek, S. - Lipka, V. - Maule, A. J.
Journal: Proc Natl Acad Sci U S A

Chitin acts as a pathogen-associated molecular pattern from fungal pathogens whose perception triggers a range of defense responses. We show that LYSIN MOTIF DOMAIN-CONTAINING GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN 2 (LYM2), the Arabidopsis homolog of a rice chitin receptor-like protein, mediates a reduction in molecular flux via plasmodesmata in the presence of chitin. For this response, lym2-1 mutants are insensitive to the presence of chitin, but not to the flagellin derivative flg22. Surprisingly, the chitin-recognition receptor CHITIN ELCITOR RECEPTOR KINASE 1 (CERK1) is not required for chitin-induced changes to plasmodesmata flux, suggesting that there are at least two chitin-activated response pathways in Arabidopsis and that LYM2 is not required for CERK1-mediated chitin-triggered defense responses, indicating that these pathways are independent. In accordance with a role in the regulation of intercellular flux, LYM2 is resident at the plasma membrane and is enriched at plasmodesmata. Chitin-triggered regulation of molecular flux between cells is required for defense responses against the fungal pathogen Botrytis cinerea, and thus we conclude that the regulation of symplastic continuity and molecular flux between cells is a vital component of chitin-triggered immunity in Arabidopsis.

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Role of a polymorphism in a Hox/Pax-responsive enhancer in the evolution of the vertebrate spine.

Publication Date: 2013 May 14 PMID: 23674686
Authors: Guerreiro, I. - Nunes, A. - Woltering, J. M. - Casaca, A. - Novoa, A. - Vinagre, T. - Hunter, M. E. - Duboule, D. - Mallo, M.
Journal: Proc Natl Acad Sci U S A

Patterning of the vertebrate skeleton requires the coordinated activity of Hox genes. In particular, Hox10 proteins are essential to set the transition from thoracic to lumbar vertebrae because of their rib-repressing activity. In snakes, however, the thoracic region extends well into Hox10-expressing areas of the embryo, suggesting that these proteins are unable to block rib formation. Here, we show that this is not a result of the loss of rib-repressing properties by the snake proteins, but rather to a single base pair change in a Hox/Paired box (Pax)-responsive enhancer, which prevents the binding of Hox proteins. This polymorphism is also found in Paenungulata, such as elephants and manatees, which have extended rib cages. In vivo, this modified enhancer failed to respond to Hox10 activity, supporting its role in the extension of rib cages. In contrast, the enhancer could still interact with Hoxb6 and Pax3 to promote rib formation. These results suggest that a polymorphism in the Hox/Pax-responsive enhancer may have played a role in the evolution of the vertebrate spine by differently modulating its response to rib-suppressing and rib-promoting Hox proteins.

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Cross-validation in cryo-EM-based structural modeling.

Publication Date: 2013 May 14 PMID: 23674685
Authors: Falkner, B. - Schroder, G. F.
Journal: Proc Natl Acad Sci U S A

Single-particle cryo-EM is a powerful approach to determine the structure of large macromolecules and assemblies thereof in many cases at subnanometer resolution. It has become popular to refine or flexibly fit atomic models into density maps derived from cryo-EM experiments. These density maps are typically significantly lower in resolution than electron density maps obtained from X-ray diffraction experiments, such that the number of parameters that need to be determined is much larger than the number of experimental observables. Overfitting and misinterpretation of the density, thus, become a serious problem. For diffraction data, a cross-validation approach was introduced almost 20 y ago; however, no such approach has been described yet for structure refinement against cryo-EM density maps, although the overfitting problem is, because of the lower resolution, significantly larger. We present a cross-validation approach for real-space refinement against cryo-EM density maps in analogy to cross-validation typically used in crystallography. Our approach is able to detect overfitting and allows for optimizing the choice of restraints used in the refinement. The approach is shown on three protein structures with simulated data and experimental data of the rotavirus double-layer particle. Because cross-validation requires splitting the dataset into at least two independent sets, we further present an approach to quantify correlations between the structure factor sets. This analysis is also helpful for other cross-validation applications, such as refinements against diffraction data or 3D reconstructions of cryo-EM density maps.

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Rapid feedback regulation of synaptic efficacy during high-frequency activity at the Drosophila larval neuromuscular junction.

Publication Date: 2013 May 14 PMID: 23674684
Authors: Kauwe, G. - Isacoff, E. Y.
Journal: Proc Natl Acad Sci U S A

High-frequency firing of neurons depresses transmitter release at many synapses. At the glutamatergic synapse of the Drosophila larval neuromuscular junction, we find that presynaptic depression is modulated by postsynaptic ionotropic glutamate receptor (iGluR) activity. Although basal release at low frequency was insensitive to postsynaptic iGluR activity, recovery from depression elicited by high-frequency presynaptic trains decreased with partial block of native iGluRs. Moreover, recovery from depression increased with optical activation of the light-gated mammalian iGluR6 (LiGluR) expressed postsynaptically. The enhancement of recovery from depression occurred within 2 min of optical activation of LiGluR and persisted for minutes after optical deactivation. This effect depended on cAMP-dependent presynaptic recruitment of vesicles from the reserve pool. Our findings reveal a unique dimension to postsynaptic iGluR activity: fast retrograde signaling that preserves transmission efficacy during high-frequency presynaptic firing.

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Host and viral features of human dengue cases shape the population of infected and infectious Aedes aegypti mosquitoes.

Publication Date: 2013 May 14 PMID: 23674683
Authors: Nguyen, N. M. - Thi Hue Kien, D. - Tuan, T. V. - Quyen, N. T. - Tran, C. N. - Vo Thi, L. - Thi, D. L. - Nguyen, H. L. - Farrar, J. J. - Holmes, E. C. - Rabaa, M. A. - Bryant, J. E. - Nguyen, T. T. - Nguyen, H. T. - Nguyen, L. T. - Pham, M. P. - Nguyen, H. T. - Luong, T. T. - Wills, B. - Nguyen, C. V. - Wolbers, M. - Simmons, C. P.
Journal: Proc Natl Acad Sci U S A

Dengue is the most prevalent arboviral disease of humans. The host and virus variables associated with dengue virus (DENV) transmission from symptomatic dengue cases (n = 208) to Aedes aegypti mosquitoes during 407 independent exposure events was defined. The 50% mosquito infectious dose for each of DENV-1-4 ranged from 6.29 to 7.52 log10 RNA copies/mL of plasma. Increasing day of illness, declining viremia, and rising antibody titers were independently associated with reduced risk of DENV transmission. High early DENV plasma viremia levels in patients were a marker of the duration of human infectiousness, and blood meals containing high concentrations of DENV were positively associated with the prevalence of infectious mosquitoes 14 d after blood feeding. Ambulatory dengue cases had lower viremia levels compared with hospitalized dengue cases but nonetheless at levels predicted to be infectious to mosquitoes. These data define serotype-specific viremia levels that vaccines or drugs must inhibit to prevent DENV transmission.

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Direct interaction between the TnsA and TnsB subunits controls the heteromeric Tn7 transposase.

Publication Date: 2013 May 14 PMID: 23674682
Authors: Choi, K. Y. - Li, Y. - Sarnovsky, R. - Craig, N. L.
Journal: Proc Natl Acad Sci U S A

The transposon Tn7 transposase that recognizes the transposon ends and mediates breakage and joining is heteromeric. It contains the Tn7-encoded proteins TnsB, which binds specifically to the transposon ends and carries out breakage and joining at the 3' ends, and TnsA, which carries out breakage at the 5' ends of Tn7. TnsA apparently does not bind specifically to DNA, and we have hypothesized that it is recruited to the ends by interaction with TnsB. In this work, we show that TnsA and TnsB interact directly and identify several TnsA and TnsB amino acids involved in this interaction. We also show that TnsA can stimulate two key activities of TnsB, specific binding to the ends and pairing of the Tn7 ends. The ends of Tn7 are structurally asymmetric (i.e., contain different numbers of TnsB-binding sites), and Tn7 also is functionally asymmetric, inserting into its specific target site, attachment site attTn7 (attTn7) in a single orientation. Moreover, Tn7 elements containing two Tn7 right ends can transpose, but elements with two Tn7 left ends cannot. We show here that TnsA + TnsB are unable to pair the ends of a Tn7 element containing two Tn7 left ends. This pairing defect likely contributes to the inability of Tn7 elements with two Tn7 left ends to transpose.

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Addressing uncertainty in adaptation planning for agriculture.

Publication Date: 2013 May 21 PMID: 23674681
Authors: Vermeulen, S. J. - Challinor, A. J. - Thornton, P. K. - Campbell, B. M. - Eriyagama, N. - Vervoort, J. M. - Kinyangi, J. - Jarvis, A. - Laderach, P. - Ramirez-Villegas, J. - Nicklin, K. J. - Hawkins, E. - Smith, D. R.
Journal: Proc Natl Acad Sci U S A

We present a framework for prioritizing adaptation approaches at a range of timeframes. The framework is illustrated by four case studies from developing countries, each with associated characterization of uncertainty. Two cases on near-term adaptation planning in Sri Lanka and on stakeholder scenario exercises in East Africa show how the relative utility of capacity vs. impact approaches to adaptation planning differ with level of uncertainty and associated lead time. An additional two cases demonstrate that it is possible to identify uncertainties that are relevant to decision making in specific timeframes and circumstances. The case on coffee in Latin America identifies altitudinal thresholds at which incremental vs. transformative adaptation pathways are robust options. The final case uses three crop-climate simulation studies to demonstrate how uncertainty can be characterized at different time horizons to discriminate where robust adaptation options are possible. We find that impact approaches, which use predictive models, are increasingly useful over longer lead times and at higher levels of greenhouse gas emissions. We also find that extreme events are important in determining predictability across a broad range of timescales. The results demonstrate the potential for robust knowledge and actions in the face of uncertainty.

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Distinct quaternary structures of the AAA+ Lon protease control substrate degradation.

Publication Date: 2013 May 14 PMID: 23674680
Authors: Vieux, E. F. - Wohlever, M. L. - Chen, J. Z. - Sauer, R. T. - Baker, T. A.
Journal: Proc Natl Acad Sci U S A

Lon is an ATPase associated with cellular activities (AAA+) protease that controls cell division in response to stress and also degrades misfolded and damaged proteins. Subunits of Lon are known to assemble into ring-shaped homohexamers that enclose an internal degradation chamber. Here, we demonstrate that hexamers of Escherichia coli Lon also interact to form a dodecamer at physiological protein concentrations. Electron microscopy of this dodecamer reveals a prolate structure with the protease chambers at the distal ends and a matrix of N domains forming an equatorial hexamer-hexamer interface, with portals of approximately 45 A providing access to the enzyme lumen. Compared with hexamers, Lon dodecamers are much less active in degrading large substrates but equally active in degrading small substrates. Our results support a unique gating mechanism that allows the repertoire of Lon substrates to be tuned by its assembly state.

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Role of aspartate 132 at the orifice of a proton pathway in cytochrome c oxidase.

Publication Date: 2013 May 14 PMID: 23674679
Authors: Johansson, A. L. - Hogbom, M. - Carlsson, J. - Gennis, R. B. - Brzezinski, P.
Journal: Proc Natl Acad Sci U S A

Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidase, one such pathway starts near a conserved Asp-132 residue. Results from earlier studies have shown that replacement of Asp-132 by, e.g., Asn, slows proton uptake by a factor of approximately 5,000. Here, we show that proton uptake at full speed ( approximately 104 s-1) can be restored in the Asp-132-Asn oxidase upon introduction of a second structural modification further inside the pathway (Asn-139-Thr) without compensating for the loss of the negative charge. This proton-uptake rate was insensitive to Zn2+ addition, which in the wild-type cytochrome c oxidase slows the reaction, indicating that Asp-132 is required for Zn2+ binding. Furthermore, in the absence of Asp-132 and with Thr at position 139, at high pH (>9), proton uptake was significantly accelerated. Thus, the data indicate that Asp-132 is not strictly required for maintaining rapid proton uptake. Furthermore, despite the rapid proton uptake in the Asn-139-Thr/Asp-132-Asn mutant cytochrome c oxidase, proton pumping was impaired, which indicates that the segment around these residues is functionally linked to pumping.

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Experimental interrogation of the path dependence and stochasticity of protein evolution using phage-assisted continuous evolution.

Publication Date: 2013 May 14 PMID: 23674678
Authors: Dickinson, B. C. - Leconte, A. M. - Allen, B. - Esvelt, K. M. - Liu, D. R.
Journal: Proc Natl Acad Sci U S A

To what extent are evolutionary outcomes determined by a population's recent environment, and to what extent do they depend on historical contingency and random chance? Here we apply a unique experimental system to investigate evolutionary reproducibility and path dependence at the protein level. We combined phage-assisted continuous evolution with high-throughput sequencing to analyze evolving protein populations as they adapted to divergent and then convergent selection pressures over hundreds of generations. Independent populations of T7 RNA polymerase genes were subjected to one of two selection histories ("pathways") demanding recognition of distinct intermediate promoters followed by a common final promoter. We observed distinct classes of solutions with unequal phenotypic activity and evolutionary potential evolve from the two pathways, as well as from replicate populations exposed to identical selection conditions. Mutational analysis revealed specific epistatic interactions that explained the observed path dependence and irreproducibility. Our results reveal in molecular detail how protein adaptation to different environments, as well as stochasticity among populations evolved in the same environment, can both generate evolutionary outcomes that preclude subsequent convergence.

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Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs.

Publication Date: 2013 May 14 PMID: 23674677
Authors: Hines, J. - Gough, J. D. - Corson, T. W. - Crews, C. M.
Journal: Proc Natl Acad Sci U S A

Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinase-signaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2alpha or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short- and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects.

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Experimental evidence that evolutionarily diverse assemblages result in higher productivity.

Publication Date: 2013 May 14 PMID: 23674676
Authors: Cadotte, M. W.
Journal: Proc Natl Acad Sci U S A

There now is ample experimental evidence that speciose assemblages are more productive and provide a greater amount of ecosystem services than depauperate ones. However, these experiments often conclude that there is a higher probability of including complementary species combinations in assemblages with more species and lack a priori prediction about which species combinations maximize function. Here, I report the results of an experiment manipulating the evolutionary relatedness of constituent plant species across a richness gradient. I show that assemblages with distantly related species contributed most to the higher biomass production in multispecies assemblages, through species complementarity. Species produced more biomass than predicted from their monocultures when they were in plots with distantly related species and produced the amount of biomass predicted from monoculture when sown with close relatives. This finding suggests that in the absence of any other information, combining distantly related species in restored or managed landscapes may serve to maximize biomass production and carbon sequestration, thus merging calls to conserve evolutionary history and maximize ecosystem function.

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Influence of organic films on the evaporation and condensation of water in aerosol.

Publication Date: 2013 May 14 PMID: 23674675
Authors: Davies, J. F. - Miles, R. E. - Haddrell, A. E. - Reid, J. P.
Journal: Proc Natl Acad Sci U S A

Uncertainties in quantifying the kinetics of evaporation and condensation of water from atmospheric aerosol are a significant contributor to the uncertainty in predicting cloud droplet number and the indirect effect of aerosols on climate. The influence of aerosol particle surface composition, particularly the impact of surface active organic films, on the condensation and evaporation coefficients remains ambiguous. Here, we report measurements of the influence of organic films on the evaporation and condensation of water from aerosol particles. Significant reductions in the evaporation coefficient are shown to result when condensed films are formed by monolayers of long-chain alcohols [CnH(2n+1)OH], with the value decreasing from 2.4 x 10-3 to 1.7 x 10-5 as n increases from 12 to 17. Temperature-dependent measurements confirm that a condensed film of long-range order must be formed to suppress the evaporation coefficient below 0.05. The condensation of water on a droplet coated in a condensed film is shown to be fast, with strong coherence of the long-chain alcohol molecules leading to islanding as the water droplet grows, opening up broad areas of uncoated surface on which water can condense rapidly. We conclude that multicomponent composition of organic films on the surface of atmospheric aerosol particles is likely to preclude the formation of condensed films and that the kinetics of water condensation during the activation of aerosol to form cloud droplets is likely to remain rapid.

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Emergence of hierarchy in cost-driven growth of spatial networks.

Publication Date: 2013 May 14 PMID: 23674674
Authors: Louf, R. - Jensen, P. - Barthelemy, M.
Journal: Proc Natl Acad Sci U S A

One of the most important features of spatial networks-such as transportation networks, power grids, the Internet, and neural networks-is the existence of a cost associated with the length of links. Such a cost has a profound influence on the global structure of these networks, which usually display a hierarchical spatial organization. The link between local constraints and large-scale structure is not elucidated, however, and we introduce here a generic model for the growth of spatial networks based on the general concept of cost-benefit analysis. This model depends essentially on a single scale and produces a family of networks that range from the star graph to the minimum spanning tree and are characterized by a continuously varying exponent. We show that spatial hierarchy emerges naturally, with structures composed of various hubs controlling geographically separated service areas, and appears as a large-scale consequence of local cost-benefit considerations. Our model thus provides the basic building blocks for a better understanding of the evolution of spatial networks and their properties. We also find that, surprisingly, the average detour is minimal in the intermediate regime as a result of a large diversity in link lengths. Finally, we estimate the important parameters for various world railway networks and find that, remarkably, they all fall in this intermediate regime, suggesting that spatial hierarchy is a crucial feature for these systems and probably possesses an important evolutionary advantage.

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In vivo imaging of CD8+ T cell-mediated elimination of malaria liver stages.

Publication Date: 2013 May 14 PMID: 23674673
Authors: Cockburn, I. A. - Amino, R. - Kelemen, R. K. - Kuo, S. C. - Tse, S. W. - Radtke, A. - Mac-Daniel, L. - Ganusov, V. V. - Zavala, F. - Menard, R.
Journal: Proc Natl Acad Sci U S A

CD8+ T cells are specialized cells of the adaptive immune system capable of finding and eliminating pathogen-infected cells. To date it has not been possible to observe the destruction of any pathogen by CD8+ T cells in vivo. Here we demonstrate a technique for imaging the killing of liver-stage malaria parasites by CD8+ T cells bearing a transgenic T cell receptor specific for a parasite epitope. We report several features that have not been described by in vitro analysis of the process, chiefly the formation of large clusters of effector CD8+ T cells around infected hepatocytes. The formation of clusters requires antigen-specific CD8+ T cells and signaling by G protein-coupled receptors, although CD8+ T cells of unrelated specificity are also recruited to clusters. By combining mathematical modeling and data analysis, we suggest that formation of clusters is mainly driven by enhanced recruitment of T cells into larger clusters. We further show various death phenotypes of the parasite, which typically follow prolonged interactions between infected hepatocytes and CD8+ T cells. These findings stress the need for intravital imaging for dissecting the fine mechanisms of pathogen recognition and killing by CD8+ T cells.

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Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens.

Publication Date: 2013 May 14 PMID: 23674672
Authors: Zupan, J. R. - Cameron, T. A. - Anderson-Furgeson, J. - Zambryski, P. C.
Journal: Proc Natl Acad Sci U S A

Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes.

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CD134 is a cellular receptor specific for human herpesvirus-6B entry.

Publication Date: 2013 May 14 PMID: 23674671
Authors: Tang, H. - Serada, S. - Kawabata, A. - Ota, M. - Hayashi, E. - Naka, T. - Yamanishi, K. - Mori, Y.
Journal: Proc Natl Acad Sci U S A

Human herpesvirus-6B (HHV-6B) is a T lymphotropic beta-herpesvirus that is clearly distinct from human herpesvirus-6A (HHV-6A) according to molecular biological features. The International Committee on Taxonomy of Viruses recently classified HHV-6B as a separate species. The primary HHV-6B infection causes exanthem subitum and is sometimes associated with severe encephalopathy. More than 90% of the general population is infected with HHV-6B during childhood, and the virus remains throughout life as a latent infection. HHV-6B reactivation causes encephalitis in immunosuppressed patients. The cellular receptor for HHV-6A entry was identified as human CD46, but the receptor for HHV-6B has not been clear. Here we found that CD134, a member of the TNF receptor superfamily, functions as a specific entry receptor for HHV-6B. A T-cell line that is normally nonpermissive for HHV-6B infection became highly susceptible to infection when CD134 was overexpressed. CD134 was down-regulated in HHV-6B-infected T cells. Soluble CD134 interacted with the HHV-6B glycoprotein complex that serves as a viral ligand for cellular receptor, which inhibited HHV-6B but not HHV-6A infection in target cells. The identification of CD134 as an HHV-6B specific entry receptor provides important insight into understanding HHV-6B entry and its pathogenesis.

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PNAS Plus Significance Statements.

Publication Date: 2013 May 14 PMID: 23674620
Authors:
Journal: Proc Natl Acad Sci U S A

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Tracking Burmese pythons.

Publication Date: 2013 May 14 PMID: 23674619
Authors: Nair, P.
Journal: Proc Natl Acad Sci U S A

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Agricultural innovation to protect the environment.

Publication Date: 2013 May 21 PMID: 23671123
Authors: Sayer, J. - Cassman, K. G.
Journal: Proc Natl Acad Sci U S A

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Transformation of polarized epithelial cells by apical mistrafficking of epiregulin.

Publication Date: 2013 May 13 PMID: 23671122
Authors: Singh, B. - Bogatcheva, G. - Washington, M. K. - Coffey, R. J.
Journal: Proc Natl Acad Sci U S A

Establishment and maintenance of apico-basolateral trafficking pathways are critical to epithelial homeostasis. Loss of polarity and trafficking fidelity are thought to occur as a consequence of transformation; however, here we report that selective mistrafficking of the epidermal growth factor receptor (EGFR) ligand epiregulin (EREG) from the basolateral to the apical cell surface drives transformation. Normally, EREG is preferentially delivered to the basolateral surface of polarized Madin-Darby canine kidney cells. EREG basolateral trafficking is regulated by a conserved tyrosine-based basolateral sorting motif in its cytoplasmic domain (YXXPhi: Y156ERV). Both Y156 and V159 are required for basolateral sorting of EREG, because Y156A and V159G substitutions redirect EREG to the apical cell surface. We also show that basolateral sorting of EREG is adaptor protein 1B-independent. Apical mistrafficking of EREG has a distinctive phenotype. In contrast to transient EGFR tyrosine phosphorylation after basolateral EREG stimulation, apical EREG leads to prolonged EGFR tyrosine phosphorylation, which may be related, at least in part, to a lack of negative regulatory Y1045 phosphorylation and subsequent ubiquitylation. Notably, Madin-Darby canine kidney cells stably expressing apically mistrafficked EREG form significantly larger, hyperproliferative, poorly differentiated, and locally invasive tumors in nude mice compared with WT EREG-expressing cells.

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Functional ecology of an Antarctic Dry Valley.

Publication Date: 2013 May 13 PMID: 23671121
Authors: Chan, Y. - Van Nostrand, J. D. - Zhou, J. - Pointing, S. B. - Farrell, R. L.
Journal: Proc Natl Acad Sci U S A

The McMurdo Dry Valleys are the largest ice-free region in Antarctica and are critically at risk from climate change. The terrestrial landscape is dominated by oligotrophic mineral soils and extensive exposed rocky surfaces where biota are largely restricted to microbial communities, although their ability to perform the majority of geobiological processes has remained largely uncharacterized. Here, we identified functional traits that drive microbial survival and community assembly, using a metagenomic approach with GeoChip-based functional gene arrays to establish metabolic capabilities in communities inhabiting soil and rock surface niches in McKelvey Valley. Major pathways in primary metabolism were identified, indicating significant plasticity in autotrophic, heterotrophic, and diazotrophic strategies supporting microbial communities. This represents a major advance beyond biodiversity surveys in that we have now identified how putative functional ecology drives microbial community assembly. Significant differences were apparent between open soil, hypolithic, chasmoendolithic, and cryptoendolithic communities. A suite of previously unappreciated Antarctic microbial stress response pathways, thermal, osmotic, and nutrient limitation responses were identified and related to environmental stressors, offering tangible clues to the mechanisms behind the enduring success of microorganisms in this seemingly inhospitable terrain. Rocky substrates exposed to larger fluctuations in environmental stress supported greater functional diversity in stress-response pathways than soils. Soils comprised a unique reservoir of genes involved in transformation of organic hydrocarbons and lignin-like degradative pathways. This has major implications for the evolutionary origin of the organisms, turnover of recalcitrant substrates in Antarctic soils, and predicting future responses to anthropogenic pollution.

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PRMT5 modulates the metabolic response to fasting signals.

Publication Date: 2013 May 13 PMID: 23671120
Authors: Tsai, W. W. - Niessen, S. - Goebel, N. - Yates, J. R. 3rd - Guccione, E. - Montminy, M.
Journal: Proc Natl Acad Sci U S A

Under fasting conditions, increases in circulating glucagon maintain glucose balance by promoting hepatic gluconeogenesis. Triggering of the cAMP pathway stimulates gluconeogenic gene expression through the PKA-mediated phosphorylation of the cAMP response element binding (CREB) protein and via the dephosphorylation of the latent cytoplasmic CREB regulated transcriptional coactivator 2 (CRTC2). CREB and CRTC2 activities are increased in insulin resistance, in which they promote hyperglycemia because of constitutive induction of the gluconeogenic program. The extent to which CREB and CRTC2 are coordinately up-regulated in response to glucagon, however, remains unclear. Here we show that, following its activation, CRTC2 enhances CREB phosphorylation through an association with the protein arginine methyltransferase 5 (PRMT5). In turn, PRMT5 was found to stimulate CREB phosphorylation via increases in histone H3 Arg2 methylation that enhanced chromatin accessibility at gluconeogenic promoters. Because depletion of PRMT5 lowers hepatic glucose production and gluconeogenic gene expression, these results demonstrate how a chromatin-modifying enzyme regulates a metabolic program through epigenetic changes that impact the phosphorylation of a transcription factor in response to hormonal stimuli.

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Essential role for Cdk2 inhibitory phosphorylation during replication stress revealed by a human Cdk2 knockin mutation.

Publication Date: 2013 May 13 PMID: 23671119
Authors: Hughes, B. T. - Sidorova, J. - Swanger, J. - J Monnat, R. Jr - Clurman, B. E.
Journal: Proc Natl Acad Sci U S A

Cyclin-dependent kinases (Cdks) coordinate cell division, and their activities are tightly controlled. Phosphorylation of threonine 14 (T14) and tyrosine 15 (Y15) inhibits Cdks and regulates their activities in numerous physiologic contexts. Although the roles of Cdk1 inhibitory phosphorylation during mitosis are well described, studies of Cdk2 inhibitory phosphorylation during S phrase have largely been indirect. To specifically study the functions of Cdk2 inhibitory phosphorylation, we used gene targeting to make an endogenous Cdk2 knockin allele in human cells, termed Cdk2AF, which prevents Cdk2 T14 and Y15 phosphorylation. Cdk2AF caused premature S-phase entry, rapid cyclin E degradation, abnormal DNA replication, and genome instability. Cdk2AF cells also exhibited strikingly abnormal responses to replication stress, accumulated irreparable DNA damage, and permanently exited the cell cycle after transient exposure to S-phase inhibitors. Our results reveal the specific and essential roles of Cdk2 inhibitory phosphorylation in the successful execution of the replication stress checkpoint response and in maintaining genome integrity.

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Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity.

Publication Date: 2013 May 13 PMID: 23671118
Authors: Levikova, M. - Klaue, D. - Seidel, R. - Cejka, P.
Journal: Proc Natl Acad Sci U S A

Dna2 is a nuclease-helicase involved in several key pathways of eukaryotic DNA metabolism. The potent nuclease activity of Saccharomyces cerevisiae Dna2 was reported to be required for all its in vivo functions tested to date. In contrast, its helicase activity was shown to be weak, and its inactivation affected only a subset of Dna2 functions. We describe here a complex interplay of the two enzymatic activities. We show that the nuclease of Dna2 inhibits its helicase by cleaving 5' flaps that are required by the helicase domain for loading onto its substrate. Mutational inactivation of Dna2 nuclease unleashes unexpectedly vigorous DNA unwinding activity, comparable with that of the most potent eukaryotic helicases. Thus, the ssDNA-specific nuclease activity of Dna2 limits and controls the enzyme's capacity to unwind dsDNA. We postulate that regulation of this interplay could modulate the biochemical properties of Dna2 and thus license it to carry out its distinct cellular functions.

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Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates.

Publication Date: 2013 May 13 PMID: 23671117
Authors: Bartlett, E. J. - Brissett, N. C. - Doherty, A. J.
Journal: Proc Natl Acad Sci U S A

Nonhomologous end-joining (NHEJ) pathways repair DNA double-strand breaks (DSBs) in eukaryotes and many prokaryotes, although it is not reported to operate in the third domain of life, archaea. Here, we describe a complete NHEJ complex, consisting of DNA ligase (Lig), polymerase (Pol), phosphoesterase (PE), and Ku from a mesophillic archaeon, Methanocella paludicola (Mpa). Mpa Lig has limited DNA nick-sealing activity but is efficient in ligating nicks containing a 3' ribonucleotide. Mpa Pol preferentially incorporates nucleoside triphosphates onto a DNA primer strand, filling DNA gaps in annealed breaks. Mpa PE sequentially removes 3' phosphates and ribonucleotides from primer strands, leaving a ligatable terminal 3' monoribonucleotide. These proteins, together with the DNA end-binding protein Ku, form a functional NHEJ break-repair apparatus that is highly homologous to the bacterial complex. Although the major roles of Pol and Lig in break repair have been reported, PE's function in NHEJ has remained obscure. We establish that PE is required for ribonucleolytic resection of RNA intermediates at annealed DSBs. Polymerase-catalyzed strand-displacement synthesis on DNA gaps can result in the formation of nonligatable NHEJ intermediates. The function of PE in NHEJ repair is to detect and remove inappropriately incorporated ribonucleotides or phosphates from 3' ends of annealed DSBs to configure the termini for ligation. Thus, PE prevents the accumulation of abortive genotoxic DNA intermediates arising from strand displacement synthesis that otherwise would be refractory to repair.

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Systematic identification of conserved bacterial c-di-AMP receptor proteins.

Publication Date: 2013 May 13 PMID: 23671116
Authors: Corrigan, R. M. - Campeotto, I. - Jeganathan, T. - Roelofs, K. G. - Lee, V. T. - Grundling, A.
Journal: Proc Natl Acad Sci U S A

Nucleotide signaling molecules are important messengers in key pathways that allow cellular responses to changing environments. Canonical secondary signaling molecules act through specific receptor proteins by direct binding to alter their activity. Cyclic diadenosine monophosphate (c-di-AMP) is an essential signaling molecule in bacteria that has only recently been discovered. Here we report on the identification of four Staphylococcus aureus c-di-AMP receptor proteins that are also widely distributed among other bacteria. Using an affinity pull-down assay we identified the potassium transporter-gating component KtrA as a c-di-AMP receptor protein, and it was further shown that this protein, together with c-di-AMP, enables S. aureus to grow in low potassium conditions. We defined the c-di-AMP binding activity within KtrA to the RCK_C (regulator of conductance of K+) domain. This domain is also found in a second S. aureus protein, a predicted cation/proton antiporter, CpaA, which as we show here also directly binds c-di-AMP. Because RCK_C domains are found in proteinaceous channels, transporters, and antiporters from all kingdoms of life, these findings have broad implications for the regulation of different pathways through nucleotide-dependent signaling. Using a genome-wide nucleotide protein interaction screen we further identified the histidine kinase protein KdpD that in many bacteria is also involved in the regulation of potassium transport and a PII-like signal transduction protein, which we renamed PstA, as c-di-AMP binding proteins. With the identification of these widely distributed c-di-AMP receptor proteins we link the c-di-AMP signaling network to a central metabolic process in bacteria.

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Huntington disease skeletal muscle is hyperexcitable owing to chloride and potassium channel dysfunction.

Publication Date: 2013 May 13 PMID: 23671115
Authors: Waters, C. W. - Varuzhanyan, G. - Talmadge, R. J. - Voss, A. A.
Journal: Proc Natl Acad Sci U S A

Huntington disease is a progressive and fatal genetic disorder with debilitating motor and cognitive defects. Chorea, rigidity, dystonia, and muscle weakness are characteristic motor defects of the disease that are commonly attributed to central neurodegeneration. However, no previous study has examined the membrane properties that control contraction in Huntington disease muscle. We show primary defects in ex vivo adult skeletal muscle from the R6/2 transgenic mouse model of Huntington disease. Action potentials in diseased fibers are more easily triggered and prolonged than in fibers from WT littermates. Furthermore, some action potentials in the diseased fibers self-trigger. These defects occur because of decreases in the resting chloride and potassium conductances. Consistent with this, the expression of the muscle chloride channel, ClC-1, in Huntington disease muscle was compromised by improper splicing and a corresponding reduction in total Clcn1 (gene for ClC-1) mRNA. Additionally, the total Kcnj2 (gene for the Kir2.1 potassium channel) mRNA was reduced in disease muscle. The resulting muscle hyperexcitability causes involuntary and prolonged contractions that may contribute to the chorea, rigidity, and dystonia that characterize Huntington disease.

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Multiple actions of {varphi}-LITX-Lw1a on ryanodine receptors reveal a functional link between scorpion DDH and ICK toxins.

Publication Date: 2013 May 13 PMID: 23671114
Authors: Smith, J. J. - Vetter, I. - Lewis, R. J. - Peigneur, S. - Tytgat, J. - Lam, A. - Gallant, E. M. - Beard, N. A. - Alewood, P. F. - Dulhunty, A. F.
Journal: Proc Natl Acad Sci U S A

We recently reported the isolation of a scorpion toxin named U1-liotoxin-Lw1a (U1-LITX-Lw1a) that adopts an unusual 3D fold termed the disulfide-directed hairpin (DDH) motif, which is the proposed evolutionary structural precursor of the three-disulfide-containing inhibitor cystine knot (ICK) motif found widely in animals and plants. Here we reveal that U1-LITX-Lw1a targets and activates the mammalian ryanodine receptor intracellular calcium release channel (RyR) with high (fM) potency and provides a functional link between DDH and ICK scorpion toxins. Moreover, U1-LITX-Lw1a, now described as varphi-liotoxin-Lw1a (varphi-LITX-Lw1a), has a similar mode of action on RyRs as scorpion calcines, although with significantly greater potency, inducing full channel openings at lower (fM) toxin concentrations whereas at higher pM concentrations increasing the frequency and duration of channel openings to a submaximal state. In addition, we show that the C-terminal residue of varphi-LITX-Lw1a is crucial for the increase in full receptor openings but not for the increase in receptor subconductance opening, thereby supporting the two-binding-site hypothesis of scorpion toxins on RyRs. varphi-LITX-Lw1a has potential both as a pharmacological tool and as a lead molecule for the treatment of human diseases that involve RyRs, such as malignant hyperthermia and polymorphic ventricular tachycardia.

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Correction for Ji et al., Molecular mechanism of quinone signaling mediated through S-quinonization of a YodB family repressor QsrR.

Publication Date: 2013 May 13 PMID: 23671113
Authors:
Journal: Proc Natl Acad Sci U S A

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Mass spectrometry-based metabolomics of single yeast cells.

Publication Date: 2013 May 13 PMID: 23671112
Authors: Ibanez, A. J. - Fagerer, S. R. - Schmidt, A. M. - Urban, P. L. - Jefimovs, K. - Geiger, P. - Dechant, R. - Heinemann, M. - Zenobi, R.
Journal: Proc Natl Acad Sci U S A

Single-cell level measurements are necessary to characterize the intrinsic biological variability in a population of cells. In this study, we demonstrate that, with the microarrays for mass spectrometry platform, we are able to observe this variability. We monitor environmentally (2-deoxy-d-glucose) and genetically (DeltaPFK2) perturbed Saccharomyces cerevisiae cells at the single-cell, few-cell, and population levels. Correlation plots between metabolites from the glycolytic pathway, as well as with the observed ATP/ADP ratio as a measure of cellular energy charge, give biological insight that is not accessible from population-level metabolomic data.

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Coevolution of farming and private property during the early Holocene.

Publication Date: 2013 May 13 PMID: 23671111
Authors: Bowles, S. - Choi, J. K.
Journal: Proc Natl Acad Sci U S A

The advent of farming around 12 millennia ago was a cultural as well as technological revolution, requiring a new system of property rights. Among mobile hunter-gatherers during the late Pleistocene, food was almost certainly widely shared as it was acquired. If a harvested crop or the meat of a domesticated animal were to have been distributed to other group members, a late Pleistocene would-be farmer would have had little incentive to engage in the required investments in clearing, cultivation, animal tending, and storage. However, the new property rights that farming required-secure individual claims to the products of one's labor-were infeasible because most of the mobile and dispersed resources of a forager economy could not cost-effectively be delimited and defended. The resulting chicken-and-egg puzzle might be resolved if farming had been much more productive than foraging, but initially it was not. Our model and simulations explain how, despite being an unlikely event, farming and a new system of farming-friendly property rights nonetheless jointly emerged when they did. This Holocene revolution was not sparked by a superior technology. It occurred because possession of the wealth of farmers-crops, dwellings, and animals-could be unambiguously demarcated and defended. This facilitated the spread of new property rights that were advantageous to the groups adopting them. Our results thus challenge unicausal models of historical dynamics driven by advances in technology, population pressure, or other exogenous changes. Our approach may be applied to other technological and institutional revolutions such as the 18th- and 19th-century industrial revolution and the information revolution today.

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Dynamic CREB family activity drives segmentation and posterior polarity specification in mammalian somitogenesis.

Publication Date: 2013 May 13 PMID: 23671110
Authors: Lopez, T. P. - Fan, C. M.
Journal: Proc Natl Acad Sci U S A

The segmented body plan of vertebrates is prefigured by reiterated embryonic mesodermal structures called somites. In the mouse embryo, timely somite formation from the presomitic mesoderm (PSM) is controlled by the "segmentation clock," a molecular oscillator that triggers progressive waves of Notch activity throughout the PSM. Notch clock activity is suppressed in the posterior PSM by FGF signaling until it crosses a determination front at which its net activity is sufficiently high to effect segmentation. Here, Notch and Wnt signaling directs somite anterior/posterior (A/P) polarity specification and boundary formation via regulation of the segmentation effector gene Mesoderm posterior 2. How Notch and Wnt signaling becomes coordinated at this front is incompletely defined. Here we show that the activity of the cAMP responsive element binding protein (CREB) family of transcription factors exhibits Wnt3a-dependent oscillatory behavior near the determination front and is in unison with Notch activity. Inhibition of CREB family in the mesoderm causes defects in somite segmentation and a loss in somite posterior polarity leading to fusions of vertebrae and ribs. Among the CREB family downstream genes, several are known to be regulated by Wnt3a. Of those, we show that the CREB family occupies a conserved binding site in the promoter region of Delta-like 1, encoding a Notch ligand, in the anterior PSM as a mechanism to specify posterior identity of somites. Together, these data support that the CREB family acts at the determination front to modulate Wnt signaling and strengthen Notch signaling as a means to orchestrate cells for somite segmentation and anterior/posterior patterning.

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Molecular evolution of peptidergic signaling systems in bilaterians.

Publication Date: 2013 May 13 PMID: 23671109
Authors: Mirabeau, O. - Joly, J. S.
Journal: Proc Natl Acad Sci U S A

Peptide hormones and their receptors are widespread in metazoans, but the knowledge we have of their evolutionary relationships remains unclear. Recently, accumulating genome sequences from many different species have offered the opportunity to reassess the relationships between protostomian and deuterostomian peptidergic systems (PSs). Here we used sequences of all human rhodopsin and secretin-type G protein-coupled receptors as bait to retrieve potential homologs in the genomes of 15 bilaterian species, including nonchordate deuterostomian and lophotrochozoan species. Our phylogenetic analysis of these receptors revealed 29 well-supported subtrees containing mixed sets of protostomian and deuterostomian sequences. This indicated that many vertebrate and arthropod PSs that were previously thought to be phyla specific are in fact of bilaterian origin. By screening sequence databases for potential peptides, we then reconstructed entire bilaterian peptide families and showed that protostomian and deuterostomian peptides that are ligands of orthologous receptors displayed some similarity at the level of their primary sequence, suggesting an ancient coevolution between peptide and receptor genes. In addition to shedding light on the function of human G protein-coupled receptor PSs, this work presents orthology markers to study ancestral neuron types that were probably present in the last common bilaterian ancestor.

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Dominant suppression of inflammation by glycan-hydrolyzed IgG.

Publication Date: 2013 May 13 PMID: 23671108
Authors: Nandakumar, K. S. - Collin, M. - Happonen, K. E. - Croxford, A. M. - Lundstrom, S. L. - Zubarev, R. A. - Rowley, M. J. - Blom, A. M. - Holmdahl, R.
Journal: Proc Natl Acad Sci U S A

A unique anti-inflammatory property of IgG, independent of antigen specificity, is described. IgG with modification of the heavy-chain glycan on asparagine 297 by the streptococcal enzyme endo-beta-N-acetylglucosaminidase (EndoS) induced a dominant suppression of immune complex (IC)-mediated inflammation, such as arthritis, through destabilization of local ICs by fragment crystallizable-fragment crystallizable (Fc-Fc) interactions. Small amounts (250 microg) of EndoS-hydrolyzed IgG were sufficient to inhibit arthritis in mice and most effective during the formation of ICs in the target tissue. The presence of EndoS-hydrolyzed IgG disrupted larger IC lattice formation both in vitro and in vivo, as visualized with anti-C3b staining. Neither complement binding in vitro nor antigen-antibody binding per se was affected.

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Role of the ubiquitin ligase E6AP/UBE3A in controlling levels of the synaptic protein Arc.

Publication Date: 2013 May 13 PMID: 23671107
Authors: Kuhnle, S. - Mothes, B. - Matentzoglu, K. - Scheffner, M.
Journal: Proc Natl Acad Sci U S A

Inactivation of the ubiquitin ligase E6 associated protein (E6AP) encoded by the UBE3A gene has been associated with development of the Angelman syndrome. Recently, it was reported that in mice, loss of E6AP expression results in increased levels of the synaptic protein Arc and a concomitant impaired synaptic function, providing an explanation for some phenotypic features of Angelman syndrome patients. Accordingly, E6AP has been shown to negatively regulate activity-regulated cytoskeleton-associated protein (Arc) and it has been suggested that E6AP targets Arc for ubiquitination and degradation. In our study, we provide evidence that Arc is not a direct substrate for E6AP and binds only weakly to E6AP, if at all. Furthermore, we show that down-regulation of E6AP expression stimulates estradiol-induced transcription of the Arc gene. Thus, we propose that Arc protein levels are controlled by E6AP at the transcriptional rather than at the posttranslational level.

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Music-color associations are mediated by emotion.

Publication Date: 2013 May 13 PMID: 23671106
Authors: Palmer, S. E. - Schloss, K. B. - Xu, Z. - Prado-Leon, L. R.
Journal: Proc Natl Acad Sci U S A

Experimental evidence demonstrates robust cross-modal matches between music and colors that are mediated by emotional associations. US and Mexican participants chose colors that were most/least consistent with 18 selections of classical orchestral music by Bach, Mozart, and Brahms. In both cultures, faster music in the major mode produced color choices that were more saturated, lighter, and yellower whereas slower, minor music produced the opposite pattern (choices that were desaturated, darker, and bluer). There were strong correlations (0.89 < r < 0.99) between the emotional associations of the music and those of the colors chosen to go with the music, supporting an emotional mediation hypothesis in both cultures. Additional experiments showed similarly robust cross-modal matches from emotionally expressive faces to colors and from music to emotionally expressive faces. These results provide further support that music-to-color associations are mediated by common emotional associations.

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Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity.

Publication Date: 2013 May 13 PMID: 23671105
Authors: Everard, A. - Belzer, C. - Geurts, L. - Ouwerkerk, J. P. - Druart, C. - Bindels, L. B. - Guiot, Y. - Derrien, M. - Muccioli, G. G. - Delzenne, N. M. - de Vos, W. M. - Cani, P. D.
Journal: Proc Natl Acad Sci U S A

Obesity and type 2 diabetes are characterized by altered gut microbiota, inflammation, and gut barrier disruption. Microbial composition and the mechanisms of interaction with the host that affect gut barrier function during obesity and type 2 diabetes have not been elucidated. We recently isolated Akkermansia muciniphila, which is a mucin-degrading bacterium that resides in the mucus layer. The presence of this bacterium inversely correlates with body weight in rodents and humans. However, the precise physiological roles played by this bacterium during obesity and metabolic disorders are unknown. This study demonstrated that the abundance of A. muciniphila decreased in obese and type 2 diabetic mice. We also observed that prebiotic feeding normalized A. muciniphila abundance, which correlated with an improved metabolic profile. In addition, we demonstrated that A. muciniphila treatment reversed high-fat diet-induced metabolic disorders, including fat-mass gain, metabolic endotoxemia, adipose tissue inflammation, and insulin resistance. A. muciniphila administration increased the intestinal levels of endocannabinoids that control inflammation, the gut barrier, and gut peptide secretion. Finally, we demonstrated that all these effects required viable A. muciniphila because treatment with heat-killed cells did not improve the metabolic profile or the mucus layer thickness. In summary, this study provides substantial insight into the intricate mechanisms of bacterial (i.e., A. muciniphila) regulation of the cross-talk between the host and gut microbiota. These results also provide a rationale for the development of a treatment that uses this human mucus colonizer for the prevention or treatment of obesity and its associated metabolic disorders.

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Effect of active water movement on energy and nutrient acquisition in coral reef-associated benthic organisms.

Publication Date: 2013 May 13 PMID: 23671104
Authors: Wild, C. - Naumann, M. S.
Journal: Proc Natl Acad Sci U S A

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Trp replacements for tightly interacting Gly-Gly pairs in LacY stabilize an outward-facing conformation.

Publication Date: 2013 May 13 PMID: 23671103
Authors: Smirnova, I. - Kasho, V. - Sugihara, J. - Kaback, H. R.
Journal: Proc Natl Acad Sci U S A

Trp replacements for conserved Gly-Gly pairs between the N- and C-terminal six-helix bundles on the periplasmic side of lactose permease (LacY) cause complete loss of transport activity with little or no effect on sugar binding. Moreover, the detergent-solubilized mutants exhibit much greater thermal stability than WT LacY. A Cys replacement for Asn245, which is inaccessible/unreactive in WT LacY, alkylates readily in the Gly-->Trp mutants, indicating that the periplasmic cavity is patent. Stopped-flow kinetic measurements of sugar binding with the Gly-->Trp mutants in detergent reveal linear dependence of binding rates on sugar concentration, as observed with WT or the C154G mutant of LacY, and are compatible with free access to the sugar-binding site in the middle of the molecule. Remarkably, after reconstitution of the Gly-->Trp mutants into proteoliposomes, the concentration dependence of sugar-binding rates increases sharply with even faster rates than measured in detergent. Such behavior is strikingly different from that observed for reconstituted WT LacY, in which sugar-binding rates are independent of sugar concentration because opening of the periplasmic cavity is limiting for sugar binding. The observations clearly indicate that Gly-->Trp replacements, which introduce bulky residues into tight Gly-Gly interdomain interactions on the periplasmic side of LacY, prevent closure of the periplasmic cavity and, as a result, shift the distribution of LacY toward an outward-open conformation.

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Significance of activity peaks in fruit flies, Drosophila melanogaster, under seminatural conditions.

Publication Date: 2013 May 13 PMID: 23671102
Authors: De, J. - Varma, V. - Saha, S. - Sheeba, V. - Sharma, V. K.
Journal: Proc Natl Acad Sci U S A

Studies on circadian entrainment have traditionally been performed under controlled laboratory conditions. Although these studies have served the purpose of providing a broad framework for our understanding of regulation of rhythmic behaviors under cyclic conditions, they do not reveal how organisms keep time in nature. Although a few recent studies have attempted to address this, it is not yet clear which environmental factors regulate rhythmic behaviors in nature and how. Here, we report the results of our studies aimed at examining (i) whether and how changes in natural light affect activity/rest rhythm and (ii) what the functional significance of this rhythmic behavior might be. We found that wild-type strains of fruit flies, Drosophila melanogaster, display morning (M), afternoon (A), and evening (E) peaks of activity under seminatural conditions (SN), whereas under constant darkness in otherwise SN, they exhibited M and E peaks, and under constant light in SN, only the E peak occurred. Unlike the A peak, which requires exposure to bright light in the afternoon, light information is dispensable for the M and E peaks. Visual examination of behaviors suggests that the M peak is associated with courtship-related locomotor activity and the A peak is due to an artifact of the experimental protocol and largely circadian clock independent.

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Palmitoylation-dependent CDKL5-PSD-95 interaction regulates synaptic targeting of CDKL5 and dendritic spine development.

Publication Date: 2013 May 13 PMID: 23671101
Authors: Zhu, Y. C. - Li, D. - Wang, L. - Lu, B. - Zheng, J. - Zhao, S. L. - Zeng, R. - Xiong, Z. Q.
Journal: Proc Natl Acad Sci U S A

The X-linked gene cyclin-dependent kinase-like 5 (CDKL5) is mutated in severe neurodevelopmental disorders, including some forms of atypical Rett syndrome, but the function and regulation of CDKL5 protein in neurons remain to be elucidated. Here, we show that CDKL5 binds to the scaffolding protein postsynaptic density (PSD)-95, and that this binding promotes the targeting of CDKL5 to excitatory synapses. Interestingly, this binding is not constitutive, but governed by palmitate cycling on PSD-95. Furthermore, pathogenic mutations that truncate the C-terminal tail of CDKL5 diminish its binding to PSD-95 and synaptic accumulation. Importantly, down-regulation of CDKL5 by RNA interference (RNAi) or interference with the CDKL5-PSD-95 interaction inhibits dendritic spine formation and growth. These results demonstrate a critical role of the palmitoylation-dependent CDKL5-PSD-95 interaction in localizing CDKL5 to synapses for normal spine development and suggest that disruption of this interaction by pathogenic mutations may be implicated in the pathogenesis of CDKL5-related disorders.

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Murine leukemia virus glycosylated Gag blocks apolipoprotein B editing complex 3 and cytosolic sensor access to the reverse transcription complex.

Publication Date: 2013 May 13 PMID: 23671100
Authors: Stavrou, S. - Nitta, T. - Kotla, S. - Ha, D. - Nagashima, K. - Rein, A. R. - Fan, H. - Ross, S. R.
Journal: Proc Natl Acad Sci U S A

Pathogenic retroviruses have evolved multiple means for evading host restriction factors such as apolipoprotein B editing complex (APOBEC3) proteins. Here, we show that murine leukemia virus (MLV) has a unique means of counteracting APOBEC3 and other cytosolic sensors of viral nucleic acid. Using virus isolated from infected WT and APOBEC3 KO mice, we demonstrate that the MLV glycosylated Gag protein (glyco-Gag) enhances viral core stability. Moreover, in vitro endogenous reverse transcription reactions of the glyco-Gag mutant virus were substantially inhibited compared with WT virus, but only in the presence of APOBEC3. Thus, glyco-Gag rendered the reverse transcription complex in the viral core resistant to APOBEC3. Glyco-Gag in the virion also rendered MLV resistant to other cytosolic sensors of viral reverse transcription products in newly infected cells. Strikingly, glyco-Gag mutant virus reverted to glyco-Gag-containing virus only in WT and not APOBEC3 KO mice, indicating that counteracting APOBEC3 is the major function of glyco-Gag. Thus, in contrast to the HIV viral infectivity factor protein, which prevents APOBEC3 packaging in the virion, the MLV glyco-Gag protein uses a unique mechanism to counteract the antiviral action of APOBEC3 in vivo-namely, protecting the reverse transcription complex in viral cores from APOBEC3. These data suggest that capsid integrity may play a critical role in virus resistance to intrinsic cellular antiviral resistance factors that act at the early stages of infection.

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Perineuronal nets protect fast-spiking interneurons against oxidative stress.

Publication Date: 2013 May 13 PMID: 23671099
Authors: Cabungcal, J. H. - Steullet, P. - Morishita, H. - Kraftsik, R. - Cuenod, M. - Hensch, T. K. - Do, K. Q.
Journal: Proc Natl Acad Sci U S A

A hallmark of schizophrenia pathophysiology is the dysfunction of cortical inhibitory GABA neurons expressing parvalbumin, which are essential for coordinating neuronal synchrony during various sensory and cognitive tasks. The high metabolic requirements of these fast-spiking cells may render them susceptible to redox dysregulation and oxidative stress. Using mice carrying a genetic redox imbalance, we demonstrate that extracellular perineuronal nets, which constitute a specialized polyanionic matrix enwrapping most of these interneurons as they mature, play a critical role in the protection against oxidative stress. These nets limit the effect of genetically impaired antioxidant systems and/or excessive reactive oxygen species produced by severe environmental insults. We observe an inverse relationship between the robustness of the perineuronal nets around parvalbumin cells and the degree of intracellular oxidative stress they display. Enzymatic degradation of the perineuronal nets renders mature parvalbumin cells and fast rhythmic neuronal synchrony more susceptible to oxidative stress. In parallel, parvalbumin cells enwrapped with mature perineuronal nets are better protected than immature parvalbumin cells surrounded by less-condensed perineuronal nets. Although the perineuronal nets act as a protective shield, they are also themselves sensitive to excess oxidative stress. The protection might therefore reflect a balance between the oxidative burden on perineuronal net degradation and the capacity of the system to maintain the nets. Abnormal perineuronal nets, as observed in the postmortem patient brain, may thus underlie the vulnerability and functional impairment of pivotal inhibitory circuits in schizophrenia.

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Dependence of hydropower energy generation on forests in the Amazon Basin at local and regional scales.

Publication Date: 2013 May 13 PMID: 23671098
Authors: Stickler, C. M. - Coe, M. T. - Costa, M. H. - Nepstad, D. C. - McGrath, D. G. - Dias, L. C. - Rodrigues, H. O. - Soares-Filho, B. S.
Journal: Proc Natl Acad Sci U S A

Tropical rainforest regions have large hydropower generation potential that figures prominently in many nations' energy growth strategies. Feasibility studies of hydropower plants typically ignore the effect of future deforestation or assume that deforestation will have a positive effect on river discharge and energy generation resulting from declines in evapotranspiration (ET) associated with forest conversion. Forest loss can also reduce river discharge, however, by inhibiting rainfall. We used land use, hydrological, and climate models to examine the local "direct" effects (through changes in ET within the watershed) and the potential regional "indirect" effects (through changes in rainfall) of deforestation on river discharge and energy generation potential for the Belo Monte energy complex, one of the world's largest hydropower plants that is currently under construction on the Xingu River in the eastern Amazon. In the absence of indirect effects of deforestation, simulated deforestation of 20% and 40% within the Xingu River basin increased discharge by 4-8% and 10-12%, with similar increases in energy generation. When indirect effects were considered, deforestation of the Amazon region inhibited rainfall within the Xingu Basin, counterbalancing declines in ET and decreasing discharge by 6-36%. Under business-as-usual projections of forest loss for 2050 (40%), simulated power generation declined to only 25% of maximum plant output and 60% of the industry's own projections. Like other energy sources, hydropower plants present large social and environmental costs. Their reliability as energy sources, however, must take into account their dependence on forests.

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Zoonosis emergence linked to agricultural intensification and environmental change.

Publication Date: 2013 May 21 PMID: 23671097
Authors: Jones, B. A. - Grace, D. - Kock, R. - Alonso, S. - Rushton, J. - Said, M. Y. - McKeever, D. - Mutua, F. - Young, J. - McDermott, J. - Pfeiffer, D. U.
Journal: Proc Natl Acad Sci U S A

A systematic review was conducted by a multidisciplinary team to analyze qualitatively best available scientific evidence on the effect of agricultural intensification and environmental changes on the risk of zoonoses for which there are epidemiological interactions between wildlife and livestock. The study found several examples in which agricultural intensification and/or environmental change were associated with an increased risk of zoonotic disease emergence, driven by the impact of an expanding human population and changing human behavior on the environment. We conclude that the rate of future zoonotic disease emergence or reemergence will be closely linked to the evolution of the agriculture-environment nexus. However, available research inadequately addresses the complexity and interrelatedness of environmental, biological, economic, and social dimensions of zoonotic pathogen emergence, which significantly limits our ability to predict, prevent, and respond to zoonotic disease emergence.

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New technologies reduce greenhouse gas emissions from nitrogenous fertilizer in China.

Publication Date: 2013 May 21 PMID: 23671096
Authors: Zhang, W. F. - Dou, Z. X. - He, P. - Ju, X. T. - Powlson, D. - Chadwick, D. - Norse, D. - Lu, Y. L. - Zhang, Y. - Wu, L. - Chen, X. P. - Cassman, K. G. - Zhang, F. S.
Journal: Proc Natl Acad Sci U S A

Synthetic nitrogen (N) fertilizer has played a key role in enhancing food production and keeping half of the world's population adequately fed. However, decades of N fertilizer overuse in many parts of the world have contributed to soil, water, and air pollution; reducing excessive N losses and emissions is a central environmental challenge in the 21st century. China's participation is essential to global efforts in reducing N-related greenhouse gas (GHG) emissions because China is the largest producer and consumer of fertilizer N. To evaluate the impact of China's use of N fertilizer, we quantify the carbon footprint of China's N fertilizer production and consumption chain using life cycle analysis. For every ton of N fertilizer manufactured and used, 13.5 tons of CO2-equivalent (eq) (t CO2-eq) is emitted, compared with 9.7 t CO2-eq in Europe. Emissions in China tripled from 1980 [131 terrogram (Tg) of CO2-eq (Tg CO2-eq)] to 2010 (452 Tg CO2-eq). N fertilizer-related emissions constitute about 7% of GHG emissions from the entire Chinese economy and exceed soil carbon gain resulting from N fertilizer use by several-fold. We identified potential emission reductions by comparing prevailing technologies and management practices in China with more advanced options worldwide. Mitigation opportunities include improving methane recovery during coal mining, enhancing energy efficiency in fertilizer manufacture, and minimizing N overuse in field-level crop production. We find that use of advanced technologies could cut N fertilizer-related emissions by 20-63%, amounting to 102-357 Tg CO2-eq annually. Such reduction would decrease China's total GHG emissions by 2-6%, which is significant on a global scale.

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Sinuous rivers.

Publication Date: 2013 May 21 PMID: 23671095
Authors: Baker, V. R.
Journal: Proc Natl Acad Sci U S A

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Millennial-scale isotope records from a wide-ranging predator show evidence of recent human impact to oceanic food webs.

Publication Date: 2013 May 13 PMID: 23671094
Authors: Wiley, A. E. - Ostrom, P. H. - Welch, A. J. - Fleischer, R. C. - Gandhi, H. - Southon, J. R. - Stafford, T. W. Jr - Penniman, J. F. - Hu, D. - Duvall, F. P. - James, H. F.
Journal: Proc Natl Acad Sci U S A

Human exploitation of marine ecosystems is more recent in oceanic than near shore regions, yet our understanding of human impacts on oceanic food webs is comparatively poor. Few records of species that live beyond the continental shelves date back more than 60 y, and the sheer size of oceanic regions makes their food webs difficult to study, even in modern times. Here, we use stable carbon and nitrogen isotopes to study the foraging history of a generalist, oceanic predator, the Hawaiian petrel (Pterodroma sandwichensis), which ranges broadly in the Pacific from the equator to near the Aleutian Islands. Our isotope records from modern and ancient, radiocarbon-dated bones provide evidence of over 3,000 y of dietary stasis followed by a decline of ca. 1.8 per thousand in delta15N over the past 100 y. Fishery-induced trophic decline is the most likely explanation for this sudden shift, which occurs in genetically distinct populations with disparate foraging locations. Our isotope records also show that coincident with the apparent decline in trophic level, foraging segregation among petrel populations decreased markedly. Because variation in the diet of generalist predators can reflect changing availability of their prey, a foraging shift in wide-ranging Hawaiian petrel populations suggests a relatively rapid change in the composition of oceanic food webs in the Northeast Pacific. Understanding and mitigating widespread shifts in prey availability may be a critical step in the conservation of endangered marine predators such as the Hawaiian petrel.

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Graphene field effect transistor without an energy gap.

Publication Date: 2013 May 13 PMID: 23671093
Authors: Jang, M. S. - Kim, H. - Son, Y. W. - Atwater, H. A. - Goddard, W. A. 3rd
Journal: Proc Natl Acad Sci U S A

Graphene is a room temperature ballistic electron conductor and also a very good thermal conductor. Thus, it has been regarded as an ideal material for postsilicon electronic applications. A major complication is that the relativistic massless electrons in pristine graphene exhibit unimpeded Klein tunneling penetration through gate potential barriers. Thus, previous efforts to realize a field effect transistor for logic applications have assumed that introduction of a band gap in graphene is a prerequisite. Unfortunately, extrinsic treatments designed to open a band gap seriously degrade device quality, yielding very low mobility and uncontrolled on/off current ratios. To solve this dilemma, we propose a gating mechanism that leads to a hundredfold enhancement in on/off transmittance ratio for normally incident electrons without any band gap engineering. Thus, our saw-shaped geometry gate potential (in place of the conventional bar-shaped geometry) leads to switching to an off state while retaining the ultrahigh electron mobility in the on state. In particular, we report that an on/off transmittance ratio of 130 is achievable for a sawtooth gate with a gate length of 80 nm. Our switching mechanism demonstrates that intrinsic graphene can be used in designing logic devices without serious alteration of the conventional field effect transistor architecture. This suggests a new variable for the optimization of the graphene-based device-geometry of the gate electrode.

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Innovative grassland management systems for environmental and livelihood benefits.

Publication Date: 2013 May 21 PMID: 23671092
Authors: Kemp, D. R. - Guodong, H. - Xiangyang, H. - Michalk, D. L. - Fujiang, H. - Jianping, W. - Yingjun, Z.
Journal: Proc Natl Acad Sci U S A

Grasslands occupy 40% of the world's land surface (excluding Antarctica and Greenland) and support diverse groups, from traditional extensive nomadic to intense livestock-production systems. Population pressures mean that many of these grasslands are in a degraded state, particularly in less-productive areas of developing countries, affecting not only productivity but also vital environmental services such as hydrology, biodiversity, and carbon cycles; livestock condition is often poor and household incomes are at or below poverty levels. The challenge is to optimize management practices that result in "win-win" outcomes for grasslands, the environment, and households. A case study is discussed from northwestern China, where it has been possible to reduce animal numbers considerably by using an energy-balance/market-based approach while improving household incomes, providing conditions within which grassland recovery is possible. This bottom-up approach was supported by informing and working with the six layers of government in China to build appropriate policies. Further policy implications are considered. Additional gains in grassland rehabilitation could be fostered through targeted environmental payment schemes. Other aspects of the livestock production system that can be modified are discussed. This work built a strategy that has implications for many other grassland areas around the world where common problems apply.

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Hypoxic and Ras-transformed cells support growth by scavenging unsaturated fatty acids from lysophospholipids.

Publication Date: 2013 May 13 PMID: 23671091
Authors: Kamphorst, J. J. - Cross, J. R. - Fan, J. - de Stanchina, E. - Mathew, R. - White, E. P. - Thompson, C. B. - Rabinowitz, J. D.
Journal: Proc Natl Acad Sci U S A

Cancer cell growth requires fatty acids to replicate cellular membranes. The kinase Akt is known to up-regulate fatty acid synthesis and desaturation, which is carried out by the oxygen-consuming enzyme stearoyl-CoA desaturase (SCD)1. We used 13C tracers and lipidomics to probe fatty acid metabolism, including desaturation, as a function of oncogene expression and oxygen availability. During hypoxia, flux from glucose to acetyl-CoA decreases, and the fractional contribution of glutamine to fatty acid synthesis increases. In addition, we find that hypoxic cells bypass de novo lipogenesis, and thus, both the need for acetyl-CoA and the oxygen-dependent SCD1-reaction, by scavenging serum fatty acids. The preferred substrates for scavenging are phospholipids with one fatty acid tail (lysophospholipids). Hypoxic reprogramming of de novo lipogenesis can be reproduced in normoxic cells by Ras activation. This renders Ras-driven cells, both in culture and in allografts, resistant to SCD1 inhibition. Thus, a mechanism by which oncogenic Ras confers metabolic robustness is through lipid scavenging.

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Specialized stem cell niche enables repetitive renewal of alligator teeth.

Publication Date: 2013 May 13 PMID: 23671090
Authors: Wu, P. - Wu, X. - Jiang, T. X. - Elsey, R. M. - Temple, B. L. - Divers, S. J. - Glenn, T. C. - Yuan, K. - Chen, M. H. - Widelitz, R. B. - Chuong, C. M.
Journal: Proc Natl Acad Sci U S A

Reptiles and fish have robust regenerative powers for tooth renewal. However, extant mammals can either renew their teeth one time (diphyodont dentition) or not at all (monophyodont dentition). Humans replace their milk teeth with permanent teeth and then lose their ability for tooth renewal. Here, we study tooth renewal in a crocodilian model, the American alligator, which has well-organized teeth similar to mammals but can still undergo life-long renewal. Each alligator tooth is a complex family unit composed of the functional tooth, successional tooth, and dental lamina. Using multiple mitotic labeling, we map putative stem cells to the distal enlarged bulge of the dental lamina that contains quiescent odontogenic progenitors that can be activated during physiological exfoliation or artificial extraction. Tooth cycle initiation correlates with beta-catenin activation and soluble frizzled-related protein 1 disappearance in the bulge. The dermal niche adjacent to the dermal lamina dynamically expresses neural cell adhesion molecule, tenascin-C, and other molecules. Furthermore, in development, asymmetric beta-catenin localization leads to the formation of a heterochronous and complex tooth family unit configuration. Understanding how these signaling molecules interact in tooth development in this model may help us to learn how to stimulate growth of adult teeth in mammals.

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Innovations in capture fisheries are an imperative for nutrition security in the developing world.

Publication Date: 2013 May 21 PMID: 23671089
Authors: Hall, S. J. - Hilborn, R. - Andrew, N. L. - Allison, E. H.
Journal: Proc Natl Acad Sci U S A

This article examines two strands of discourse on wild capture fisheries; one that focuses on resource sustainability and environmental impacts, another related to food and nutrition security and human well-being. Available data and research show that, for countries most dependent on fish to meet the nutritional requirements of their population, wild capture fisheries remain the dominant supplier. Although, contrary to popular narratives, the sustainability of these fisheries is not always and everywhere in crisis, securing their sustainability is essential and requires considerable effort across a broad spectrum of fishery systems. An impediment to achieving this is that the current research and policy discourses on environmental sustainability of fisheries and food security remain only loosely and superficially linked. Overcoming this requires adoption of a broader sustainability science paradigm to help harness synergies and negotiate tradeoffs between food security, resource conservation, and macroeconomic development goals. The way society chooses to govern fisheries is, however, an ethical choice, not just a technical one, and we recommend adding an ethical dimension to sustainability science as applied to fisheries.

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Distinct preplay of multiple novel spatial experiences in the rat.

Publication Date: 2013 May 13 PMID: 23671088
Authors: Dragoi, G. - Tonegawa, S.
Journal: Proc Natl Acad Sci U S A

The activity of ensembles of hippocampal place cells represents a hallmark of an animal's spatial experience. The neuronal mechanisms that enable the rapid expression of novel place cell sequences are not entirely understood. Here we report that during sleep or rest, distinct sets of hippocampal temporal sequences in the rat preplay multiple corresponding novel spatial experiences with high specificity. These findings suggest that the place cell sequence of a novel spatial experience is determined, in part, by an online selection of a subset of cellular firing sequences from a larger repertoire of preexisting temporal firing sequences in the hippocampal cellular assembly network that become rapidly bound to the novel experience. We estimate that for the given context, the recorded hippocampal network activity has the capacity to preplay an extended repertoire of at least 15 future spatial experiences of similar distinctiveness and complexity.

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High regional climate sensitivity over continental China constrained by glacial-recent changes in temperature and the hydrological cycle.

Publication Date: 2013 May 13 PMID: 23671087
Authors: Eagle, R. A. - Risi, C. - Mitchell, J. L. - Eiler, J. M. - Seibt, U. - Neelin, J. D. - Li, G. - Tripati, A. K.
Journal: Proc Natl Acad Sci U S A

The East Asian monsoon is one of Earth's most significant climatic phenomena, and numerous paleoclimate archives have revealed that it exhibits variations on orbital and suborbital time scales. Quantitative constraints on the climate changes associated with these past variations are limited, yet are needed to constrain sensitivity of the region to changes in greenhouse gas levels. Here, we show central China is a region that experienced a much larger temperature change since the Last Glacial Maximum than typically simulated by climate models. We applied clumped isotope thermometry to carbonates from the central Chinese Loess Plateau to reconstruct temperature and water isotope shifts from the Last Glacial Maximum to present. We find a summertime temperature change of 6-7 degrees C that is reproduced by climate model simulations presented here. Proxy data reveal evidence for a shift to lighter isotopic composition of meteoric waters in glacial times, which is also captured by our model. Analysis of model outputs suggests that glacial cooling over continental China is significantly amplified by the influence of stationary waves, which, in turn, are enhanced by continental ice sheets. These results not only support high regional climate sensitivity in Central China but highlight the fundamental role of planetary-scale atmospheric dynamics in the sensitivity of regional climates to continental glaciation, changing greenhouse gas levels, and insolation.

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Green Revolution research saved an estimated 18 to 27 million hectares from being brought into agricultural production.

Publication Date: 2013 May 21 PMID: 23671086
Authors: Stevenson, J. R. - Villoria, N. - Byerlee, D. - Kelley, T. - Maredia, M.
Journal: Proc Natl Acad Sci U S A

New estimates of the impacts of germplasm improvement in the major staple crops between 1965 and 2004 on global land-cover change are presented, based on simulations carried out using a global economic model (Global Trade Analysis Project Agro-Ecological Zone), a multicommodity, multiregional computable general equilibrium model linked to a global spatially explicit database on land use. We estimate the impact of removing the gains in cereal productivity attributed to the widespread adoption of improved varieties in developing countries. Here, several different effects-higher yields, lower prices, higher land rents, and trade effects-have been incorporated in a single model of the impact of Green Revolution research (and subsequent advances in yields from crop germplasm improvement) on land-cover change. Our results generally support the Borlaug hypothesis that increases in cereal yields as a result of widespread adoption of improved crop germplasm have saved natural ecosystems from being converted to agriculture. However, this relationship is complex, and the net effect is of a much smaller magnitude than Borlaug proposed. We estimate that the total crop area in 2004 would have been between 17.9 and 26.7 million hectares larger in a world that had not benefited from crop germplasm improvement since 1965. Of these hectares, 12.0-17.7 million would have been in developing countries, displacing pastures and resulting in an estimated 2 million hectares of additional deforestation. However, the negative impacts of higher food prices on poverty and hunger under this scenario would likely have dwarfed the welfare effects of agricultural expansion.

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Cyclophilin 20-3 relays a 12-oxo-phytodienoic acid signal during stress responsive regulation of cellular redox homeostasis.

Publication Date: 2013 May 13 PMID: 23671085
Authors: Park, S. W. - Li, W. - Viehhauser, A. - He, B. - Kim, S. - Nilsson, A. K. - Andersson, M. X. - Kittle, J. D. - Ambavaram, M. M. - Luan, S. - Esker, A. R. - Tholl, D. - Cimini, D. - Ellerstrom, M. - Coaker, G. - Mitchell, T. K. - Pereira, A. - Dietz, K. J. - Lawrence, C. B.
Journal: Proc Natl Acad Sci U S A

The jasmonate family of phytohormones plays central roles in plant development and stress acclimation. However, the architecture of their signaling circuits remains largely unknown. Here we describe a jasmonate family binding protein, cyclophilin 20-3 (CYP20-3), which regulates stress-responsive cellular redox homeostasis. (+)-12-oxo-phytodienoic acid (OPDA) binding promotes CYP20-3 to form a complex with serine acetyltransferase 1, which triggers the formation of a hetero-oligomeric cysteine synthase complex with O-acetylserine(thiol)lyase B in chloroplasts. The cysteine synthase complex formation then activates sulfur assimilation that leads to increased levels of thiol metabolites and the buildup of cellular reduction potential. The enhanced redox capacity in turn coordinates the expression of a subset of OPDA-responsive genes. Thus, we conclude that CYP20-3 is a key effector protein that links OPDA signaling to amino acid biosynthesis and cellular redox homeostasis in stress responses.

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Calcium signaling mediates cold sensing in insect tissues.

Publication Date: 2013 May 13 PMID: 23671084
Authors: Teets, N. M. - Yi, S. X. - Lee, R. E. Jr - Denlinger, D. L.
Journal: Proc Natl Acad Sci U S A

The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 degrees C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms.

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Biodegradable synthetic high-density lipoprotein nanoparticles for atherosclerosis.

Publication Date: 2013 May 13 PMID: 23671083
Authors: Marrache, S. - Dhar, S.
Journal: Proc Natl Acad Sci U S A

Atherosclerosis remains one of the most common causes of death in the United States and throughout the world because of the lack of early detection. Macrophage apoptosis is a major contributor to the instability of atherosclerotic lesions. Development of an apoptosis targeted high-density lipoprotein (HDL)-mimicking nanoparticle (NP) to carry contrast agents for early detection of vulnerable plaques and the initiation of preventative therapies that exploit the vascular protective effects of HDL can be attractive for atherosclerosis. Here, we report the construction of a synthetic, biodegradable HDL-NP platform for detection of vulnerable plaques by targeting the collapse of mitochondrial membrane potential that occurs during apoptosis. This HDL mimic contains a core of biodegradable poly(lactic-co-glycolic acid), cholesteryl oleate, and a phospholipid bilayer coat that is decorated with triphenylphosphonium (TPP) cations for detection of mitochondrial membrane potential collapse. The lipid layer provides the surface for adsorption of apolipoprotein (apo) A-I mimetic 4F peptide, and the core contains diagnostically active quantum dots (QDs) for optical imaging. In vitro uptake, detection of apoptosis, and cholesterol binding studies indicated promising detection ability and therapeutic potential of TPP-HDL-apoA-I-QD NPs. In vitro studies indicated the potential of these NPs in reverse cholesterol transport. In vivo biodistribution and pharmacokinetics indicated favorable tissue distribution, controlled pharmacokinetic parameters, and significant triglyceride reduction for i.v.-injected TPP-HDL-apoA-I-QD NPs in rats. These HDL NPs demonstrate excellent biocompatibility, stability, nontoxic, and nonimmunogenic properties, which prove to be promising for future translation in early plaque diagnosis and might find applications to prevent vulnerable plaque progression.

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Integrating the HIV-1 assembly/maturation pathway.

Publication Date: 2013 May 21 PMID: 23671082
Authors: Potempa, M. - Swanstrom, R.
Journal: Proc Natl Acad Sci U S A

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Molecular layer perforant path-associated cells contribute to feed-forward inhibition in the adult dentate gyrus.

Publication Date: 2013 May 13 PMID: 23671081
Authors: Li, Y. - Stam, F. J. - Aimone, J. B. - Goulding, M. - Callaway, E. M. - Gage, F. H.
Journal: Proc Natl Acad Sci U S A

New neurons, which have been implicated in pattern separation, are continually generated in the dentate gyrus in the adult hippocampus. Using a genetically modified rabies virus, we demonstrated that molecular layer perforant pathway (MOPP) cells innervated newborn granule neurons in adult mouse brain. Stimulating the perforant pathway resulted in the activation of MOPP cells before the activation of dentate granule neurons. Moreover, activation of MOPP cells by focal uncaging of glutamate induced strong inhibition of granule cells. Together, these results indicate that MOPP cells located in the molecular layer of the dentate gyrus contribute to feed-forward inhibition of granule cells via perforant pathway activation.

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Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers.

Publication Date: 2013 May 13 PMID: 23671080
Authors: Rakowska, P. D. - Jiang, H. - Ray, S. - Pyne, A. - Lamarre, B. - Carr, M. - Judge, P. J. - Ravi, J. - M Gerling, U. I. - Koksch, B. - Martyna, G. J. - Hoogenboom, B. W. - Watts, A. - Crain, J. - Grovenor, C. R. - Ryadnov, M. G.
Journal: Proc Natl Acad Sci U S A

Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.

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Early hominin auditory ossicles from South Africa.

Publication Date: 2013 May 13 PMID: 23671079
Authors: Quam, R. M. - de Ruiter, D. J. - Masali, M. - Arsuaga, J. L. - Martinez, I. - Moggi-Cecchi, J.
Journal: Proc Natl Acad Sci U S A

The middle ear ossicles are only rarely preserved in fossil hominins. Here, we report the discovery of a complete ossicular chain (malleus, incus, and stapes) of Paranthropus robustus as well as additional ear ossicles from Australopithecus africanus. The malleus in both early hominin taxa is clearly human-like in the proportions of the manubrium and corpus, whereas the incus and stapes resemble African and Asian great apes more closely. A deep phylogenetic origin is proposed for the derived malleus morphology, and this may represent one of the earliest human-like features to appear in the fossil record. The anatomical differences found in the early hominin incus and stapes, along with other aspects of the outer, middle, and inner ear, are consistent with the suggestion of different auditory capacities in these early hominin taxa compared with modern humans.

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Mannodendrimers prevent acute lung inflammation by inhibiting neutrophil recruitment.

Publication Date: 2013 May 13 PMID: 23671078
Authors: Blattes, E. - Vercellone, A. - Eutamene, H. - Turrin, C. O. - Theodorou, V. - Majoral, J. P. - Caminade, A. M. - Prandi, J. - Nigou, J. - Puzo, G.
Journal: Proc Natl Acad Sci U S A

Mycobacterium tuberculosis mannose-capped lipoarabinomannan inhibits the release of proinflammatory cytokines by LPS-stimulated human dendritic cells (DCs) via targeting the C-type lectin receptor DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN). With the aim of mimicking the bioactive supramolecular structure of mannose-capped lipoarabinomannan, we designed and synthesized a set of poly(phosphorhydrazone) dendrimers grafted with mannose units, called mannodendrimers, that differed by size and the number and length of their (alpha1-->2)-oligommanoside caps. A third-generation dendrimer bearing 48 trimannoside caps (3T) and a fourth-generation dendrimer bearing 96 dimannosides (4D) displayed the highest binding avidity for DC-SIGN. Moreover, these dendrimers inhibited proinflammatory cytokines, including TNF-alpha, production by LPS-stimulated DCs in a DC-SIGN-dependent fashion. Finally, in a model of acute lung inflammation in which mice were exposed to aerosolized LPS, per os administration of 3T mannodendrimer was found to significantly reduce neutrophil influx via targeting the DC-SIGN murine homolog SIGN-related 1. The 3T mannodendrimer therefore represents an innovative fully synthetic compound for the treatment of lung inflammatory diseases.

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182Hf-182W age dating of a 26Al-poor inclusion and implications for the origin of short-lived radioisotopes in the early Solar System.

Publication Date: 2013 May 13 PMID: 23671077
Authors: Holst, J. C. - Olsen, M. B. - Paton, C. - Nagashima, K. - Schiller, M. - Wielandt, D. - Larsen, K. K. - Connelly, J. N. - Jorgensen, J. K. - Krot, A. N. - Nordlund, A. - Bizzarro, M.
Journal: Proc Natl Acad Sci U S A

Refractory inclusions [calcium-aluminum-rich inclusions, (CAIs)] represent the oldest Solar System solids and provide information regarding the formation of the Sun and its protoplanetary disk. CAIs contain evidence of now extinct short-lived radioisotopes (e.g., 26Al, 41Ca, and 182Hf) synthesized in one or multiple stars and added to the protosolar molecular cloud before or during its collapse. Understanding how and when short-lived radioisotopes were added to the Solar System is necessary to assess their validity as chronometers and constrain the birthplace of the Sun. Whereas most CAIs formed with the canonical abundance of 26Al corresponding to 26Al/27Al of approximately 5 x 10-5, rare CAIs with fractionation and unidentified nuclear isotope effects (FUN CAIs) record nucleosynthetic isotopic heterogeneity and 26Al/27Al of <5 x 10-6, possibly reflecting their formation before canonical CAIs. Thus, FUN CAIs may provide a unique window into the earliest Solar System, including the origin of short-lived radioisotopes. However, their chronology is unknown. Using the 182Hf-182W chronometer, we show that a FUN CAI recording a condensation origin from a solar gas formed coevally with canonical CAIs, but with 26Al/27Al of approximately 3 x 10-6. The decoupling between 182Hf and 26Al requires distinct stellar origins: steady-state galactic stellar nucleosynthesis for 182Hf and late-stage contamination of the protosolar molecular cloud by a massive star(s) for 26Al. Admixing of stellar-derived 26Al to the protoplanetary disk occurred during the epoch of CAI formation and, therefore, the 26Al-26Mg systematics of CAIs cannot be used to define their formation interval. In contrast, our results support 182Hf homogeneity and chronological significance of the 182Hf-182W clock.

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Concordant mast cell and basophil production by individual hematopoietic blast colony-forming cells.

Publication Date: 2013 May 13 PMID: 23671076
Authors: Metcalf, D. - Ng, A. P. - Baldwin, T. M. - Di Rago, L. - Mifsud, S.
Journal: Proc Natl Acad Sci U S A

Previous studies have shown that mouse bone marrow cells can produce mast cells when stimulated in vitro by stem cell factor (SCF) and interleukin-3 (IL-3). Experiments to define the marrow cells able to generate mast cells showed that the most active subpopulations were the Kit+ Sca1- progenitor cell fraction and the more ancestral Kit+ Sca1+ blast colony-forming cell fraction. In clonal cultures, up to 64% of blast colony-forming cells were able to generate mast cells when stimulated by SCF and IL-3, and, of these, the most active were those in the CD34- Flt3R- long-term repopulating cell fraction. Basophils, identified by the monoclonal antibody mMCP-8 to mouse mast cell serine protease-8, were also produced by 50% of blast colony-forming cells with a strong concordance in the production of both cell types by individual blast colony-forming cells. Enriched populations of marrow-derived basophils were shown to generate variable numbers of mast cells after a further incubation with SCF and IL-3. The data extend the repertoire of lineage-committed cells able to be produced by multipotential hematopoietic blast colony-forming cells and show that basophils and mast cells can have common ancestral cells and that basophils can probably generate mast cells at least under defined in vitro conditions.

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Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX.

Publication Date: 2013 May 13 PMID: 23671075
Authors: Laptenok, S. P. - Bouzhir-Sima, L. - Lambry, J. C. - Myllykallio, H. - Liebl, U. - Vos, M. H.
Journal: Proc Natl Acad Sci U S A

In many bacteria the flavoenzyme thymidylate synthase ThyX produces the DNA nucleotide deoxythymidine monophosphate from dUMP, using methylenetetrahydrofolate as carbon donor and NADPH as hydride donor. Because all three substrates bind in close proximity to the catalytic flavin adenine dinucleotide group, substantial flexibility of the ThyX active site has been hypothesized. Using femtosecond time-resolved fluorescence spectroscopy, we have studied the conformational heterogeneity and the conformational interconversion dynamics in real time in ThyX from the hyperthermophilic bacterium Thermotoga maritima. The dynamics of electron transfer to excited flavin adenine dinucleotide from a neighboring tyrosine residue are used as a sensitive probe of the functional dynamics of the active site. The fluorescence decay spanned a full three orders of magnitude, demonstrating a very wide range of conformations. In particular, at physiological temperatures, multiple angstrom cofactor-residue displacements occur on the picoseconds timescale. These experimental findings are supported by molecular dynamics simulations. Binding of the dUMP substrate abolishes this flexibility and stabilizes the active site in a configuration where dUMP closely interacts with the flavin cofactor and very efficiently quenches fluorescence itself. Our results indicate a dynamic selected-fit mechanism where binding of the first substrate dUMP at high temperature stabilizes the enzyme in a configuration favorable for interaction with the second substrate NADPH, and more generally have important implications for the role of active site flexibility in enzymes interacting with multiple poly-atom substrates and products. Moreover, our data provide the basis for exploring the effect of inhibitor molecules on the active site dynamics of ThyX and other multisubstrate flavoenzymes.

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Human frontal lobes are not relatively large.

Publication Date: 2013 May 13 PMID: 23671074
Authors: Barton, R. A. - Venditti, C.
Journal: Proc Natl Acad Sci U S A

One of the most pervasive assumptions about human brain evolution is that it involved relative enlargement of the frontal lobes. We show that this assumption is without foundation. Analysis of five independent data sets using correctly scaled measures and phylogenetic methods reveals that the size of human frontal lobes, and of specific frontal regions, is as expected relative to the size of other brain structures. Recent claims for relative enlargement of human frontal white matter volume, and for relative enlargement shared by all great apes, seem to be mistaken. Furthermore, using a recently developed method for detecting shifts in evolutionary rates, we find that the rate of change in relative frontal cortex volume along the phylogenetic branch leading to humans was unremarkable and that other branches showed significantly faster rates of change. Although absolute and proportional frontal region size increased rapidly in humans, this change was tightly correlated with corresponding size increases in other areas and whole brain size, and with decreases in frontal neuron densities. The search for the neural basis of human cognitive uniqueness should therefore focus less on the frontal lobes in isolation and more on distributed neural networks.

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Interactive effects among ecosystem services and management practices on crop production: Pollination in coffee agroforestry systems.

Publication Date: 2013 May 21 PMID: 23671073
Authors: Boreux, V. - Kushalappa, C. G. - Vaast, P. - Ghazoul, J.
Journal: Proc Natl Acad Sci U S A

Crop productivity is improved by ecosystem services, including pollination, but this should be set in the context of trade-offs among multiple management practices. We investigated the impact of pollination services on coffee production, considering variation in fertilization, irrigation, shade cover, and environmental variables such as rainfall (which stimulates coffee flowering across all plantations), soil pH, and nitrogen availability. After accounting for management interventions, bee abundance improved coffee production (number of berries harvested). Some management interventions, such as irrigation, used once to trigger asynchronous flowering, dramatically increased bee abundance at coffee trees. Others, such as the extent and type of tree cover, revealed interacting effects on pollination and, ultimately, crop production. The effects of management interventions, notably irrigation and addition of lime, had, however, far more substantial positive effects on coffee production than tree cover. These results suggest that pollination services matter, but managing the asynchrony of flowering was a more effective tool for securing good pollination than maintaining high shade tree densities as pollinator habitat. Complex interactions across farm and landscape scales, including both management practices and environmental conditions, shape pollination outcomes. Effective production systems therefore require the integrated consideration of management practices in the context of the surrounding habitat structure. This paper points toward a more strategic use of ecosystem services in agricultural systems, where ecosystem services are shaped by the coupling of management interventions and environmental variables.

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Nucleotide specificity in bacterial mRNA recycling.

Publication Date: 2013 May 13 PMID: 23671072
Authors: Bechhofer, D. H.
Journal: Proc Natl Acad Sci U S A

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Scope for improved eco-efficiency varies among diverse cropping systems.

Publication Date: 2013 May 21 PMID: 23671071
Authors: Carberry, P. S. - Liang, W. L. - Twomlow, S. - Holzworth, D. P. - Dimes, J. P. - McClelland, T. - Huth, N. I. - Chen, F. - Hochman, Z. - Keating, B. A.
Journal: Proc Natl Acad Sci U S A

Global food security requires eco-efficient agriculture to produce the required food and fiber products concomitant with ecologically efficient use of resources. This eco-efficiency concept is used to diagnose the state of agricultural production in China (irrigated wheat-maize double-cropping systems), Zimbabwe (rainfed maize systems), and Australia (rainfed wheat systems). More than 3,000 surveyed crop yields in these three countries were compared against simulated grain yields at farmer-specified levels of nitrogen (N) input. Many Australian commercial wheat farmers are both close to existing production frontiers and gain little prospective return from increasing their N input. Significant losses of N from their systems, either as nitrous oxide emissions or as nitrate leached from the soil profile, are infrequent and at low intensities relative to their level of grain production. These Australian farmers operate close to eco-efficient frontiers in regard to N, and so innovations in technologies and practices are essential to increasing their production without added economic or environmental risks. In contrast, many Chinese farmers can reduce N input without sacrificing production through more efficient use of their fertilizer input. In fact, there are real prospects for the double-cropping systems on the North China Plain to achieve both production increases and reduced environmental risks. Zimbabwean farmers have the opportunity for significant production increases by both improving their technical efficiency and increasing their level of input; however, doing so will require improved management expertise and greater access to institutional support for addressing the higher risks. This paper shows that pathways for achieving improved eco-efficiency will differ among diverse cropping systems.

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Circadian patterns of gene expression in the human brain and disruption in major depressive disorder.

Publication Date: 2013 May 13 PMID: 23671070
Authors: Li, J. Z. - Bunney, B. G. - Meng, F. - Hagenauer, M. H. - Walsh, D. M. - Vawter, M. P. - Evans, S. J. - Choudary, P. V. - Cartagena, P. - Barchas, J. D. - Schatzberg, A. F. - Jones, E. G. - Myers, R. M. - Watson, S. J. Jr - Akil, H. - Bunney, W. E.
Journal: Proc Natl Acad Sci U S A

A cardinal symptom of major depressive disorder (MDD) is the disruption of circadian patterns. However, to date, there is no direct evidence of circadian clock dysregulation in the brains of patients who have MDD. Circadian rhythmicity of gene expression has been observed in animals and peripheral human tissues, but its presence and variability in the human brain were difficult to characterize. Here, we applied time-of-death analysis to gene expression data from high-quality postmortem brains, examining 24-h cyclic patterns in six cortical and limbic regions of 55 subjects with no history of psychiatric or neurological illnesses ("controls") and 34 patients with MDD. Our dataset covered approximately 12,000 transcripts in the dorsolateral prefrontal cortex, anterior cingulate cortex, hippocampus, amygdala, nucleus accumbens, and cerebellum. Several hundred transcripts in each region showed 24-h cyclic patterns in controls, and >100 transcripts exhibited consistent rhythmicity and phase synchrony across regions. Among the top-ranked rhythmic genes were the canonical clock genes BMAL1(ARNTL), PER1-2-3, NR1D1(REV-ERBa), DBP, BHLHE40 (DEC1), and BHLHE41(DEC2). The phasing of known circadian genes was consistent with data derived from other diurnal mammals. Cyclic patterns were much weaker in the brains of patients with MDD due to shifted peak timing and potentially disrupted phase relationships between individual circadian genes. This transcriptome-wide analysis of the human brain demonstrates a rhythmic rise and fall of gene expression in regions outside of the suprachiasmatic nucleus in control subjects. The description of its breakdown in MDD suggests potentially important molecular targets for treatment of mood disorders.

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Visualization of pinholin lesions in vivo.

Publication Date: 2013 May 13 PMID: 23671069
Authors: Pang, T. - Fleming, T. C. - Pogliano, K. - Young, R.
Journal: Proc Natl Acad Sci U S A

Lambdoid phage 21 uses a pinholin-signal anchor release endolysin strategy to effect temporally regulated host lysis. In this strategy, the pinholin S2168 accumulates harmlessly in the bilayer until suddenly triggering to form lethal membrane lesions, consisting of S2168 heptamers with central pores <2 nm in diameter. The membrane depolarization caused by these pores activates the muralytic endolysin, R21, leading immediately to peptidoglycan degradation. The lethal S2168 complexes have been designated as pinholes to distinguish from the micrometer-scale holes formed by canonical holins. Here, we used GFP fusions of WT and mutant forms of S2168 to show that the holin accumulates uniformly throughout the membrane until the time of triggering, when it suddenly redistributes into numerous small foci (rafts). Raft formation correlates with the depletion of the proton motive force, which is indicated by the potential-sensitive dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol. By contrast, GFP fusions of either antiholin variant irsS2168, which only forms inactive dimers, or nonlethal mutant S2168S44C, which is blocked at an activated dimer stage of the pinhole formation pathway, were both blocked in a state of uniform distribution. In addition, fluorescence recovery after photobleaching revealed that, although the antiholin irsS2168-GFP fusion was highly mobile in the membrane (even when the proton motive force was depleted), more than one-half of the S2168-GFP molecules were immobile, and the rest were in mobile states with a much lower diffusion rate than the rate of irsS2168-GFP. These results suggest a model in which, after transiting into an oligomeric state, S2168 migrates into rafts with heterogeneous sizes, within which the final pinholes form.

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PI3Kalpha activates integrin alpha4beta1 to establish a metastatic niche in lymph nodes.

Publication Date: 2013 May 13 PMID: 23671068
Authors: Garmy-Susini, B. - Avraamides, C. J. - Desgrosellier, J. S. - Schmid, M. C. - Foubert, P. - Ellies, L. G. - Lowy, A. M. - Blair, S. L. - Vandenberg, S. R. - Datnow, B. - Wang, H. Y. - Cheresh, D. A. - Varner, J.
Journal: Proc Natl Acad Sci U S A

Lymph nodes are initial sites of tumor metastasis, yet whether the lymph node microenvironment actively promotes tumor metastasis remains unknown. We show here that VEGF-C/PI3Kalpha-driven remodeling of lymph nodes promotes tumor metastasis by activating integrin alpha4beta1 on lymph node lymphatic endothelium. Activated integrin alpha4beta1 promotes expansion of the lymphatic endothelium in lymph nodes and serves as an adhesive ligand that captures vascular cell adhesion molecule 1 (VCAM-1)+ metastatic tumor cells, thereby promoting lymph node metastasis. Experimental induction of alpha4beta1 expression in lymph nodes is sufficient to promote tumor cell adhesion to lymphatic endothelium and lymph node metastasis in vivo, whereas genetic or pharmacological blockade of integrin alpha4beta1 or VCAM-1 inhibits it. As lymph node metastases accurately predict poor disease outcome, and integrin alpha4beta1 is a biomarker of lymphatic endothelium in tumor-draining lymph nodes from animals and patients, these results indicate that targeting integrin alpha4beta1 or VCAM to inhibit the interactions of tumor cells with the lymph node microenvironment may be an effective strategy to suppress tumor metastasis.

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Reinstatement of nicotine seeking is mediated by glutamatergic plasticity.

Publication Date: 2013 May 13 PMID: 23671067
Authors: Gipson, C. D. - Reissner, K. J. - Kupchik, Y. M. - Smith, A. C. - Stankeviciute, N. - Hensley-Simon, M. E. - Kalivas, P. W.
Journal: Proc Natl Acad Sci U S A

Nicotine abuse and addiction is a major health liability. Nicotine, an active alkaloid in tobacco, is self-administered by animals and produces cellular adaptations in brain regions associated with drug reward, such as the nucleus accumbens. However, it is unknown whether, akin to illicit drugs of abuse such as cocaine or heroin, the adaptations endure and contribute to the propensity to relapse after discontinuing nicotine use. Using a rat model of cue-induced relapse, we made morphological and electrophysiological measures of synaptic plasticity, as well as quantified glutamate overflow, in the accumbens after 2 wk of withdrawal with extinction training. We found an enduring basal increase in dendritic spine head diameter and in the ratio of AMPA to NMDA currents in accumbens spiny neurons compared with yoked saline animals at 2 wk after the last nicotine self-administration session. This synaptic potentiation was associated with an increase in both AMPA (GluA1) and NMDA (GluN2A and GluN2B) receptor subunits, and a reduction in the glutamate transporter-1 (GLT-1). When nicotine seeking was reinstated by presentation of conditioned cues, there were parallel increases in behavioral responding, extracellular glutamate, and further increases in dendritic spine head diameter and ratio of AMPA to NMDA currents within 15 min. These findings suggest that targeting glutamate transmission might inhibit cue-induced nicotine seeking. In support of this hypothesis, we found that pharmacological inhibition of GluN2A with 3-Chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulfo namide (TCN-201) or GluN2B with ifenprodil abolished reinstated nicotine seeking. These results indicate that up-regulated GluN2A, GluN2B, and rapid synaptic potentiation in the accumbens contribute to cue-induced relapse to nicotine use.

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Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo.

Publication Date: 2013 May 13 PMID: 23671066
Authors: Davis, S. C. - Samkoe, K. S. - Tichauer, K. M. - Sexton, K. J. - Gunn, J. R. - Deharvengt, S. J. - Hasan, T. - Pogue, B. W.
Journal: Proc Natl Acad Sci U S A

The up-regulation of cell surface receptors has become a central focus in personalized cancer treatment; however, because of the complex nature of contrast agent pharmacokinetics in tumor tissue, methods to quantify receptor binding in vivo remain elusive. Here, we present a dual-tracer optical technique for noninvasive estimation of specific receptor binding in cancer. A multispectral MRI-coupled fluorescence molecular tomography system was used to image the uptake kinetics of two fluorescent tracers injected simultaneously, one tracer targeted to the receptor of interest and the other tracer a nontargeted reference. These dynamic tracer data were then fit to a dual-tracer compartmental model to estimate the density of receptors available for binding in the tissue. Applying this approach to mice with deep-seated gliomas that overexpress the EGF receptor produced an estimate of available receptor density of 2.3 +/- 0.5 nM (n = 5), consistent with values estimated in comparative invasive imaging and ex vivo studies.

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Docosahexaenoic acid ethyl esters ineffective?

Publication Date: 2013 May 13 PMID: 23671065
Authors: Harris, W. S. - De Caterina, R. - Marik, P. E.
Journal: Proc Natl Acad Sci U S A

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Is Toso/IgM Fc receptor (FcmuR) expressed by innate immune cells?

Publication Date: 2013 May 13 PMID: 23671064
Authors: Honjo, K. - Kubagawa, Y. - Kubagawa, H.
Journal: Proc Natl Acad Sci U S A

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Nitrogen cycling, forest canopy reflectance, and emergent properties of ecosystems.

Publication Date: 2013 May 13 PMID: 23671063
Authors: Ollinger, S. V. - Reich, P. B. - Frolking, S. - Lepine, L. C. - Hollinger, D. Y. - Richardson, A. D.
Journal: Proc Natl Acad Sci U S A

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More mixotrophy in the marine microbial mix.

Publication Date: 2013 May 21 PMID: 23667152
Authors: Moore, L. R.
Journal: Proc Natl Acad Sci U S A

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Using natural variation in Drosophila to discover previously unknown endoplasmic reticulum stress genes.

Publication Date: 2013 May 10 PMID: 23667151
Authors: Chow, C. Y. - Wolfner, M. F. - Clark, A. G.
Journal: Proc Natl Acad Sci U S A

Natural genetic variation is a rich resource for identifying novel elements of cellular pathways such as endoplasmic reticulum (ER) stress. ER stress occurs when misfolded proteins accumulate in the ER and cells respond with the conserved unfolded protein response (UPR), which includes large-scale gene expression changes. Although ER stress can be a cause or a modifying factor of human disease, little is known of the amount of variation in the response to ER stress and the genes contributing to such variation. To study natural variation in ER stress response in a model system, we measured the survival time in response to tunicamycin-induced ER stress in flies from 114 lines from the sequenced Drosophila Genetic Reference Panel of wild-derived inbred strains. These lines showed high heterogeneity in survival time under ER stress conditions. To identify the genes that may be driving this phenotypic variation, we profiled ER stress-induced gene expression and performed an association study. Microarray analysis identified variation in transcript levels of numerous known and previously unknown ER stress-responsive genes. Survival time was significantly associated with polymorphisms in candidate genes with known (i.e., Xbp1) and unknown roles in ER stress. Functional testing found that 17 of 25 tested candidate genes from the association study have putative roles in ER stress. In both approaches, one-third of ER stress genes had human orthologs that contribute to human disease. This study establishes Drosophila as a useful model for studying variation in ER stress and identifying ER stress genes that may contribute to human disease.

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Single-agent combinatorial cancer therapy.

Publication Date: 2013 May 21 PMID: 23667150
Authors: Stritzker, J. - Szalay, A. A.
Journal: Proc Natl Acad Sci U S A

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Energetic basis of catalytic activity of layered nanophase calcium manganese oxides for water oxidation.

Publication Date: 2013 May 10 PMID: 23667149
Authors: Birkner, N. - Nayeri, S. - Pashaei, B. - Najafpour, M. M. - Casey, W. H. - Navrotsky, A.
Journal: Proc Natl Acad Sci U S A

Previous measurements show that calcium manganese oxide nanoparticles are better water oxidation catalysts than binary manganese oxides (Mn3O4, Mn2O3, and MnO2). The probable reasons for such enhancement involve a combination of factors: The calcium manganese oxide materials have a layered structure with considerable thermodynamic stability and a high surface area, their low surface energy suggests relatively loose binding of H2O on the internal and external surfaces, and they possess mixed-valent manganese with internal oxidation enthalpy independent of the Mn3+/Mn4+ ratio and much smaller in magnitude than the Mn2O3-MnO2 couple. These factors enhance catalytic ability by providing easy access for solutes and water to active sites and facile electron transfer between manganese in different oxidation states.

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Species richness can decrease with altitude but not with habitat diversity.

Publication Date: 2013 May 9 PMID: 23661060
Authors: Hortal, J. - Carrascal, L. M. - Triantis, K. A. - Thebault, E. - Meiri, S. - Sfenthourakis, S.
Journal: Proc Natl Acad Sci U S A

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Robust measurement of telomere length in single cells.

Publication Date: 2013 May 21 PMID: 23661059
Authors: Wang, F. - Pan, X. - Kalmbach, K. - Seth-Smith, M. L. - Ye, X. - Antumes, D. M. - Yin, Y. - Liu, L. - Keefe, D. L. - Weissman, S. M.
Journal: Proc Natl Acad Sci U S A

Measurement of telomere length currently requires a large population of cells, which masks telomere length heterogeneity in single cells, or requires FISH in metaphase arrested cells, posing technical challenges. A practical method for measuring telomere length in single cells has been lacking. We established a simple and robust approach for single-cell telomere length measurement (SCT-pqPCR). We first optimized a multiplex preamplification specific for telomeres and reference genes from individual cells, such that the amplicon provides a consistent ratio (T/R) of telomeres (T) to the reference genes (R) by quantitative PCR (qPCR). The average T/R ratio of multiple single cells corresponded closely to that of a given cell population measured by regular qPCR, and correlated with those of telomere restriction fragments (TRF) and quantitative FISH measurements. Furthermore, SCT-pqPCR detected the telomere length for quiescent cells that are inaccessible by quantitative FISH. The reliability of SCT-pqPCR also was confirmed using sister cells from two cell embryos. Telomere length heterogeneity was identified by SCT-pqPCR among cells of various human and mouse cell types. We found that the T/R values of human fibroblasts at later passages and from old donors were lower and more heterogeneous than those of early passages and from young donors, that cancer cell lines show heterogeneous telomere lengths, that human oocytes and polar bodies have nearly identical telomere lengths, and that the telomere lengths progressively increase from the zygote, two-cell to four-cell embryo. This method will facilitate understanding of telomere heterogeneity and its role in tumorigenesis, aging, and associated diseases.

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Methanol incorporation in clathrate hydrates and the implications for oil and gas pipeline flow assurance and icy planetary bodies.

Publication Date: 2013 May 21 PMID: 23661058
Authors: Shin, K. - Udachin, K. A. - Moudrakovski, I. L. - Leek, D. M. - Alavi, S. - Ratcliffe, C. I. - Ripmeester, J. A.
Journal: Proc Natl Acad Sci U S A

One of the best-known uses of methanol is as antifreeze. Methanol is used in large quantities in industrial applications to prevent methane clathrate hydrate blockages from forming in oil and gas pipelines. Methanol is also assigned a major role as antifreeze in giving icy planetary bodies (e.g., Titan) a liquid subsurface ocean and/or an atmosphere containing significant quantities of methane. In this work, we reveal a previously unverified role for methanol as a guest in clathrate hydrate cages. X-ray diffraction (XRD) and NMR experiments showed that at temperatures near 273 K, methanol is incorporated in the hydrate lattice along with other guest molecules. The amount of included methanol depends on the preparative method used. For instance, single-crystal XRD shows that at low temperatures, the methanol molecules are hydrogen-bonded in 4.4% of the small cages of tetrahydrofuran cubic structure II hydrate. At higher temperatures, NMR spectroscopy reveals a number of methanol species incorporated in hydrocarbon hydrate lattices. At temperatures characteristic of icy planetary bodies, vapor deposits of methanol, water, and methane or xenon show that the presence of methanol accelerates hydrate formation on annealing and that there is unusually complex phase behavior as revealed by powder XRD and NMR spectroscopy. The presence of cubic structure I hydrate was confirmed and a unique hydrate phase was postulated to account for the data. Molecular dynamics calculations confirmed the possibility of methanol incorporation into the hydrate lattice and show that methanol can favorably replace a number of methane guests.

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Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag.

Publication Date: 2013 May 9 PMID: 23661057
Authors: Schneider, W. M. - Brzezinski, J. D. - Aiyer, S. - Malani, N. - Gyuricza, M. - Bushman, F. D. - Roth, M. J.
Journal: Proc Natl Acad Sci U S A

The p12 protein of murine leukemia virus (MuLV) group-specific antigen (Gag) is associated with the preintegration complex, and mutants of p12 (PM14) show defects in nuclear entry or retention. Here we show that p12 proteins engineered to encode peptide sequences derived from known viral tethering proteins can direct chromatin binding during the early phase of viral replication and rescue a lethal p12-PM14 mutant. Peptides studied included segments of Kaposi sarcoma herpesvirus latency-associated nuclear antigen (LANA)1-23, human papillomavirus 8 E2, and prototype foamy virus chromatin-binding sequences. Amino acid substitutions in Kaposi sarcoma herpesvirus LANA and prototype foamy virus chromatin-binding sequences that blocked nucleosome association failed to rescue MuLV p12-PM14. Rescue by a larger LANA peptide, LANA1-32, required second-site mutations that are predicted to reduce peptide binding affinity to chromosomes, suggesting that excessively high binding affinity interfered with Gag/p12 function. This is supported by confocal microscopy of chimeric p12-GFP fusion constructs showing the reverted proteins had weaker association to condensed mitotic chromosomes. Analysis of the integration-site selection of these chimeric viruses showed no significant change in integration profile compared with wild-type MuLV, suggesting release of the tethered p12 post mitosis, before viral integration.

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Vaccine-induced plasma IgA specific for the C1 region of the HIV-1 envelope blocks binding and effector function of IgG.

Publication Date: 2013 May 9 PMID: 23661056
Authors: Tomaras, G. D. - Ferrari, G. - Shen, X. - Alam, S. M. - Liao, H. X. - Pollara, J. - Bonsignori, M. - Moody, M. A. - Fong, Y. - Chen, X. - Poling, B. - Nicholson, C. O. - Zhang, R. - Lu, X. - Parks, R. - Kaewkungwal, J. - Nitayaphan, S. - Pitisuttithum, P. - Rerks-Ngarm, S. - Gilbert, P. B. - Kim, J. H. - Michael, N. L. - Montefiori, D. C. - Haynes, B. F.
Journal: Proc Natl Acad Sci U S A

Analysis of correlates of risk of infection in the RV144 HIV-1 vaccine efficacy trial demonstrated that plasma IgG against the HIV-1 envelope (Env) variable region 1 and 2 inversely correlated with risk, whereas HIV-1 Env-specific plasma IgA responses directly correlated with risk. In the secondary analysis, antibody-dependent cellular cytotoxicity (ADCC) was another inverse correlate of risk, but only in the presence of low plasma IgA Env-specific antibodies. Thus, we investigated the hypothesis that IgA could attenuate the protective effect of IgG responses through competition for the same Env binding sites. We report that Env-specific plasma IgA/IgG ratios are higher in infected than in uninfected vaccine recipients in RV144. Moreover, Env-specific IgA antibodies from RV144 vaccinees blocked the binding of ADCC-mediating mAb to HIV-1 Env glycoprotein 120 (gp120). An Env-specific monomeric IgA mAb isolated from an RV144 vaccinee also inhibited the ability of natural killer cells to kill HIV-1-infected CD4+ T cells coated with RV144-induced IgG antibodies. We show that monomeric Env-specific IgA, as part of postvaccination polyclonal antibody response, may modulate vaccine-induced immunity by diminishing ADCC effector function.

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Solution coating around ice particles of incipient cirrus clouds.

Publication Date: 2013 May 9 PMID: 23661055
Authors: Bogdan, A. - Molina, M. J. - Kulmala, M. - Tenhu, H. - Loerting, T.
Journal: Proc Natl Acad Sci U S A

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No evidence of continuously advanced green-up dates in the Tibetan Plateau over the last decade.

Publication Date: 2013 May 9 PMID: 23661054
Authors: Shen, M. - Sun, Z. - Wang, S. - Zhang, G. - Kong, W. - Chen, A. - Piao, S.
Journal: Proc Natl Acad Sci U S A

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Caution needed when linking weather extremes to amplified planetary waves.

Publication Date: 2013 May 8 PMID: 23657013
Authors: Screen, J. A. - Simmonds, I.
Journal: Proc Natl Acad Sci U S A

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Palb2 synergizes with Trp53 to suppress mammary tumor formation in a model of inherited breast cancer.

Publication Date: 2013 May 21 PMID: 23657012
Authors: Bowman-Colin, C. - Xia, B. - Bunting, S. - Klijn, C. - Drost, R. - Bouwman, P. - Fineman, L. - Chen, X. - Culhane, A. C. - Cai, H. - Rodig, S. J. - Bronson, R. T. - Jonkers, J. - Nussenzweig, A. - Kanellopoulou, C. - Livingston, D. M.
Journal: Proc Natl Acad Sci U S A

Germ-line mutations in PALB2 lead to a familial predisposition to breast and pancreatic cancer or to Fanconi Anemia subtype N. PALB2 performs its tumor suppressor role, at least in part, by supporting homologous recombination-type double strand break repair (HR-DSBR) through physical interactions with BRCA1, BRCA2, and RAD51. To further understand the mechanisms underlying PALB2-mediated DNA repair and tumor suppression functions, we targeted Palb2 in the mouse. Palb2-deficient murine ES cells recapitulated DNA damage defects caused by PALB2 depletion in human cells, and germ-line deletion of Palb2 led to early embryonic lethality. Somatic deletion of Palb2 driven by K14-Cre led to mammary tumor formation with long latency. Codeletion of both Palb2 and Tumor protein 53 (Trp53) accelerated mammary tumor formation. Like BRCA1 and BRCA2 mutant breast cancers, these tumors were defective in RAD51 focus formation, reflecting a defect in Palb2 HR-DSBR function, a strongly suspected contributor to Brca1, Brca2, and Palb2 mammary tumor development. However, unlike the case of Brca1-mutant cells, Trp53bp1 deletion failed to rescue the genomic instability of Palb2- or Brca2-mutant primary lymphocytes. Therefore, Palb2-driven DNA damage control is, in part, distinct from that executed by Brca1 and more similar to that of Brca2. The mechanisms underlying Palb2 mammary tumor suppression functions can now be explored genetically in vivo.

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Reciprocal regulation of PKA and Rac signaling.

Publication Date: 2013 May 21 PMID: 23657011
Authors: Bachmann, V. A. - Riml, A. - Huber, R. G. - Baillie, G. S. - Liedl, K. R. - Valovka, T. - Stefan, E.
Journal: Proc Natl Acad Sci U S A

Activated G protein-coupled receptors (GPCRs) and receptor tyrosine kinases relay extracellular signals through spatial and temporal controlled kinase and GTPase entities. These enzymes are coordinated by multifunctional scaffolding proteins for precise intracellular signal processing. The cAMP-dependent protein kinase A (PKA) is the prime example for compartmentalized signal transmission downstream of distinct GPCRs. A-kinase anchoring proteins tether PKA to specific intracellular sites to ensure precision and directionality of PKA phosphorylation events. Here, we show that the Rho-GTPase Rac contains A-kinase anchoring protein properties and forms a dynamic cellular protein complex with PKA. The formation of this transient core complex depends on binary interactions with PKA subunits, cAMP levels and cellular GTP-loading accounting for bidirectional consequences on PKA and Rac downstream signaling. We show that GTP-Rac stabilizes the inactive PKA holoenzyme. However, beta-adrenergic receptor-mediated activation of GTP-Rac-bound PKA routes signals to the Raf-Mek-Erk cascade, which is critically implicated in cell proliferation. We describe a further mechanism of how cAMP enhances nuclear Erk1/2 signaling: It emanates from transphosphorylation of p21-activated kinases in their evolutionary conserved kinase-activation loop through GTP-Rac compartmentalized PKA activities. Sole transphosphorylation of p21-activated kinases is not sufficient to activate Erk1/2. It requires complex formation of both kinases with GTP-Rac1 to unleash cAMP-PKA-boosted activation of Raf-Mek-Erk. Consequently GTP-Rac functions as a dual kinase-tuning scaffold that favors the PKA holoenzyme and contributes to potentiate Erk1/2 signaling. Our findings offer additional mechanistic insights how beta-adrenergic receptor-controlled PKA activities enhance GTP-Rac-mediated activation of nuclear Erk1/2 signaling.

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The age-specific force of natural selection and biodemographic walls of death.

Publication Date: 2013 May 8 PMID: 23657010
Authors: Wachter, K. W. - Evans, S. N. - Steinsaltz, D.
Journal: Proc Natl Acad Sci U S A

W. D. Hamilton's celebrated formula for the age-specific force of natural selection furnishes predictions for senescent mortality due to mutation accumulation, at the price of reliance on a linear approximation. Applying to Hamilton's setting the full nonlinear demographic model for mutation accumulation recently developed by Evans, Steinsaltz, and Wachter, we find surprising differences. Nonlinear interactions cause the collapse of Hamilton-style predictions in the most commonly studied case, refine predictions in other cases, and allow walls of death at ages before the end of reproduction. Haldane's principle for genetic load has an exact but unfamiliar generalization.

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Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function.

Publication Date: 2013 May 21 PMID: 23657009
Authors: Goutte-Gattat, D. - Shuaib, M. - Ouararhni, K. - Gautier, T. - Skoufias, D. A. - Hamiche, A. - Dimitrov, S.
Journal: Proc Natl Acad Sci U S A

The role of the mitotic phosphorylation of the amino (NH2) terminus of Centromere Protein A (CENP-A), the histone variant epigenetic centromeric marker, remains elusive. Here, we show that the NH2 terminus of human CENP-A is essential for mitotic progression and that localization of CENP-C, another key centromeric protein, requires only phosphorylation of the CENP-A NH2 terminus, and is independent of the CENP-A NH2 terminus length and amino acid sequence. Mitotic CENP-A nucleosomal complexes contain CENP-C and phosphobinding 14-3-3 proteins. In contrast, mitotic nucleosomal complexes carrying nonphosphorylatable CENP-A-S7A contained only low levels of CENP-C and no detectable 14-3-3 proteins. Direct interactions between the phosphorylated form of CENP-A and 14-3-3 proteins as well as between 14-3-3 proteins and CENP-C were demonstrated. Taken together, our results reveal that 14-3-3 proteins could act as specific mitotic "bridges," linking phosphorylated CENP-A and CENP-C, which are necessary for the platform function of CENP-A centromeric chromatin in the assembly and maintenance of active kinetochores.

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Correction for Ndeffo Mbah et al., Cost-effectiveness of a community-based intervention for reducing the transmission of Schistosoma haematobium and HIV in Africa.

Publication Date: 2013 May 21 PMID: 23653482
Authors:
Journal: Proc Natl Acad Sci U S A

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A Huygens principle for diffusion and anomalous diffusion in spatially extended systems.

Publication Date: 2013 May 21 PMID: 23653481
Authors: Gottwald, G. A. - Melbourne, I.
Journal: Proc Natl Acad Sci U S A

We present a universal view on diffusive behavior in chaotic spatially extended systems for anisotropic and isotropic media. For anisotropic systems, strong chaos leads to diffusive behavior (Brownian motion with drift) and weak chaos leads to superdiffusive behavior (Levy processes with drift). For isotropic systems, the drift term vanishes and strong chaos again leads to Brownian motion. We establish the existence of a nonlinear Huygens principle for weakly chaotic systems in isotropic media whereby the dynamics behaves diffusively in even space dimension and exhibits superdiffusive behavior in odd space dimensions.

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Engineering bone tissue substitutes from human induced pluripotent stem cells.

Publication Date: 2013 May 21 PMID: 23653480
Authors: de Peppo, G. M. - Marcos-Campos, I. - Kahler, D. J. - Alsalman, D. - Shang, L. - Vunjak-Novakovic, G. - Marolt, D.
Journal: Proc Natl Acad Sci U S A

Congenital defects, trauma, and disease can compromise the integrity and functionality of the skeletal system to the extent requiring implantation of bone grafts. Engineering of viable bone substitutes that can be personalized to meet specific clinical needs represents a promising therapeutic alternative. The aim of our study was to evaluate the utility of human-induced pluripotent stem cells (hiPSCs) for bone tissue engineering. We first induced three hiPSC lines with different tissue and reprogramming backgrounds into the mesenchymal lineages and used a combination of differentiation assays, surface antigen profiling, and global gene expression analysis to identify the lines exhibiting strong osteogenic differentiation potential. We then engineered functional bone substitutes by culturing hiPSC-derived mesenchymal progenitors on osteoconductive scaffolds in perfusion bioreactors and confirmed their phenotype stability in a subcutaneous implantation model for 12 wk. Molecular analysis confirmed that the maturation of bone substitutes in perfusion bioreactors results in global repression of cell proliferation and an increased expression of lineage-specific genes. These results pave the way for growing patient-specific bone substitutes for reconstructive treatments of the skeletal system and for constructing qualified experimental models of development and disease.

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Evidence for orphan nuclear receptor TR4 in the etiology of Cushing disease.

Publication Date: 2013 May 21 PMID: 23653479
Authors: Du, L. - Bergsneider, M. - Mirsadraei, L. - Young, S. H. - Jonker, J. W. - Downes, M. - Yong, W. H. - Evans, R. M. - Heaney, A. P.
Journal: Proc Natl Acad Sci U S A

Cushing disease (CD) is a life-threatening disorder attributed to excess pituitary tumor-derived adrenocorticotrophic hormone (ACTH) and adrenal steroid secretion caused by pituitary tumors. Whereas CD was first described in 1932, the underlying genetic basis driving tumor growth and ACTH secretion remains unsolved. Here, we show that testicular orphan nuclear receptor 4 (TR4, nuclear receptor subfamily 2, group C, member 2) is overexpressed in human corticotroph tumors as well as in human and mouse corticotroph tumor cell lines. Forced overexpression of TR4 in both human and murine tumor cells increased proopiomelanocortin transcription, ACTH secretion, cellular proliferation, and tumor invasion rates in vitro. Conversely, knockdown of TR4 expression reversed all phenotypes. Mechanistically, we show that TR4 transcriptionally activates proopiomelanocortin through binding of a direct repeat 1 response element in the promoter, and that this is enhanced by MAPK-mediated TR4 phosphorylation. In vivo, TR4 overexpression promotes murine corticotroph tumor growth as well as enhances ACTH and corticosterone production, whereas TR4 knockdown decreases circulating ACTH and corticosterone levels in mice harboring ACTH-secreting tumors. Our findings directly link TR4 to the etiology of corticotroph tumors, hormone secretion, and cell growth as well as identify it as a potential target in the treatment of CD.

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Correction for Sievert et al., Paradoxical activation and RAF inhibitor resistance of BRAF protein kinase fusions characterizing pediatric astrocytomas.

Publication Date: 2013 May 21 PMID: 23653478
Authors:
Journal: Proc Natl Acad Sci U S A

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QnAs with Randolph Blake.

Publication Date: 2013 May 21 PMID: 23653477
Authors: Azar, B.
Journal: Proc Natl Acad Sci U S A

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Stat2 loss leads to cytokine-independent, cell-mediated lethality in LPS-induced sepsis.

Publication Date: 2013 May 21 PMID: 23653476
Authors: Alazawi, W. - Heath, H. - Waters, J. A. - Woodfin, A. - O'Brien, A. J. - Scarzello, A. J. - Ma, B. - Lopez-Otalora, Y. - Jacobs, M. - Petts, G. - Goldin, R. D. - Nourshargh, S. - Gamero, A. M. - Foster, G. R.
Journal: Proc Natl Acad Sci U S A

Deregulated Toll-like receptor (TLR)-triggered inflammatory responses that depend on NF-kappaB are detrimental to the host via excessive production of proinflammatory cytokines, including TNF-alpha. Stat2 is a critical component of type I IFN signaling, but it is not thought to participate in TLR signaling. Our study shows that LPS-induced lethality in Stat2(-/-) mice is accelerated as a result of increased cellular transmigration. Blocking intercellular adhesion molecule-1 prevents cellular egress and confers survival of Stat2(-/-) mice. The main determinant of cellular egress in Stat2(-/-) mice is the genotype of the host and not the circulating leukocyte. Surprisingly, lethality and cellular egress observed on Stat2(-/-) mice are not associated with excessive increases in classical sepsis cytokines or chemokines. Indeed, in the absence of Stat2, cytokine production in response to multiple TLR agonists is reduced. We find that Stat2 loss leads to reduced expression of NF-kappaB target genes by affecting nuclear translocation of NF-kappaB. Thus, our data reveal the existence of a different mechanism of LPS-induced lethality that is independent of NF-kappaB triggered cytokine storm but dependent on cellular egress.

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PNAS Plus Significance Statements.

Publication Date: 2013 May 7 PMID: 23653475
Authors:
Journal: Proc Natl Acad Sci U S A

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How a nested framework illuminates the challenges of comparative environmental analysis.

Publication Date: 2013 May 7 PMID: 23653474
Authors: Bors, E. K. - Solomon, S.
Journal: Proc Natl Acad Sci U S A

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Growth differentiation factor 9:bone morphogenetic protein 15 (GDF9:BMP15) synergism and protein heterodimerization.

Publication Date: 2013 May 6 PMID: 23650403
Authors: Mottershead, D. G. - Harrison, C. A. - Mueller, T. D. - Stanton, P. G. - Gilchrist, R. B. - McNatty, K. P.
Journal: Proc Natl Acad Sci U S A

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History, novelty, and emergence of an infectious amphibian disease.

Publication Date: 2013 May 6 PMID: 23650402
Authors: Collins, J. P.
Journal: Proc Natl Acad Sci U S A

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Climate change frames debate over the extinction of megafauna in Sahul (Pleistocene Australia-New Guinea).

Publication Date: 2013 May 6 PMID: 23650401
Authors: Wroe, S. - Field, J. H. - Archer, M. - Grayson, D. K. - Price, G. J. - Louys, J. - Faith, J. T. - Webb, G. E. - Davidson, I. - Mooney, S. D.
Journal: Proc Natl Acad Sci U S A

Around 88 large vertebrate taxa disappeared from Sahul sometime during the Pleistocene, with the majority of losses (54 taxa) clearly taking place within the last 400,000 years. The largest was the 2.8-ton browsing Diprotodon optatum, whereas the approximately 100- to 130-kg marsupial lion, Thylacoleo carnifex, the world's most specialized mammalian carnivore, and Varanus priscus, the largest lizard known, were formidable predators. Explanations for these extinctions have centered on climatic change or human activities. Here, we review the evidence and arguments for both. Human involvement in the disappearance of some species remains possible but unproven. Mounting evidence points to the loss of most species before the peopling of Sahul (circa 50-45 ka) and a significant role for climate change in the disappearance of the continent's megafauna.

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The posttranslational modification cascade to the thiopeptide berninamycin generates linear forms and altered macrocyclic scaffolds.

Publication Date: 2013 May 21 PMID: 23650400
Authors: Malcolmson, S. J. - Young, T. S. - Ruby, J. G. - Skewes-Cox, P. - Walsh, C. T.
Journal: Proc Natl Acad Sci U S A

Berninamycin is a member of the pyridine-containing thiopeptide class of antibiotics that undergoes massive posttranslational modifications from ribosomally generated preproteins. Berninamycin has a 2-oxazolyl-3-thiazolyl-pyridine core embedded in a 35-atom macrocycle rather than typical trithiazolylpyridine cores embedded in 26-atom and 29-atom peptide macrocycles. We describe the cloning of an 11-gene berninamycin cluster from Streptomyces bernensis UC 5144, its heterologous expression in Streptomyces lividans TK24 and Streptomyces venezuelae ATCC 10712, and detection of variant and incompletely processed scaffolds. Posttranslational maturation in S. lividans of both the wild-type berninamycin prepeptide (BerA) and also a T3A mutant generates macrocyclic compounds as well as linear variants, which have failed to form the pyridine and the macrocycle. Expression of the gene cluster in S. venezuelae generates a variant of the 35-atom skeleton of berninamycin, containing a methyloxazoline in the place of a methyloxazole within the macrocyclic framework.

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Motile invaded neutrophils in the small intestine of Toxoplasma gondii-infected mice reveal a potential mechanism for parasite spread.

Publication Date: 2013 May 21 PMID: 23650399
Authors: Coombes, J. L. - Charsar, B. A. - Han, S. J. - Halkias, J. - Chan, S. W. - Koshy, A. A. - Striepen, B. - Robey, E. A.
Journal: Proc Natl Acad Sci U S A

Toxoplasma gondii infection occurs through the oral route, but we lack important information about how the parasite interacts with the host immune system in the intestine. We used two-photon laser-scanning microscopy in conjunction with a mouse model of oral T. gondii infection to address this issue. T. gondii established discrete foci of infection in the small intestine, eliciting the recruitment and transepithelial migration of neutrophils and inflammatory monocytes. Neutrophils accounted for a high proportion of actively invaded cells, and we provide evidence for a role for transmigrating neutrophils and other immune cells in the spread of T. gondii infection through the lumen of the intestine. Our data identify neutrophils as motile reservoirs of T. gondii infection and suggest a surprising retrograde pathway for parasite spread in the intestine.

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Impaired learning resulting from Respiratory Syncytial Virus infection.

Publication Date: 2013 May 6 PMID: 23650398
Authors: Espinoza, J. A. - Bohmwald, K. - Cespedes, P. F. - Gomez, R. S. - Riquelme, S. A. - Cortes, C. M. - Valenzuela, J. A. - Sandoval, R. A. - Pancetti, F. C. - Bueno, S. M. - Riedel, C. A. - Kalergis, A. M.
Journal: Proc Natl Acad Sci U S A

Respiratory syncytial virus (RSV) is the major cause of respiratory illness in infants worldwide. Neurologic alterations, such as seizures and ataxia, have been associated with RSV infection. We demonstrate the presence of RSV proteins and RNA in zones of the brain-such as the hippocampus, ventromedial hypothalamic nucleus, and brainstem-of infected mice. One month after disease resolution, rodents showed behavioral and cognitive impairment in marble burying (MB) and Morris water maze (MWM) tests. Our data indicate that the learning impairment caused by RSV is a result of a deficient induction of long-term potentiation in the hippocampus of infected animals. In addition, immunization with recombinant bacillus Calmette-Guerin (BCG) expressing RSV nucleoprotein prevented behavioral disorders, corroborating the specific effect of RSV infection over the central nervous system. Our findings provide evidence that RSV can spread from the airways to the central nervous system and cause functional alterations to the brain, both of which can be prevented by proper immunization against RSV.

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Role for the kinase SGK1 in stress, depression, and glucocorticoid effects on hippocampal neurogenesis.

Publication Date: 2013 May 21 PMID: 23650397
Authors: Anacker, C. - Cattaneo, A. - Musaelyan, K. - Zunszain, P. A. - Horowitz, M. - Molteni, R. - Luoni, A. - Calabrese, F. - Tansey, K. - Gennarelli, M. - Thuret, S. - Price, J. - Uher, R. - Riva, M. A. - Pariante, C. M.
Journal: Proc Natl Acad Sci U S A

Stress and glucocorticoid hormones regulate hippocampal neurogenesis, but the molecular mechanisms mediating these effects are poorly understood. Here we identify the glucocorticoid receptor (GR) target gene, serum- and glucocorticoid-inducible kinase 1 (SGK1), as one such mechanism. Using a human hippocampal progenitor cell line, we found that a small molecule inhibitor for SGK1, GSK650394, counteracted the cortisol-induced reduction in neurogenesis. Moreover, gene expression and pathway analysis showed that inhibition of the neurogenic Hedgehog pathway by cortisol was SGK1-dependent. SGK1 also potentiated and maintained GR activation in the presence of cortisol, and even after cortisol withdrawal, by increasing GR phosphorylation and GR nuclear translocation. Experiments combining the inhibitor for SGK1, GSK650394, with the GR antagonist, RU486, demonstrated that SGK1 was involved in the cortisol-induced reduction in progenitor proliferation both downstream of GR, by regulating relevant target genes, and upstream of GR, by increasing GR function. Corroborating the relevance of these findings in clinical and rodent settings, we also observed a significant increase of SGK1 mRNA in peripheral blood of drug-free depressed patients, as well as in the hippocampus of rats subjected to either unpredictable chronic mild stress or prenatal stress. Our findings identify SGK1 as a mediator for the effects of cortisol on neurogenesis and GR function, with particular relevance to stress and depression.

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Biopolymer-reinforced synthetic granular nanocomposites for affordable point-of-use water purification.

Publication Date: 2013 May 21 PMID: 23650396
Authors: Sankar, M. U. - Aigal, S. - Maliyekkal, S. M. - Chaudhary, A. - Anshup - Kumar, A. A. - Chaudhari, K. - Pradeep, T.
Journal: Proc Natl Acad Sci U S A

Creation of affordable materials for constant release of silver ions in water is one of the most promising ways to provide microbially safe drinking water for all. Combining the capacity of diverse nanocomposites to scavenge toxic species such as arsenic, lead, and other contaminants along with the above capability can result in affordable, all-inclusive drinking water purifiers that can function without electricity. The critical problem in achieving this is the synthesis of stable materials that can release silver ions continuously in the presence of complex species usually present in drinking water that deposit and cause scaling on nanomaterial surfaces. Here we show that such constant release materials can be synthesized in a simple and effective fashion in water itself without the use of electrical power. The nanocomposite exhibits river sand-like properties, such as higher shear strength in loose and wet forms. These materials have been used to develop an affordable water purifier to deliver clean drinking water at US $2.5/y per family. The ability to prepare nanostructured compositions at near ambient temperature has wide relevance for adsorption-based water purification.

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Phosphatidylinositol 4,5-bisphosphate alters pharmacological selectivity for epilepsy-causing KCNQ potassium channels.

Publication Date: 2013 May 21 PMID: 23650395
Authors: Zhou, P. - Yu, H. - Gu, M. - Nan, F. J. - Gao, Z. - Li, M.
Journal: Proc Natl Acad Sci U S A

Pharmacological augmentation of neuronal KCNQ muscarinic (M) currents by drugs such as retigabine (RTG) represents a first-in-class therapeutic to treat certain hyperexcitatory diseases by dampening neuronal firing. Whereas all five potassium channel subtypes (KCNQ1-KCNQ5) are found in the nervous system, KCNQ2 and KCNQ3 are the primary players that mediate M currents. We investigated the plasticity of subtype selectivity by two M current effective drugs, retigabine and zinc pyrithione (ZnPy). Retigabine is more effective on KCNQ3 than KCNQ2, whereas ZnPy is more effective on KCNQ2 with no detectable effect on KCNQ3. In neurons, activation of muscarinic receptor signaling desensitizes effects by retigabine but not ZnPy. Importantly, reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) causes KCNQ3 to become sensitive to ZnPy but lose sensitivity to retigabine. The dynamic shift of pharmacological selectivity caused by PIP2 may be induced orthogonally by voltage-sensitive phosphatase, or conversely, abolished by mutating a PIP2 site within the S4-S5 linker of KCNQ3. Therefore, whereas drug-channel binding is a prerequisite, the drug selectivity on M current is dynamic and may be regulated by receptor signaling pathways via PIP2.

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Structural plasticity of the cellular prion protein and implications in health and disease.

Publication Date: 2013 May 21 PMID: 23650394
Authors: Christen, B. - Damberger, F. F. - Perez, D. R. - Hornemann, S. - Wuthrich, K.
Journal: Proc Natl Acad Sci U S A

Two lines of transgenic mice expressing mouse/elk and mouse/horse prion protein (PrP) hybrids, which both form a well-structured beta2-alpha2 loop in the NMR structures at 20 degrees C termed rigid-loop cellular prion proteins (RL-PrP(C)), presented with accumulation of the aggregated scrapie form of PrP in brain tissue, and the mouse/elk hybrid has also been shown to develop a spontaneous transmissible spongiform encephalopathy. Independently, there is in vitro evidence for correlations between the amino acid sequence in the beta2-alpha2 loop and the propensity for conformational transitions to disease-related forms of PrP. To further contribute to the structural basis for these observations, this paper presents a detailed characterization of RL-PrP(C) conformations in solution. A dynamic local conformational polymorphism involving the beta2-alpha2 loop was found to be evolutionarily preserved among all mammalian species, including those species for which the WT PrP forms an RL-PrP(C). The interconversion between two ensembles of PrP(C) conformers that contain, respectively, a 310-helix turn or a type I beta-turn structure of the beta2-alpha2 loop, exposes two different surface epitopes, which are analyzed for their possible roles in the still evasive function of PrP(C) in healthy organisms and/or at the onset of a transmissible spongiform encephalopathy.

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Lymphatic abnormalities are associated with RASA1 gene mutations in mouse and man.

Publication Date: 2013 May 21 PMID: 23650393
Authors: Burrows, P. E. - Gonzalez-Garay, M. L. - Rasmussen, J. C. - Aldrich, M. B. - Guilliod, R. - Maus, E. A. - Fife, C. E. - Kwon, S. - Lapinski, P. E. - King, P. D. - Sevick-Muraca, E. M.
Journal: Proc Natl Acad Sci U S A

Mutations in gene RASA1 have been historically associated with capillary malformation-arteriovenous malformation, but sporadic reports of lymphatic involvement have yet to be investigated in detail. To investigate the impact of RASA1 mutations in the lymphatic system, we performed investigational near-infrared fluorescence lymphatic imaging and confirmatory radiographic lymphangiography in a Parkes-Weber syndrome (PKWS) patient with suspected RASA1 mutations and correlated the lymphatic abnormalities against that imaged in an inducible Rasa1 knockout mouse. Whole-exome sequencing (WES) analysis and validation by Sanger sequencing of DNA from the patient and unaffected biological parents enabled us to identify an early-frameshift deletion in RASA1 that was shared with the father, who possessed a capillary stain but otherwise no overt disease phenotype. Abnormal lymphatic vasculature was imaged in both affected and unaffected legs of the PKWS subject that transported injected indocyanine green dye to the inguinal lymph node and drained atypically into the abdomen and into dermal lymphocele-like vesicles on the groin. Dermal lymphatic hyperplasia and dilated vessels were observed in Rasa1-deficient mice, with subsequent development of chylous ascites. WES analyses did not identify potential gene modifiers that could explain the variability of penetrance between father and son. Nonetheless, we conclude that the RASA1 mutation is responsible for the aberrant lymphatic architecture and functional abnormalities, as visualized in the PKWS subject and in the animal model. Our unique method to combine investigatory near-infrared fluorescence lymphatic imaging and WES for accurate phenoptyping and unbiased genotyping allows the study of molecular mechanisms of lymphatic involvement of hemovascular disorders.

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Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice.

Publication Date: 2013 May 21 PMID: 23650392
Authors: Martinod, K. - Demers, M. - Fuchs, T. A. - Wong, S. L. - Brill, A. - Gallant, M. - Hu, J. - Wang, Y. - Wagner, D. D.
Journal: Proc Natl Acad Sci U S A

Deep vein thrombosis and pulmonary embolism are major health problems associated with high mortality. Recently, DNA-based neutrophil extracellular traps (NETs) resulting from the release of decondensed chromatin, were found to be part of the thrombus scaffold and to promote coagulation. However, the significance of nuclear decondensation and NET generation in thrombosis is largely unknown. To address this, we adopted a stenosis model of deep vein thrombosis and analyzed venous thrombi in peptidylarginine deiminase 4 (PAD4)-deficient mice that cannot citrullinate histones, a process required for chromatin decondensation and NET formation. Intriguingly, less than 10% of PAD4(-/-) mice produced a thrombus 48 h after inferior vena cava stenosis whereas 90% of wild-type mice did. Neutrophils were abundantly present in thrombi formed in both groups, whereas extracellular citrullinated histones were seen only in thrombi from wild-type mice. Bone marrow chimera experiments indicated that PAD4 in hematopoietic cells was the source of the prothrombotic effect in deep vein thrombosis. Thrombosis could be rescued by infusion of wild-type neutrophils, suggesting that neutrophil PAD4 was important and sufficient. Endothelial activation and platelet aggregation were normal in PAD4(-/-) mice, as was hemostatic potential determined by bleeding time and platelet plug formation after venous injury. Our results show that PAD4-mediated chromatin decondensation in the neutrophil is crucial for pathological venous thrombosis and present neutrophil activation and PAD4 as potential drug targets for deep vein thrombosis.

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Mesenchymal glioma stem cells are maintained by activated glycolytic metabolism involving aldehyde dehydrogenase 1A3.

Publication Date: 2013 May 21 PMID: 23650391
Authors: Mao, P. - Joshi, K. - Li, J. - Kim, S. H. - Li, P. - Santana-Santos, L. - Luthra, S. - Chandran, U. R. - Benos, P. V. - Smith, L. - Wang, M. - Hu, B. - Cheng, S. Y. - Sobol, R. W. - Nakano, I.
Journal: Proc Natl Acad Sci U S A

Tumor heterogeneity of high-grade glioma (HGG) is recognized by four clinically relevant subtypes based on core gene signatures. However, molecular signaling in glioma stem cells (GSCs) in individual HGG subtypes is poorly characterized. Here we identified and characterized two mutually exclusive GSC subtypes with distinct dysregulated signaling pathways. Analysis of mRNA profiles distinguished proneural (PN) from mesenchymal (Mes) GSCs and revealed a pronounced correlation with the corresponding PN or Mes HGGs. Mes GSCs displayed more aggressive phenotypes in vitro and as intracranial xenografts in mice. Further, Mes GSCs were markedly resistant to radiation compared with PN GSCs. The glycolytic pathway, comprising aldehyde dehydrogenase (ALDH) family genes and in particular ALDH1A3, were enriched in Mes GSCs. Glycolytic activity and ALDH activity were significantly elevated in Mes GSCs but not in PN GSCs. Expression of ALDH1A3 was also increased in clinical HGG compared with low-grade glioma or normal brain tissue. Moreover, inhibition of ALDH1A3 attenuated the growth of Mes but not PN GSCs. Last, radiation treatment of PN GSCs up-regulated Mes-associated markers and down-regulated PN-associated markers, whereas inhibition of ALDH1A3 attenuated an irradiation-induced gain of Mes identity in PN GSCs. Taken together, our data suggest that two subtypes of GSCs, harboring distinct metabolic signaling pathways, represent intertumoral glioma heterogeneity and highlight previously unidentified roles of ALDH1A3-associated signaling that promotes aberrant proliferation of Mes HGGs and GSCs. Inhibition of ALDH1A3-mediated pathways therefore might provide a promising therapeutic approach for a subset of HGGs with the Mes signature.

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Ultraconserved words point to deep language ancestry across Eurasia.

Publication Date: 2013 May 21 PMID: 23650390
Authors: Pagel, M. - Atkinson, Q. D. - S Calude, A. - Meade, A.
Journal: Proc Natl Acad Sci U S A

The search for ever deeper relationships among the World's languages is bedeviled by the fact that most words evolve too rapidly to preserve evidence of their ancestry beyond 5,000 to 9,000 y. On the other hand, quantitative modeling indicates that some "ultraconserved" words exist that might be used to find evidence for deep linguistic relationships beyond that time barrier. Here we use a statistical model, which takes into account the frequency with which words are used in common everyday speech, to predict the existence of a set of such highly conserved words among seven language families of Eurasia postulated to form a linguistic superfamily that evolved from a common ancestor around 15,000 y ago. We derive a dated phylogenetic tree of this proposed superfamily with a time-depth of approximately 14,450 y, implying that some frequently used words have been retained in related forms since the end of the last ice age. Words used more than once per 1,000 in everyday speech were 7- to 10-times more likely to show deep ancestry on this tree. Our results suggest a remarkable fidelity in the transmission of some words and give theoretical justification to the search for features of language that might be preserved across wide spans of time and geography.

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Cross-talk between MET and EGFR in non-small cell lung cancer involves miR-27a and Sprouty2.

Publication Date: 2013 May 21 PMID: 23650389
Authors: Acunzo, M. - Romano, G. - Palmieri, D. - Lagana, A. - Garofalo, M. - Balatti, V. - Drusco, A. - Chiariello, M. - Nana-Sinkam, P. - Croce, C. M.
Journal: Proc Natl Acad Sci U S A

In the past decade, we have observed exciting advances in lung cancer therapy, including the development of targeted therapies. However, additional strategies for early detection and tumor-based therapy are still essential in improving patient outcomes. EGF receptor (EGFR) and MET (the receptor tyrosine kinase for hepatocyte growth factors) are cell-surface tyrosine kinase receptors that have been implicated in diverse cellular processes and as regulators of several microRNAs (miRNAs), thus contributing to tumor progression. Here, we demonstrate a biological link between EGFR, MET, and the miRNA cluster 23a approximately 27a approximately 24-2. We show that miR-27a regulates MET, EGFR, and Sprouty2 in lung cancer. In addition, we identify both direct and indirect mechanisms by which miR-27a can regulate both MET and EGFR. Thus, we propose a mechanism for MET and EGFR axis regulation that may lead to the development of therapeutics in lung cancer.

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Interventions for avian influenza A (H5N1) risk management in live bird market networks.

Publication Date: 2013 May 6 PMID: 23650388
Authors: Fournie, G. - Guitian, J. - Desvaux, S. - Cuong, V. C. - Dung, D. H. - Pfeiffer, D. U. - Mangtani, P. - Ghani, A. C.
Journal: Proc Natl Acad Sci U S A

Highly pathogenic avian influenza virus subtype H5N1 is endemic in Asia, with live bird trade as a major disease transmission pathway. A cross-sectional survey was undertaken in northern Vietnam to investigate the structure of the live bird market (LBM) contact network and the implications for virus spread. Based on the movements of traders between LBMs, weighted and directed networks were constructed and used for social network analysis and individual-based modeling. Most LBMs were connected to one another, suggesting that the LBM network may support large-scale disease spread. Because of cross-border trade, it also may promote transboundary virus circulation. However, opportunities for disease control do exist. The implementation of thorough, daily disinfection of the market environment as well as of traders' vehicles and equipment in only a small number of hubs can disconnect the network dramatically, preventing disease spread. These targeted interventions would be an effective alternative to the current policy of a complete ban of LBMs in some areas. Some LBMs that have been banned still are very active, and they likely have a substantial impact on disease dynamics, exhibiting the highest levels of susceptibility and infectiousness. The number of trader visits to markets, information that can be collected quickly and easily, may be used to identify LBMs suitable for implementing interventions. This would not require prior knowledge of the force of infection, for which laboratory-confirmed surveillance would be necessary. These findings are of particular relevance for policy development in resource-scarce settings.

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Kif7 is required for the patterning and differentiation of the diaphragm in a model of syndromic congenital diaphragmatic hernia.

Publication Date: 2013 May 21 PMID: 23650387
Authors: Coles, G. L. - Ackerman, K. G.
Journal: Proc Natl Acad Sci U S A

Congenital diaphragmatic hernia (CDH) is a common birth defect that results in a high degree of neonatal morbidity and mortality, but its pathological mechanisms are largely unknown. Therefore, we performed a forward genetic screen in mice to identify unique genes, models, and mechanisms of abnormal diaphragm development. We identified a mutant allele of kinesin family member 7 (Kif7), the disorganized diaphragm (dd). Embryos homozygous for the dd allele possess communicating diaphragmatic hernias, central tendon patterning defects, and increased cell proliferation with diaphragmatic tissue hyperplasia. Because the patterning of the central tendon is undescribed, we analyzed the expression of genes regulating tendonogenesis in dd/dd mutant embryos, and we determined that retinoic acid (RA) signaling was misregulautted. To further investigate the role of Kif7 and RA signaling in the development of the embryonic diaphragm, we established primary mesenchymal cultures of WT embryonic day 13.5 diaphragmatic cells. We determined that RA signaling is necessary for the expression of tendon markers as well as the expression of other CDH-associated genes. Knockdown of Kif7, and retinoic acid receptors alpha (Rara), beta (Rarb), and gamma (Rarg) indicated that RA signaling is dependent on these genes to promote tendonogenesis within the embryonic diaphragm. Taken together, our results provide evidence for a model in which inhibition of RA receptor signaling promotes CDH pathogenesis through a complex gene network.

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Persistence and origin of the lunar core dynamo.

Publication Date: 2013 May 21 PMID: 23650386
Authors: Suavet, C. - Weiss, B. P. - Cassata, W. S. - Shuster, D. L. - Gattacceca, J. - Chan, L. - Garrick-Bethell, I. - Head, J. W. - Grove, T. L. - Fuller, M. D.
Journal: Proc Natl Acad Sci U S A

The lifetime of the ancient lunar core dynamo has implications for its power source and the mechanism of field generation. Here, we report analyses of two 3.56-Gy-old mare basalts demonstrating that they were magnetized in a stable and surprisingly intense dynamo magnetic field of at least approximately 13 muT. These data extend the known lifetime of the lunar dynamo by approximately 160 My and indicate that the field was likely continuously active until well after the final large basin-forming impact. This likely excludes impact-driven changes in rotation rate as the source of the dynamo at this time in lunar history. Rather, our results require a persistent power source like precession of the lunar mantle or a compositional convection dynamo.

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Structural basis of the relaxed state of a Ca2+-regulated myosin filament and its evolutionary implications.

Publication Date: 2013 May 21 PMID: 23650385
Authors: Woodhead, J. L. - Zhao, F. Q. - Craig, R.
Journal: Proc Natl Acad Sci U S A

Myosin filaments of muscle are regulated either by phosphorylation of their regulatory light chains or Ca(2+) binding to the essential light chains, contributing to on-off switching or modulation of contraction. Phosphorylation-regulated filaments in the relaxed state are characterized by an asymmetric interaction between the two myosin heads, inhibiting their actin binding or ATPase activity. Here, we have tested whether a similar interaction switches off activity in myosin filaments regulated by Ca(2+) binding. Cryo-electron microscopy and single-particle image reconstruction of Ca(2+)-regulated (scallop) filaments reveals a helical array of myosin head-pair motifs above the filament surface. Docking of atomic models of scallop myosin head domains into the motifs reveals that the heads interact in a similar way to those in phosphorylation-regulated filaments. The results imply that the two major evolutionary branches of myosin regulation-involving phosphorylation or Ca(2+) binding-share a common structural mechanism for switching off thick-filament activity in relaxed muscle. We suggest that the Ca(2+)-binding mechanism evolved from the more ancient phosphorylation-based system to enable rapid response of myosin-regulated muscles to activation. Although the motifs are similar in both systems, the scallop structure is more tilted and higher above the filament backbone, leading to different intermolecular interactions. The reconstruction reveals how the myosin tail emerges from the motif, connecting the heads to the filament backbone, and shows that the backbone is built from supramolecular assemblies of myosin tails. The reconstruction provides a native structural context for understanding past biochemical and biophysical studies of this model Ca(2+)-regulated myosin.

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Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites.

Publication Date: 2013 May 21 PMID: 23650384
Authors: Bally, I. - Ancelet, S. - Moriscot, C. - Gonnet, F. - Mantovani, A. - Daniel, R. - Schoehn, G. - Arlaud, G. J. - Thielens, N. M.
Journal: Proc Natl Acad Sci U S A

Complement C1q is a hexameric molecule assembled from 18 polypeptide chains of three different types encoded by three genes. This versatile recognition protein senses a wide variety of immune and nonimmune ligands, including pathogens and altered self components, and triggers the classical complement pathway through activation of its associated proteases C1r and C1s. We report a method for expression of recombinant full-length human C1q involving stable transfection of HEK 293-F mammalian cells and fusion of an affinity tag to the C-terminal end of the C chain. The resulting recombinant (r) C1q molecule is similar to serum C1q as judged from biochemical and structural analyses and exhibits the characteristic shape of a bunch of flowers. Analysis of its interaction properties by surface plasmon resonance shows that rC1q retains the ability of serum C1q to associate with the C1s-C1r-C1r-C1s tetramer, to recognize physiological C1q ligands such as IgG and pentraxin 3, and to trigger C1r and C1s activation. Functional analysis of rC1q variants carrying mutations of LysA59, LysB61, and/or LysC58, in the collagen-like stems, demonstrates that LysB61 and LysC58 each play a key role in the interaction with C1s-C1r-C1r-C1s, with LysA59 being involved to a lesser degree. We propose that LysB61 and LysC58 both form salt bridges with outer acidic Ca(2+) ligands of the C1r and C1s CUB (complement C1r/C1s, Uegf, bone morphogenetic protein) domains. The expression method reported here opens the way for deciphering the molecular basis of the unusual binding versatility of C1q by mapping the residues involved in the sensing of its targets and the binding of its receptors.

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Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation.

Publication Date: 2013 May 21 PMID: 23650383
Authors: Dubiella, U. - Seybold, H. - Durian, G. - Komander, E. - Lassig, R. - Witte, C. P. - Schulze, W. X. - Romeis, T.
Journal: Proc Natl Acad Sci U S A

In animals and plants, pathogen recognition triggers the local activation of intracellular signaling that is prerequisite for mounting systemic defenses in the whole organism. We identified that Arabidopsis thaliana isoform CPK5 of the plant calcium-dependent protein kinase family becomes rapidly biochemically activated in response to pathogen-associated molecular pattern (PAMP) stimulation. CPK5 signaling resulted in enhanced salicylic acid-mediated resistance to the bacterial pathogen Pst DC3000, differential plant defense gene expression, and synthesis of reactive oxygen species (ROS). Using selected reaction monitoring MS, we identified the plant NADPH oxidase, respiratory burst oxidase homolog D (RBOHD), as an in vivo phosphorylation target of CPK5. Remarkably, CPK5-dependent in vivo phosphorylation of RBOHD occurs on both PAMP- and ROS stimulation. Furthermore, rapid CPK5-dependent biochemical and transcriptional activation of defense reactions at distal sites is compromised in cpk5 and rbohd mutants. Our data not only identify CPK5 as a key regulator of innate immune responses in plants but also support a model of ROS-mediated cell-to-cell communication, where a self-propagating mutual activation circuit consisting of the protein kinase, CPK5, and the NADPH oxidase RBOHD facilitates rapid signal propagation as a prerequisite for defense response activation at distal sites within the plant.

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Specialized bat tongue is a hemodynamic nectar mop.

Publication Date: 2013 May 6 PMID: 23650382
Authors: Harper, C. J. - Swartz, S. M. - Brainerd, E. L.
Journal: Proc Natl Acad Sci U S A

Nectarivorous birds and bats have evolved highly specialized tongues to gather nectar from flowers. Here, we show that a nectar-feeding bat, Glossophaga soricina, uses dynamic erectile papillae to collect nectar. In G. soricina, the tip of the tongue is covered with long filamentous papillae and resembles a brush or mop. During nectar feeding, blood vessels within the tongue tip become engorged with blood and the papillae become erect. Tumescence and papilla erection persist throughout tongue retraction, and nectar, trapped between the rows of erect papillae, is carried into the mouth. The tongue tip does not increase in overall volume as it elongates, suggesting that muscle contraction against the tongue's fixed volume (i.e., a muscular hydrostat) is primarily responsible for tip elongation, whereas papilla erection is a hydraulic process driven by blood flow. The hydraulic system is embedded within the muscular hydrostat, and, thus, intrinsic muscle contraction may simultaneously increase the length of the tongue and displace blood into the tip. The tongue of G. soricina, together with the tongues of nectar-feeding bees and hummingbirds, which also have dynamic surfaces, could serve as valuable models for developing miniature surgical robots that are both protrusible and have highly dynamic surface configurations.

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Visualizing cellular interactions with a generalized proximity reporter.

Publication Date: 2013 May 21 PMID: 23650381
Authors: Sellmyer, M. A. - Bronsart, L. - Imoto, H. - Contag, C. H. - Wandless, T. J. - Prescher, J. A.
Journal: Proc Natl Acad Sci U S A

Interactions among neighboring cells underpin many physiological processes ranging from early development to immune responses. When these interactions do not function properly, numerous pathologies, including infection and cancer, can result. Molecular imaging technologies, especially optical imaging, are uniquely suited to illuminate complex cellular interactions within the context of living tissues in the body. However, no tools yet exist that allow the detection of microscopic events, such as two cells coming into close proximity, on a global, whole-animal scale. We report here a broadly applicable, longitudinal strategy for probing interactions among cells in living subjects. This approach relies on the generation of bioluminescent light when two distinct cell populations come into close proximity, with the intensity of the optical signal correlating with relative cellular location. We demonstrate the ability of this reporter strategy to gauge cell-cell proximity in culture models in vitro and then evaluate this approach for imaging tumor-immune cell interactions using a murine breast cancer model. In these studies, our imaging strategy enabled the facile visualization of features that are otherwise difficult to observe with conventional imaging techniques, including detection of micrometastatic lesions and potential sites of tumor immunosurveillance. This proximity reporter will facilitate probing of numerous types of cell-cell interactions and will stimulate the development of similar techniques to detect rare events and pathological processes in live animals.

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Dynamic subunit stoichiometry confers a progressive continuum of pharmacological sensitivity by KCNQ potassium channels.

Publication Date: 2013 May 21 PMID: 23650380
Authors: Yu, H. - Lin, Z. - Mattmann, M. E. - Zou, B. - Terrenoire, C. - Zhang, H. - Wu, M. - McManus, O. B. - Kass, R. S. - Lindsley, C. W. - Hopkins, C. R. - Li, M.
Journal: Proc Natl Acad Sci U S A

Voltage-gated KCNQ1 (Kv7.1) potassium channels are expressed abundantly in heart but they are also found in multiple other tissues. Differential coassembly with single transmembrane KCNE beta subunits in different cell types gives rise to a variety of biophysical properties, hence endowing distinct physiological roles for KCNQ1-KCNEx complexes. Mutations in either KCNQ1 or KCNE1 genes result in diseases in brain, heart, and the respiratory system. In addition to complexities arising from existence of five KCNE subunits, KCNE1 to KCNE5, recent studies in heterologous systems suggest unorthodox stoichiometric dynamics in subunit assembly is dependent on KCNE expression levels. The resultant KCNQ1-KCNE channel complexes may have a range of zero to two or even up to four KCNE subunits coassembling per KCNQ1 tetramer. These findings underscore the need to assess the selectivity of small-molecule KCNQ1 modulators on these different assemblies. Here we report a unique small-molecule gating modulator, ML277, that potentiates both homomultimeric KCNQ1 channels and unsaturated heteromultimeric (KCNQ1)4(KCNE1)n (n < 4) channels. Progressive increase of KCNE1 or KCNE3 expression reduces efficacy of ML277 and eventually abolishes ML277-mediated augmentation. In cardiomyocytes, the slowly activating delayed rectifier potassium current, or IKs, is believed to be a heteromultimeric combination of KCNQ1 and KCNE1, but it is not entirely clear whether IKs is mediated by KCNE-saturated KCNQ1 channels or by channels with intermediate stoichiometries. We found ML277 effectively augments IKs current of cultured human cardiomyocytes and shortens action potential duration. These data indicate that unsaturated heteromultimeric (KCNQ1)4(KCNE1)n channels are present as components of IKs and are pharmacologically distinct from KCNE-saturated KCNQ1-KCNE1 channels.

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Parkin overexpression during aging reduces proteotoxicity, alters mitochondrial dynamics, and extends lifespan.

Publication Date: 2013 May 21 PMID: 23650379
Authors: Rana, A. - Rera, M. - Walker, D. W.
Journal: Proc Natl Acad Sci U S A

Aberrant protein aggregation and mitochondrial dysfunction have each been linked to aging and a number of age-onset neurodegenerative disorders, including Parkinson disease. Loss-of-function mutations in parkin, an E3 ubiquitin ligase that functions to promote the ubiquitin-proteasome system of protein degradation and also in mitochondrial quality control, have been implicated in heritable forms of Parkinson disease. The question of whether parkin can modulate aging or positively impact longevity, however, has not been addressed. Here, we show that ubiquitous or neuron-specific up-regulation of Parkin, in adult Drosophila melanogaster, increases both mean and maximum lifespan without reducing reproductive output, physical activity, or food intake. Long-lived Parkin-overexpressing flies display an increase in K48-linked polyubiquitin and reduced levels of protein aggregation during aging. Recent evidence suggests that Parkin interacts with the mitochondrial fission/fusion machinery to mediate the turnover of dysfunctional mitochondria. However, the relationships between parkin gene activity, mitochondrial dynamics, and aging have not been explored. We show that the mitochondrial fusion-promoting factor Drosophila Mitofusin, a Parkin substrate, increases in abundance during aging. Parkin overexpression results in reduced Drosophila Mitofusin levels in aging flies, with concomitant changes in mitochondrial morphology and an increase in mitochondrial activity. Together, these findings reveal roles for Parkin in modulating organismal aging and provide insight into the molecular mechanisms linking aging to neurodegeneration.

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Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS-NO-nitrite signaling.

Publication Date: 2013 May 6 PMID: 23650378
Authors: Yazji, I. - Sodhi, C. P. - Lee, E. K. - Good, M. - Egan, C. E. - Afrazi, A. - Neal, M. D. - Jia, H. - Lin, J. - Ma, C. - Branca, M. F. - Prindle, T. - Richardson, W. M. - Ozolek, J. - Billiar, T. R. - Binion, D. G. - Gladwin, M. T. - Hackam, D. J.
Journal: Proc Natl Acad Sci U S A

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS-/- mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate-a precursor for enteral generation of nitrite and nitric oxide-and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate-nitrite-NO signaling.

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Mechanism for nitrogen isotope fractionation during ammonium assimilation by Escherichia coli K12.

Publication Date: 2013 May 21 PMID: 23650377
Authors: Vo, J. - Inwood, W. - Hayes, J. M. - Kustu, S.
Journal: Proc Natl Acad Sci U S A

Organisms that use ammonium as the sole nitrogen source discriminate between [(15)N] and [(14)N] ammonium. This selectivity leaves an isotopic signature in their biomass that depends on the external concentration of ammonium. To dissect how differences in discrimination arise molecularly, we examined a wild-type (WT) strain of Escherichia coli K12 and mutant strains with lesions affecting ammonium-assimilatory proteins. We used isotope ratio mass spectrometry (MS) to assess the nitrogen isotopic composition of cell material when the strains were grown in batch culture at either high or low external concentrations of NH3 (achieved by controlling total NH4Cl and pH of the medium). At high NH3 (>/=0.89 microM), discrimination against the heavy isotope by the WT strain (-19.2 per thousand) can be accounted for by the equilibrium isotope effect for dissociation of NH4(+) to NH3 + H(+). NH3 equilibrates across the cytoplasmic membrane, and glutamine synthetase does not manifest an isotope effect in vivo. At low NH3 (
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Presynaptic maturation in auditory hair cells requires a critical period of sensory-independent spiking activity.

Publication Date: 2013 May 21 PMID: 23650376
Authors: Johnson, S. L. - Kuhn, S. - Franz, C. - Ingham, N. - Furness, D. N. - Knipper, M. - Steel, K. P. - Adelman, J. P. - Holley, M. C. - Marcotti, W.
Journal: Proc Natl Acad Sci U S A

The development of neural circuits relies on spontaneous electrical activity that occurs during immature stages of development. In the developing mammalian auditory system, spontaneous calcium action potentials are generated by inner hair cells (IHCs), which form the primary sensory synapse. It remains unknown whether this electrical activity is required for the functional maturation of the auditory system. We found that sensory-independent electrical activity controls synaptic maturation in IHCs. We used a mouse model in which the potassium channel SK2 is normally overexpressed, but can be modulated in vivo using doxycycline. SK2 overexpression affected the frequency and duration of spontaneous action potentials, which prevented the development of the Ca(2+)-sensitivity of vesicle fusion at IHC ribbon synapses, without affecting their morphology or general cell development. By manipulating the in vivo expression of SK2 channels, we identified the "critical period" during which spiking activity influences IHC synaptic maturation. Here we provide direct evidence that IHC development depends upon a specific temporal pattern of calcium spikes before sound-driven neuronal activity.

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Structural snapshots reveal distinct mechanisms of procaspase-3 and -7 activation.

Publication Date: 2013 May 21 PMID: 23650375
Authors: Thomsen, N. D. - Koerber, J. T. - Wells, J. A.
Journal: Proc Natl Acad Sci U S A

Procaspase-3 (P3) and procaspase-7 (P7) are activated through proteolytic maturation to form caspase-3 (C3) and caspase-7 (C7), respectively, which serve overlapping but nonredundant roles as the executioners of apoptosis in humans. However, it is unclear if differences in P3 and P7 maturation mechanisms underlie their unique biological functions, as the structure of P3 remains unknown. Here, we report structures of P3 in a catalytically inactive conformation, structures of P3 and P7 bound to covalent peptide inhibitors that reveal the active conformation of the zymogens, and the structure of a partially matured C7:P7 heterodimer. Along with a biochemical analysis, we show that P3 is catalytically inactive and matures through a symmetric all-or-nothing process. In contrast, P7 contains latent catalytic activity and matures through an asymmetric and tiered mechanism, suggesting a lower threshold for activation. Finally, we use our structures to design a selection strategy for conformation specific antibody fragments that stimulate procaspase activity, showing that executioner procaspase conformational equilibrium can be rationally modulated. Our studies provide a structural framework that may help to explain the unique roles of these important proapoptotic enzymes, and suggest general strategies for the discovery of proenzyme activators.

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Transcytosis and brain uptake of transferrin-containing nanoparticles by tuning avidity to transferrin receptor.

Publication Date: 2013 May 21 PMID: 23650374
Authors: Wiley, D. T. - Webster, P. - Gale, A. - Davis, M. E.
Journal: Proc Natl Acad Sci U S A

Receptor-mediated transcytosis across the blood-brain barrier (BBB) may be a useful way to transport therapeutics into the brain. Here we report that transferrin (Tf)-containing gold nanoparticles can reach the brain parenchyma from systemic administration in mice through a receptor-mediated transcytosis pathway. This transport is aided by tuning the nanoparticle avidity to Tf receptor (TfR), which is correlated with nanoparticle size and total amount of Tf decorating the nanoparticle surface. Nanoparticles of both 45 nm and 80 nm diameter reach the brain parenchyma, and their accumulation there (visualized by silver enhancement light microscopy in combination with transmission electron microscopy imaging) is observed to be dependent on Tf content (avidity); nanoparticles with large amounts of Tf remain strongly attached to brain endothelial cells, whereas those with less Tf are capable of both interacting with TfR on the luminal side of the BBB and detaching from TfR on the brain side of the BBB. The requirement of proper avidity for nanoparticles to reach the brain parenchyma is consistent with recent behavior observed with transcytosing antibodies that bind to TfR.

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Profile of vivian w.-W. Yam.

Publication Date: 2013 May 14 PMID: 23650373
Authors: Ahmed, F.
Journal: Proc Natl Acad Sci U S A

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Weather and anomalous heat flow occurring near absolute zero.

Publication Date: 2013 May 14 PMID: 23650372
Authors: Niemela, J. J.
Journal: Proc Natl Acad Sci U S A

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CaBP1, a neuronal Ca2+ sensor protein, inhibits inositol trisphosphate receptors by clamping intersubunit interactions.

Publication Date: 2013 May 21 PMID: 23650371
Authors: Li, C. - Enomoto, M. - Rossi, A. M. - Seo, M. D. - Rahman, T. - Stathopulos, P. B. - Taylor, C. W. - Ikura, M. - Ames, J. B.
Journal: Proc Natl Acad Sci U S A

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca(2+) channels, including inositol 1,4,5-trisphosphate receptors (InsP3Rs). CaBP1 alone does not affect InsP3R activity, but it inhibits InsP3-evoked Ca(2+) release by slowing the rate of InsP3R opening. The inhibition is enhanced by Ca(2+) binding to both the InsP3R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1-604) of InsP3R1. NMR paramagnetic relaxation enhancement analysis demonstrates that a cluster of hydrophobic residues (V101, L104, and V162) within the C lobe of CaBP1 that are exposed after Ca(2+) binding interact with a complementary cluster of hydrophobic residues (L302, I364, and L393) in the beta-domain of the InsP3-binding core. These residues are essential for CaBP1 binding to the NT and for inhibition of InsP3R activity by CaBP1. Docking analyses and paramagnetic relaxation enhancement structural restraints suggest that CaBP1 forms an extended tetrameric turret attached by the tetrameric NT to the cytosolic vestibule of the InsP3R pore. InsP3 activates InsP3Rs by initiating conformational changes that lead to disruption of an intersubunit interaction between a "hot-spot" loop in the suppressor domain (residues 1-223) and the InsP3-binding core beta-domain. Targeted cross-linking of residues that contribute to this interface show that InsP3 attenuates cross-linking, whereas CaBP1 promotes it. We conclude that CaBP1 inhibits InsP3R activity by restricting the intersubunit movements that initiate gating.

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Farnesylation of lamin B1 is important for retention of nuclear chromatin during neuronal migration.

Publication Date: 2013 May 21 PMID: 23650370
Authors: Jung, H. J. - Nobumori, C. - Goulbourne, C. N. - Tu, Y. - Lee, J. M. - Tatar, A. - Wu, D. - Yoshinaga, Y. - de Jong, P. J. - Coffinier, C. - Fong, L. G. - Young, S. G.
Journal: Proc Natl Acad Sci U S A

The role of protein farnesylation in lamin A biogenesis and the pathogenesis of progeria has been studied in considerable detail, but the importance of farnesylation for the B-type lamins, lamin B1 and lamin B2, has received little attention. Lamins B1 and B2 are expressed in nearly every cell type from the earliest stages of development, and they have been implicated in a variety of functions within the cell nucleus. To assess the importance of protein farnesylation for B-type lamins, we created knock-in mice expressing nonfarnesylated versions of lamin B1 and lamin B2. Mice expressing nonfarnesylated lamin B2 developed normally and were free of disease. In contrast, mice expressing nonfarnesylated lamin B1 died soon after birth, with severe neurodevelopmental defects and striking nuclear abnormalities in neurons. The nuclear lamina in migrating neurons was pulled away from the chromatin so that the chromatin was left "naked" (free from the nuclear lamina). Thus, farnesylation of lamin B1-but not lamin B2-is crucial for brain development and for retaining chromatin within the bounds of the nuclear lamina during neuronal migration.

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Study of the fluoro- and chlorodimethylbutyl cations.

Publication Date: 2013 May 21 PMID: 23650369
Authors: Olah, G. A. - Prakash, G. K. - Rasul, G.
Journal: Proc Natl Acad Sci U S A

A comparative study of the 2,3-dimethyl-3-fluoro-2-butyl cation and its chloro analog was carried out by the ab initio/GIAO-CCSD(T) (gauge invariant atomic orbital-coupled cluster with single, double, and perturbative triple excitation) method. The structures and (13)C NMR chemical shifts of the cations were calculated at the GIAO-CCSD(T)/tzp/dz//MP2/cc-pVTZ level. Bridged fluoronium ion 1, carbenium ion 2, and fluorocarbenium ion 3 were found to be minima on the potential energy surface. Bridged fluoronium ion 1, although a minimum on the potential energy surface, is 12.8 kcal/mol less stable than the open chain fluorobutyl cation 3. In contrast to the fluorinated ion, bridged chloronium ion 5 was found to be the lowest energy minimum being 10.6 kcal/mol more stable than ion 6 and 7.4 kcal/mol more stable than ion 7.

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X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification.

Publication Date: 2013 May 21 PMID: 23650368
Authors: Goldman, P. J. - Grove, T. L. - Sites, L. A. - McLaughlin, M. I. - Booker, S. J. - Drennan, C. L.
Journal: Proc Natl Acad Sci U S A

Arylsulfatases require a maturating enzyme to perform a co- or posttranslational modification to form a catalytically essential formylglycine (FGly) residue. In organisms that live aerobically, molecular oxygen is used enzymatically to oxidize cysteine to FGly. Under anaerobic conditions, S-adenosylmethionine (AdoMet) radical chemistry is used. Here we present the structures of an anaerobic sulfatase maturating enzyme (anSME), both with and without peptidyl-substrates, at 1.6-1.8 A resolution. We find that anSMEs differ from their aerobic counterparts in using backbone-based hydrogen-bonding patterns to interact with their peptidyl-substrates, leading to decreased sequence specificity. These anSME structures from Clostridium perfringens are also the first of an AdoMet radical enzyme that performs dehydrogenase chemistry. Together with accompanying mutagenesis data, a mechanistic proposal is put forth for how AdoMet radical chemistry is coopted to perform a dehydrogenation reaction. In the oxidation of cysteine or serine to FGly by anSME, we identify D277 and an auxiliary [4Fe-4S] cluster as the likely acceptor of the final proton and electron, respectively. D277 and both auxiliary clusters are housed in a cysteine-rich C-terminal domain, termed SPASM domain, that contains homology to approximately 1,400 other unique AdoMet radical enzymes proposed to use [4Fe-4S] clusters to ligate peptidyl-substrates for subsequent modification. In contrast to this proposal, we find that neither auxiliary cluster in anSME bind substrate, and both are fully ligated by cysteine residues. Instead, our structural data suggest that the placement of these auxiliary clusters creates a conduit for electrons to travel from the buried substrate to the protein surface.

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Direct observation of intermediates formed during steady-state electrocatalytic O2 reduction by iron porphyrins.

Publication Date: 2013 May 21 PMID: 23650367
Authors: Sengupta, K. - Chatterjee, S. - Samanta, S. - Dey, A.
Journal: Proc Natl Acad Sci U S A

Heme/porphyrin-based electrocatalysts (both synthetic and natural) have been known to catalyze electrochemical O2, H(+), and CO2 reduction for more than five decades. So far, no direct spectroscopic investigations of intermediates formed on the electrodes during these processes have been reported; and this has limited detailed understanding of the mechanism of these catalysts, which is key to their development. Rotating disk electrochemistry coupled to resonance Raman spectroscopy is reported for iron porphyrin electrocatalysts that reduce O2 in buffered aqueous solutions. Unlike conventional single-turnover intermediate trapping experiments, these experiments probe the system while it is under steady state. A combination of oxidation and spin-state marker bands and metal ligand vibrations (identified using isotopically enriched substrates) allow in situ identification of O2-derived intermediates formed on the electrode surface. This approach, combining dynamic electrochemistry with resonance Raman spectroscopy, may be routinely used to investigate a plethora of metalloporphyrin complexes and heme enzymes used as electrocatalysts for small-molecule activation.

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Optimal fold symmetry of LH2 rings on a photosynthetic membrane.

Publication Date: 2013 May 21 PMID: 23650366
Authors: Cleary, L. - Chen, H. - Chuang, C. - Silbey, R. J. - Cao, J.
Journal: Proc Natl Acad Sci U S A

An intriguing observation of photosynthetic light-harvesting systems is the N-fold symmetry of light-harvesting complex 2 (LH2) of purple bacteria. We calculate the optimal rotational configuration of N-fold rings on a hexagonal lattice and establish two related mechanisms for the promotion of maximum excitation energy transfer (EET). (i) For certain fold numbers, there exist optimal basis cells with rotational symmetry, extendable to the entire lattice for the global optimization of the EET network. (ii) The type of basis cell can reduce or remove the frustration of EET rates across the photosynthetic network. We find that the existence of a basis cell and its type are directly related to the number of matching points S between the fold symmetry and the hexagonal lattice. The two complementary mechanisms provide selection criteria for the fold number and identify groups of consecutive numbers. Remarkably, one such group consists of the naturally occurring 8-, 9-, and 10-fold rings. By considering the inter-ring distance and EET rate, we demonstrate that this group can achieve minimal rotational sensitivity in addition to an optimal packing density, achieving robust and efficient EET. This corroborates our findings i and ii and, through their direct relation to S, suggests the design principle of matching the internal symmetry with the lattice order.

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Complex history of the amphibian-killing chytrid fungus revealed with genome resequencing data.

Publication Date: 2013 May 6 PMID: 23650365
Authors: Rosenblum, E. B. - James, T. Y. - Zamudio, K. R. - Poorten, T. J. - Ilut, D. - Rodriguez, D. - Eastman, J. M. - Richards-Hrdlicka, K. - Joneson, S. - Jenkinson, T. S. - Longcore, J. E. - Parra Olea, G. - Toledo, L. F. - Arellano, M. L. - Medina, E. M. - Restrepo, S. - Flechas, S. V. - Berger, L. - Briggs, C. J. - Stajich, J. E.
Journal: Proc Natl Acad Sci U S A

Understanding the evolutionary history of microbial pathogens is critical for mitigating the impacts of emerging infectious diseases on economically and ecologically important host species. We used a genome resequencing approach to resolve the evolutionary history of an important microbial pathogen, the chytrid Batrachochytrium dendrobatidis (Bd), which has been implicated in amphibian declines worldwide. We sequenced the genomes of 29 isolates of Bd from around the world, with an emphasis on North, Central, and South America because of the devastating effect that Bd has had on amphibian populations in the New World. We found a substantial amount of evolutionary complexity in Bd with deep phylogenetic diversity that predates observed global amphibian declines. By investigating the entire genome, we found that even the most recently evolved Bd clade (termed the global panzootic lineage) contained more genetic variation than previously reported. We also found dramatic differences among isolates and among genomic regions in chromosomal copy number and patterns of heterozygosity, suggesting complex and heterogeneous genome dynamics. Finally, we report evidence for selection acting on the Bd genome, supporting the hypothesis that protease genes are important in evolutionary transitions in this group. Bd is considered an emerging pathogen because of its recent effects on amphibians, but our data indicate that it has a complex evolutionary history that predates recent disease outbreaks. Therefore, it is important to consider the contemporary effects of Bd in a broader evolutionary context and identify specific mechanisms that may have led to shifts in virulence in this system.

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Fluorescence thermometry enhanced by the quantum coherence of single spins in diamond.

Publication Date: 2013 May 21 PMID: 23650364
Authors: Toyli, D. M. - de Las Casas, C. F. - Christle, D. J. - Dobrovitski, V. V. - Awschalom, D. D.
Journal: Proc Natl Acad Sci U S A

We demonstrate fluorescence thermometry techniques with sensitivities approaching 10 mKHz(-1/2) based on the spin-dependent photoluminescence of nitrogen vacancy (NV) centers in diamond. These techniques use dynamical decoupling protocols to convert thermally induced shifts in the NV center's spin resonance frequencies into large changes in its fluorescence. By mitigating interactions with nearby nuclear spins and facilitating selective thermal measurements, these protocols enhance the spin coherence times accessible for thermometry by 45-fold, corresponding to a 7-fold improvement in the NV center's temperature sensitivity. Moreover, we demonstrate these techniques can be applied over a broad temperature range and in both finite and near-zero magnetic field environments. This versatility suggests that the quantum coherence of single spins could be practically leveraged for sensitive thermometry in a wide variety of biological and microscale systems.

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Skeletal muscle PGC-1alpha controls whole-body lactate homeostasis through estrogen-related receptor alpha-dependent activation of LDH B and repression of LDH A.

Publication Date: 2013 May 21 PMID: 23650363
Authors: Summermatter, S. - Santos, G. - Perez-Schindler, J. - Handschin, C.
Journal: Proc Natl Acad Sci U S A

The peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) controls metabolic adaptations. We now show that PGC-1alpha in skeletal muscle drives the expression of lactate dehydrogenase (LDH) B in an estrogen-related receptor-alpha-dependent manner. Concomitantly, PGC-1alpha reduces the expression of LDH A and one of its regulators, the transcription factor myelocytomatosis oncogene. PGC-1alpha thereby coordinately alters the composition of the LDH complex and prevents the increase in blood lactate during exercise. Our results show how PGC-1alpha actively coordinates lactate homeostasis and provide a unique molecular explanation for PGC-1alpha-mediated muscle adaptations to training that ultimately enhance exercise performance and improve metabolic health.

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Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor.

Publication Date: 2013 May 21 PMID: 23650362
Authors: Lee, J. - Kim, J. H. - Biter, A. B. - Sielaff, B. - Lee, S. - Tsai, F. T.
Journal: Proc Natl Acad Sci U S A

Heat shock protein (Hsp) 104 is a ring-forming, protein-remodeling machine that harnesses the energy of ATP binding and hydrolysis to drive protein disaggregation. Although Hsp104 is an active ATPase, the recovery of functional protein requires the species-specific cooperation of the Hsp70 system. However, like Hsp104, Hsp70 is an active ATPase, which recognizes aggregated and aggregation-prone proteins, making it difficult to differentiate the mechanistic roles of Hsp104 and Hsp70 during protein disaggregation. Mapping the Hsp70-binding sites in yeast Hsp104 using peptide array technology and photo-cross-linking revealed a striking conservation of the primary Hsp70-binding motifs on the Hsp104 middle-domain across species, despite lack of sequence identity. Remarkably, inserting a Strep-Tactin binding motif at the spatially conserved Hsp70-binding site elicits the Hsp104 protein disaggregating activity that now depends on Strep-Tactin but no longer requires Hsp70/40. Consistent with a Strep-Tactin-dependent activation step, we found that full-length Hsp70 on its own could activate the Hsp104 hexamer by promoting intersubunit coordination, suggesting that Hsp70 is an activator of the Hsp104 motor.

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Activated microglia enhance neurogenesis via trypsinogen secretion.

Publication Date: 2013 May 21 PMID: 23650361
Authors: Nikolakopoulou, A. M. - Dutta, R. - Chen, Z. - Miller, R. H. - Trapp, B. D.
Journal: Proc Natl Acad Sci U S A

White matter neurons in multiple sclerosis brains are destroyed during demyelination and then replaced in some chronic multiple sclerosis lesions that exhibit a morphologically distinct population of activated microglia [Chang A, et al. (2008) Brain 131(Pt 9):2366-2375]. Here we investigated whether activated microglia secrete factors that promote the generation of neurons from white matter cells. Adult rat brain microglia (resting or activated with lipopolysaccharide) were isolated by flow cytometry and cocultured with neonatal rat optic nerve cells in separate but media-connected chambers. Optic nerve cells cocultured with activated microglia showed a significant increase in the number of cells of neuronal phenotype, identified by neuron-specific class III beta-tubulin (TUJ-1) labeling, compared with cultures with resting microglia. To investigate the possible source of the TUJ-1-positive cells, A2B5-positive oligodendrocyte progenitor cells and A2B5-negative cells were isolated and cocultured with resting and activated microglia. Significantly more TUJ-1-positive cells were generated from A2B5-negative cells ( approximately 70%) than from A2B5-positive cells ( approximately 30%). Mass spectrometry analysis of microglia culture media identified protease serine 2 (PRSS2) as a factor secreted by activated, but not resting, microglia. When added to optic nerve cultures, PRSS2 significantly increased neurogenesis, whereas the serine protease inhibitor, secretory leukocyte protease inhibitor, decreased activated microglia-induced neurogenesis. Collectively our data provide evidence that activated microglia increase neurogenesis through secretion of PRSS2.

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The phase transition of matrix recovery from Gaussian measurements matches the minimax MSE of matrix denoising.

Publication Date: 2013 May 21 PMID: 23650360
Authors: Donoho, D. L. - Gavish, M. - Montanari, A.
Journal: Proc Natl Acad Sci U S A

Let be an unknown M by N matrix. In matrix recovery, one takes linear measurements of , where and each is an M by N matrix. A popular approach for matrix recovery is nuclear norm minimization (NNM): solving the convex optimization problem for all , where denotes the nuclear norm, namely, the sum of singular values. Empirical work reveals a phase transition curve, stated in terms of the undersampling fraction , rank fraction , and aspect ratio . Specifically when the measurement matrices Ai have independent standard Gaussian random entries, a curve exists such that, if , NNM typically succeeds for large M,N, whereas if , it typically fails. An apparently quite different problem is matrix denoising in Gaussian noise, in which an unknown M by N matrix is to be estimated based on direct noisy measurements , where the matrix Z has independent and identically distributed Gaussian entries. A popular matrix denoising scheme solves the unconstrained optimization problem . When optimally tuned, this scheme achieves the asymptotic minimax mean-squared error , where . We report extensive experiments showing that the phase transition in the first problem, matrix recovery from Gaussian measurements, coincides with the minimax risk curve in the second problem, matrix denoising in Gaussian noise: , for any rank fraction (at each common aspect ratio beta). Our experiments considered matrices belonging to two constraint classes: real M by N matrices, of various ranks and aspect ratios, and real symmetric positive-semidefinite N by N matrices, of various ranks.

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WRKY8 transcription factor functions in the TMV-cg defense response by mediating both abscisic acid and ethylene signaling in Arabidopsis.

Publication Date: 2013 May 21 PMID: 23650359
Authors: Chen, L. - Zhang, L. - Li, D. - Wang, F. - Yu, D.
Journal: Proc Natl Acad Sci U S A

WRKY transcription factors are key players in the plant immune response, but less is known about their involvement in antiviral defense than about their roles in defense against bacterial or fungi pathogens. Here, we report that Arabidopsis thaliana WRKY DNA-binding protein 8 (WRKY8) has a role in mediating the long-distance movement of crucifer-infecting tobacco mosaic virus (TMV-cg). The expression of WRKY8 was inhibited by TMV-cg infection, and mutation of WRKY8 accelerated the accumulation of TMV-cg in systemically infected leaves. Quantitative RT-PCR analysis showed that the expression of ABA insensitive 4 (ABI4) was reduced and the expression of 1-aminocyclopropane-1-carboxylic acid synthase 6 (ACS6) and ethylene response factor 104 (ERF104) was enhanced in the systemically infected leaves of wrky8. Immunoprecipitation assays demonstrated that WRKY8 could bind selectively to putative W-boxes of the ABI4, ACS6, and ERF104 promoters. Furthermore, TMV-cg infection enhanced WRKY8 binding to the ABI4 promoter but reduced the binding of WRKY8 to the ACS6 and ERF104 promoters, indicating that regulation of ABI4, ACS6, and ERF104 by WRKY8 is at least partially dependent on TMV-cg. Exogenous applications of abscisic acid (ABA) reduced the systemic accumulation of TMV-cg. Mutations in ABA deficient 1, ABA deficient 2, ABA deficient 3, or abi4 accelerated systemic TMV-cg accumulation. In contrast, exogenous application of aminocyclopropane-1-carboxylic acid enhanced the systemic accumulation of TMV-cg, but mutations in acs6, erf104, or an octuple acs mutant inhibited systemic TMV-cg accumulation. Our results demonstrate that WRKY8 is involved in the defense response against TMV-cg through the direct regulation of the expression of ABI4, ACS6, and ERF104 and may mediate the crosstalk between ABA and ethylene signaling during the TMV-cg-Arabidopsis interaction.

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Heterochromatin protein Sir3 induces contacts between the amino terminus of histone H4 and nucleosomal DNA.

Publication Date: 2013 May 21 PMID: 23650358
Authors: Wang, F. - Li, G. - Altaf, M. - Lu, C. - Currie, M. A. - Johnson, A. - Moazed, D.
Journal: Proc Natl Acad Sci U S A

The regulated binding of effector proteins to the nucleosome plays a central role in the activation and silencing of eukaryotic genes. How this binding changes the properties of chromatin to mediate gene activation or silencing is not fully understood. Here we provide evidence that association of the budding yeast silent information regulator 3 (Sir3) silencing protein with the nucleosome induces a conformational change in the amino terminus of histone H4 that promotes interactions between the conserved H4 arginines 17 and 19 (R17 and R19) and nucleosomal DNA. Substitutions of H4R17 and R19 with alanine abolish silencing in vivo, but have little or no effect on binding of Sir3 to nucleosomes or histone H4 peptides in vitro. Furthermore, in both the previously reported crystal structure of the Sir3-bromo adjacent homology (BAH) domain bound to the Xenopus laevis nucleosome core particle and the crystal structure of the Sir3-BAH domain bound to the yeast nucleosome core particle described here, H4R17 and R19 make contacts with nucleosomal DNA rather than with Sir3. These results suggest that Sir3 binding generates a more stable nucleosome by clamping H4R17 and R19 to nucleosomal DNA, and raise the possibility that such induced changes in histone-DNA contacts play major roles in the regulation of chromatin structure.

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Combined effect of a peptide-morpholino oligonucleotide conjugate and a cell-penetrating peptide as an antibiotic.

Publication Date: 2013 May 21 PMID: 23650357
Authors: Wesolowski, D. - Alonso, D. - Altman, S.
Journal: Proc Natl Acad Sci U S A

A cell-penetrating peptide (CPP)-morpholino oligonucleotide (MO) conjugate (PMO) that has an antibiotic effect in culture had some contaminating CPPs in earlier preparations. The mixed conjugate had gene-specific and gene-nonspecific effects. An improved purification procedure separates the PMO from the free CPP and MO. The gene-specific effects are a result of the PMO, and the nonspecific effects are a result of the unlinked, unreacted CPP. The PMO and the CPP can be mixed together, as has been shown previously in earlier experiments, and have a combined effect as an antibiotic. Kinetic analysis of these effects confirm this observation. The effect of the CPP is bacteriostatic. The effect of the PMO appears to be bacteriocidal. An assay for mutations that would alter the ability of these agents to affect bacterial viability is negative.

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Gene regulatory network for neurogenesis in a sea star embryo connects broad neural specification and localized patterning.

Publication Date: 2013 May 21 PMID: 23650356
Authors: Yankura, K. A. - Koechlein, C. S. - Cryan, A. F. - Cheatle, A. - Hinman, V. F.
Journal: Proc Natl Acad Sci U S A

A great challenge in development biology is to understand how interacting networks of regulatory genes can direct the often highly complex patterning of cells in a 3D embryo. Here, we detail the gene regulatory network that describes the distribution of ciliary band-associated neurons in the bipinnaria larva of the sea star. This larva, typically for the ancestral deuterostome dipleurula larval type that it represents, forms two loops of ciliary bands that extend across much of the anterior-posterior and dorsal-ventral ectoderm. We show that the sea star first likely uses maternally inherited factors and the Wnt and Delta pathways to distinguish neurogenic ectoderm from endomesoderm. The broad neurogenic potential of the ectoderm persists throughout much of gastrulation. Nodal, bone morphogenetic protein 2/4 (Bmp2/4), and Six3-dependent pathways then sculpt a complex ciliary band territory that is defined by the expression of the forkhead transcription factor, foxg. Foxg is needed to define two molecularly distinct ectodermal domains, and for the formation of differentiated neurons along the edge of these two territories. Thus, significantly, Bmp2/4 signaling in sea stars does not distinguish differentiated neurons from nonneuronal ectoderm as it does in many other animals, but instead contributes to the patterning of an ectodermal territory, which then, in turn, provides cues to permit the final steps of neuronal differentiation. The modularity between specification and patterning likely reflects the evolutionary history of this gene regulatory network, in which an ancient module for specification of a broad neurogenic potential ectoderm was subsequently overlaid with a module for patterning.

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Salts drive controllable multilayered upright assembly of amyloid-like peptides at mica/water interface.

Publication Date: 2013 May 21 PMID: 23650355
Authors: Dai, B. - Kang, S. G. - Huynh, T. - Lei, H. - Castelli, M. - Hu, J. - Zhang, Y. - Zhou, R.
Journal: Proc Natl Acad Sci U S A

Surface-assisted self-assembly of amyloid-like peptides has received considerable interest in both amyloidosis research and nanotechnology in recent years. Despite extensive studies, some controlling factors, such as salts, are still not well understood, even though it is known that some salts can promote peptide self-assemblies through the so-called "salting-out" effect. However, they are usually noncontrollable, disordered, amorphous aggregates. Here, we show via a combined experimental and theoretical approach that a conserved consensus peptide NH2-VGGAVVAGV-CONH2 (GAV-9) (from representative amyloidogenic proteins) can self-assemble into highly ordered, multilayered nanofilaments, with surprising all-upright conformations, under high-salt concentrations. Our atomic force microscopy images also demonstrate that the vertical stacking of multiple layers is highly controllable by tuning the ionic strength, such as from 0 mM (monolayer) to 100 mM (mainly double layer), and to 250 mM MgCl2 (double, triple, quadruple, and quintuple layers). Our atomistic molecular dynamics simulations then reveal that these individual layers have very different internal nanostructures, with parallel beta-sheets in the first monolayer but antiparallel beta-sheets in the subsequent upper layers due to their different microenvironment. Further studies show that the growth of multilayered, all-upright nanostructures is a common phenomenon for GAV-9 at the mica/water interface, under a variety of salt types and a wide range of salt concentrations.

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Dynamic vaccine blocks relapse to compulsive intake of heroin.

Publication Date: 2013 May 6 PMID: 23650354
Authors: Schlosburg, J. E. - Vendruscolo, L. F. - Bremer, P. T. - Lockner, J. W. - Wade, C. L. - Nunes, A. A. - Stowe, G. N. - Edwards, S. - Janda, K. D. - Koob, G. F.
Journal: Proc Natl Acad Sci U S A

Heroin addiction, a chronic relapsing disorder characterized by excessive drug taking and seeking, requires constant psychotherapeutic and pharmacotherapeutic interventions to minimize the potential for further abuse. Vaccine strategies against many drugs of abuse are being developed that generate antibodies that bind drug in the bloodstream, preventing entry into the brain and nullifying psychoactivity. However, this strategy is complicated by heroin's rapid metabolism to 6-acetylmorphine and morphine. We recently developed a "dynamic" vaccine that creates antibodies against heroin and its psychoactive metabolites by presenting multihaptenic structures to the immune system that match heroin's metabolism. The current study presents evidence of effective and continuous sequestration of brain-permeable constituents of heroin in the bloodstream following vaccination. The result is efficient blockade of heroin activity in treated rats, preventing various features of drugs of abuse: heroin reward, drug-induced reinstatement of drug seeking, and reescalation of compulsive heroin self-administration following abstinence in dependent rats. The dynamic vaccine shows the capability to significantly devalue the reinforcing and motivating properties of heroin, even in subjects with a history of dependence. In addition, targeting a less brain-permeable downstream metabolite, morphine, is insufficient to prevent heroin-induced activity in these models, suggesting that heroin and 6-acetylmorphine are critical players in heroin's psychoactivity. Because the heroin vaccine does not target opioid receptors or common opioid pharmacotherapeutics, it can be used in conjunction with available treatment options. Thus, our vaccine represents a promising adjunct therapy for heroin addiction, providing continuous heroin antagonism, requiring minimal medical monitoring and patient compliance.

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Frequent adaptation and the McDonald-Kreitman test.

Publication Date: 2013 May 21 PMID: 23650353
Authors: Messer, P. W. - Petrov, D. A.
Journal: Proc Natl Acad Sci U S A

Population genomic studies have shown that genetic draft and background selection can profoundly affect the genome-wide patterns of molecular variation. We performed forward simulations under realistic gene-structure and selection scenarios to investigate whether such linkage effects impinge on the ability of the McDonald-Kreitman (MK) test to infer the rate of positive selection (alpha) from polymorphism and divergence data. We find that in the presence of slightly deleterious mutations, MK estimates of alpha severely underestimate the true rate of adaptation even if all polymorphisms with population frequencies under 50% are excluded. Furthermore, already under intermediate rates of adaptation, genetic draft substantially distorts the site frequency spectra at neutral and functional sites from the expectations under mutation-selection-drift balance. MK-type approaches that first infer demography from synonymous sites and then use the inferred demography to correct the estimation of alpha obtain almost the correct alpha in our simulations. However, these approaches typically infer a severe past population expansion although there was no such expansion in the simulations, casting doubt on the accuracy of methods that infer demography from synonymous polymorphism data. We propose a simple asymptotic extension of the MK test that yields accurate estimates of alpha in our simulations and should provide a fruitful direction for future studies.

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Control of slippage with tunable bubble mattresses.

Publication Date: 2013 May 21 PMID: 23650352
Authors: Karatay, E. - Haase, A. S. - Visser, C. W. - Sun, C. - Lohse, D. - Tsai, P. A. - Lammertink, R. G.
Journal: Proc Natl Acad Sci U S A

Tailoring the hydrodynamic boundary condition is essential for both applied and fundamental aspects of drag reduction. Hydrodynamic friction on superhydrophobic substrates providing gas-liquid interfaces can potentially be optimized by controlling the interface geometry. Therefore, establishing stable and optimal interfaces is crucial but rather challenging. Here we present unique superhydrophobic microfluidic devices that allow the presence of stable and controllable microbubbles at the boundary of microchannels. We experimentally and numerically examine the effect of microbubble geometry on the slippage at high resolution. The effective slip length is obtained for a wide range of protrusion angles, theta, of the microbubbles into the flow, using a microparticle image velocimetry technique. Our numerical results reveal a maximum effective slip length, corresponding to a 23% drag reduction at an optimal theta approximately 10 degrees . In agreement with the simulation results, our measurements correspond to up to 21% drag reduction when theta is in the range of -2 degrees to 12 degrees . The experimental and numerical results reveal a decrease in slip length with increasing protrusion angles when theta greater, similar 10 degrees . Such microfluidic devices with tunable slippage are essential for the amplified interfacial transport of fluids and particles.

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Evolution of the plankton paleome in the Black Sea from the Deglacial to Anthropocene.

Publication Date: 2013 May 21 PMID: 23650351
Authors: Coolen, M. J. - Orsi, W. D. - Balkema, C. - Quince, C. - Harris, K. - Sylva, S. P. - Filipova-Marinova, M. - Giosan, L.
Journal: Proc Natl Acad Sci U S A

The complex interplay of climate shifts over Eurasia and global sea level changes modulates freshwater and saltwater inputs to the Black Sea. The dynamics of the hydrologic changes from the Late Glacial into the Holocene remain a matter of debate, and information on how these changes affected the ecology of the Black Sea is sparse. Here we used Roche 454 next-generation pyrosequencing of sedimentary 18S rRNA genes to reconstruct the plankton community structure in the Black Sea over the last ca. 11,400 y. We found that 150 of 2,710 species showed a statistically significant response to four environmental stages. Freshwater chlorophytes were the best indicator species for lacustrine conditions (>9.0 ka B.P.), although the copresence of previously unidentified marine taxa indicated that the Black Sea might have been influenced to some extent by the Marmara Sea since at least 9.6 ka calendar (cal) B.P. Dinoflagellates, cercozoa, eustigmatophytes, and haptophytes responded most dramatically to the gradual increase in salinity after the latest marine reconnection and during the warm and moist mid-Holocene climatic optimum. According to paired analysis of deuterium/hydrogen (D/H) isotope ratios in fossil alkenones, salinity increased rapidly with the onset of the dry Subboreal after approximately 5.2 ka B.P., leading to an increase in marine fungi and the first occurrence of marine copepods. A gradual succession of dinoflagellates, diatoms, and chrysophytes occurred during the refreshening after approximately 2.5 ka cal B.P. with the onset of the cool and wet Subatlantic climate and recent anthropogenic perturbations.

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Modeling nuclear volume isotope effects in crystals.

Publication Date: 2013 May 6 PMID: 23650350
Authors: Schauble, E. A.
Journal: Proc Natl Acad Sci U S A

Mass-independent isotope fractionations driven by differences in volumes and shapes of nuclei (the field shift effect) are known in several elements and are likely to be found in more. All-electron relativistic electronic structure calculations can predict this effect but at present are computationally intensive and limited to modeling small gas phase molecules and clusters. Density functional theory, using the projector augmented wave method (DFT-PAW), has advantages in greater speed and compatibility with a three-dimensional periodic boundary condition while preserving information about the effects of chemistry on electron densities within nuclei. These electron density variations determine the volume component of the field shift effect. In this study, DFT-PAW calculations are calibrated against all-electron, relativistic Dirac-Hartree-Fock, and coupled-cluster with single, double (triple) excitation methods for estimating nuclear volume isotope effects. DFT-PAW calculations accurately reproduce changes in electron densities within nuclei in typical molecules, when PAW datasets constructed with finite nuclei are used. Nuclear volume contributions to vapor-crystal isotope fractionation are calculated for elemental cadmium and mercury, showing good agreement with experiments. The nuclear-volume component of mercury and cadmium isotope fractionations between atomic vapor and montroydite (HgO), cinnabar (HgS), calomel (Hg2Cl2), monteponite (CdO), and the CdS polymorphs hawleyite and greenockite are calculated, indicating preferential incorporation of neutron-rich isotopes in more oxidized, ionically bonded phases. Finally, field shift energies are related to Mossbauer isomer shifts, and equilibrium mass-independent fractionations for several tin-bearing crystals are calculated from 119Sn spectra. Isomer shift data should simplify calculations of mass-independent isotope fractionations in other elements with Mossbauer isotopes, such as platinum and uranium.

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Phosphorylation-dependent conformational changes and domain rearrangements in Staphylococcus aureus VraR activation.

Publication Date: 2013 May 21 PMID: 23650349
Authors: Leonard, P. G. - Golemi-Kotra, D. - Stock, A. M.
Journal: Proc Natl Acad Sci U S A

Staphylococcus aureus VraR, a vancomycin-resistance-associated response regulator, activates a cell-wall-stress stimulon in response to antibiotics that inhibit cell wall formation. X-ray crystal structures of VraR in both unphosphorylated and beryllofluoride-activated states have been determined, revealing a mechanism of phosphorylation-induced dimerization that features a deep hydrophobic pocket at the center of the receiver domain interface. Unphosphorylated VraR exists in a closed conformation that inhibits dimer formation. Phosphorylation at the active site promotes conformational changes that are propagated throughout the receiver domain, promoting the opening of a hydrophobic pocket that is essential for homodimer formation and enhanced DNA-binding activity. This prominent feature in the VraR dimer can potentially be exploited for the development of novel therapeutics to counteract antibiotic resistance in this important pathogen.

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True scale-invariant random spatial networks.

Publication Date: 2013 May 6 PMID: 23650348
Authors: Aldous, D. - Ganesan, K.
Journal: Proc Natl Acad Sci U S A

Some aspects of real-world road networks seem to have an approximate scale invariance property, motivating study of mathematical models of random networks whose distributions are exactly invariant under Euclidean scaling. This requires working in the continuum plane, so making a precise definition is not trivial. We introduce an axiomatization of a class of processes we call scale-invariant random spatial networks, whose primitives are routes between each pair of points in the plane. One concrete model, based on minimum-time routes in a binary hierarchy of roads with different speed limits, has been shown to satisfy the axioms, and two other constructions (based on Poisson line processes and on dynamic proximity graphs) are expected also to do so. We initiate study of structure theory and summary statistics for general processes in the class. Many questions arise in this setting via analogies with diverse existing topics, from geodesics in first-passage percolation to transit node-based route-finding algorithms.

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Retargeting of the Bacillus thuringiensis toxin Cyt2Aa against hemipteran insect pests.

Publication Date: 2013 May 21 PMID: 23650347
Authors: Chougule, N. P. - Li, H. - Liu, S. - Linz, L. B. - Narva, K. E. - Meade, T. - Bonning, B. C.
Journal: Proc Natl Acad Sci U S A

Although transgenic crops expressing Bacillus thuringiensis (Bt) toxins have been used successfully for management of lepidopteran and coleopteran pest species, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. To overcome this limitation, we demonstrate that addition of a short peptide sequence selected for binding to the gut of the targeted pest species serves to increase toxicity against said pest. Insertion of a 12-aa pea aphid gut-binding peptide by adding to or replacing amino acids in one of three loops of the Bt cytolytic toxin, Cyt2Aa, resulted in enhanced binding and toxicity against both the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. This strategy may allow for transgenic plant-mediated suppression of other hemipteran pests, which include some of the most important pests of global agriculture.

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Sulfur isotopes in coal constrain the evolution of the Phanerozoic sulfur cycle.

Publication Date: 2013 May 21 PMID: 23650346
Authors: Canfield, D. E.
Journal: Proc Natl Acad Sci U S A

Sulfate is the second most abundant anion (behind chloride) in modern seawater, and its cycling is intimately coupled to the cycling of organic matter and oxygen at the Earth's surface. For example, the reduction of sulfide by microbes oxidizes vast amounts of organic carbon and the subsequent reaction of sulfide with iron produces pyrite whose burial in sediments is an important oxygen source to the atmosphere. The concentrations of seawater sulfate and the operation of sulfur cycle have experienced dynamic changes through Earth's history, and our understanding of this history is based mainly on interpretations of the isotope record of seawater sulfates and sedimentary pyrites. The isotope record, however, does not give a complete picture of the ancient sulfur cycle. This is because, in standard isotope mass balance models, there are more variables than constraints. Typically, in interpretations of the isotope record and in the absence of better information, one assumes that the isotopic composition of the input sulfate to the oceans has remained constant through time. It is argued here that this assumption has a constraint over the last 390 Ma from the isotopic composition of sulfur in coal. Indeed, these compositions do not deviate substantially from the modern surface-water input to the oceans. When applied to mass balance models, these results support previous interpretations of sulfur cycle operation and counter recent suggestions that sulfate has been a minor player in sulfur cycling through the Phanerozoic Eon.

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Mycobacterial toxin MazF-mt6 inhibits translation through cleavage of 23S rRNA at the ribosomal A site.

Publication Date: 2013 May 21 PMID: 23650345
Authors: Schifano, J. M. - Edifor, R. - Sharp, J. D. - Ouyang, M. - Konkimalla, A. - Husson, R. N. - Woychik, N. A.
Journal: Proc Natl Acad Sci U S A

The Mycobacterium tuberculosis genome contains an unusually high number of toxin-antitoxin modules, some of which have been suggested to play a role in the establishment and maintenance of latent tuberculosis. Nine of these toxin-antitoxin loci belong to the mazEF family, encoding the intracellular toxin MazF and its antitoxin inhibitor MazE. Nearly every MazF ortholog recognizes a unique three- or five-base RNA sequence and cleaves mRNA. As a result, these toxins selectively target a subset of the transcriptome for degradation and are known as "mRNA interferases." Here we demonstrate that a MazF family member from M. tuberculosis, MazF-mt6, has an additional role-inhibiting translation through targeted cleavage of 23S rRNA in the evolutionarily conserved helix/loop 70. We first determined that MazF-mt6 cleaves mRNA at (5)(')UU downward arrowCCU(3') sequences. We then discovered that MazF-mt6 also cleaves M. tuberculosis 23S rRNA at a single UUCCU in the ribosomal A site that contacts tRNA and ribosome recycling factor. To gain further mechanistic insight, we demonstrated that MazF-mt6-mediated cleavage of rRNA can inhibit protein synthesis in the absence of mRNA cleavage. Finally, consistent with the position of 23S rRNA cleavage, MazF-mt6 destabilized 50S-30S ribosomal subunit association. Collectively, these results show that MazF toxins do not universally act as mRNA interferases, because MazF-mt6 inhibits protein synthesis by cleaving 23S rRNA in the ribosome active center.

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Healing power of honey.

Publication Date: 2013 May 3 PMID: 23645636
Authors: Leal, W. S.
Journal: Proc Natl Acad Sci U S A

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Flow induces epithelial-mesenchymal transition, cellular heterogeneity and biomarker modulation in 3D ovarian cancer nodules.

Publication Date: 2013 May 3 PMID: 23645635
Authors: Rizvi, I. - Gurkan, U. A. - Tasoglu, S. - Alagic, N. - Celli, J. P. - Mensah, L. B. - Mai, Z. - Demirci, U. - Hasan, T.
Journal: Proc Natl Acad Sci U S A

Seventy-five percent of patients with epithelial ovarian cancer present with advanced-stage disease that is extensively disseminated intraperitoneally and prognosticates the poorest outcomes. Primarily metastatic within the abdominal cavity, ovarian carcinomas initially spread to adjacent organs by direct extension and then disseminate via the transcoelomic route to distant sites. Natural fluidic streams of malignant ascites triggered by physiological factors, including gravity and negative subdiaphragmatic pressure, carry metastatic cells throughout the peritoneum. We investigated the role of fluidic forces as modulators of metastatic cancer biology in a customizable microfluidic platform using 3D ovarian cancer nodules. Changes in the morphological, genetic, and protein profiles of biomarkers associated with aggressive disease were evaluated in the 3D cultures grown under controlled and continuous laminar flow. A modulation of biomarker expression and tumor morphology consistent with increased epithelial-mesenchymal transition, a critical step in metastatic progression and an indicator of aggressive disease, is observed because of hydrodynamic forces. The increase in epithelial-mesenchymal transition is driven in part by a posttranslational up-regulation of epidermal growth factor receptor (EGFR) expression and activation, which is associated with the worst prognosis in ovarian cancer. A flow-induced, transcriptionally regulated decrease in E-cadherin protein expression and a simultaneous increase in vimentin is observed, indicating increased metastatic potential. These findings demonstrate that fluidic streams induce a motile and aggressive tumor phenotype. The microfluidic platform developed here potentially provides a flow-informed framework complementary to conventional mechanism-based therapeutic strategies, with broad applicability to other lethal malignancies.

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Chloride transport-driven alveolar fluid secretion is a major contributor to cardiogenic lung edema.

Publication Date: 2013 May 3 PMID: 23645634
Authors: Solymosi, E. A. - Kaestle-Gembardt, S. M. - Vadasz, I. - Wang, L. - Neye, N. - Chupin, C. J. - Rozowsky, S. - Ruehl, R. - Tabuchi, A. - Schulz, H. - Kapus, A. - Morty, R. E. - Kuebler, W. M.
Journal: Proc Natl Acad Sci U S A

Alveolar fluid clearance driven by active epithelial Na+ and secondary Cl- absorption counteracts edema formation in the intact lung. Recently, we showed that impairment of alveolar fluid clearance because of inhibition of epithelial Na+ channels (ENaCs) promotes cardiogenic lung edema. Concomitantly, we observed a reversal of alveolar fluid clearance, suggesting that reversed transepithelial ion transport may promote lung edema by driving active alveolar fluid secretion. We, therefore, hypothesized that alveolar ion and fluid secretion may constitute a pathomechanism in lung edema and aimed to identify underlying molecular pathways. In isolated perfused lungs, alveolar fluid clearance and secretion were determined by a double-indicator dilution technique. Transepithelial Cl- secretion and alveolar Cl- influx were quantified by radionuclide tracing and alveolar Cl- imaging, respectively. Elevated hydrostatic pressure induced ouabain-sensitive alveolar fluid secretion that coincided with transepithelial Cl- secretion and alveolar Cl- influx. Inhibition of either cystic fibrosis transmembrane conductance regulator (CFTR) or Na+-K+-Cl- cotransporters (NKCC) blocked alveolar fluid secretion, and lungs of CFTR-/- mice were protected from hydrostatic edema. Inhibition of ENaC by amiloride reproduced alveolar fluid and Cl- secretion that were again CFTR-, NKCC-, and Na+-K+-ATPase-dependent. Our findings show a reversal of transepithelial Cl- and fluid flux from absorptive to secretory mode at hydrostatic stress. Alveolar Cl- and fluid secretion are triggered by ENaC inhibition and mediated by NKCC and CFTR. Our results characterize an innovative mechanism of cardiogenic edema formation and identify NKCC1 as a unique therapeutic target in cardiogenic lung edema.

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Biomineralization toolkit: The importance of sample cleaning prior to the characterization of biomineral proteomes.

Publication Date: 2013 May 3 PMID: 23645633
Authors: Ramos-Silva, P. - Marin, F. - Kaandorp, J. - Marie, B.
Journal: Proc Natl Acad Sci U S A

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Correction for Schulten et al., Previously undescribed grass pollen antigens are the major inducers of T helper 2 cytokine-producing T cells in allergic individuals.

Publication Date: 2013 May 21 PMID: 23641005
Authors:
Journal: Proc Natl Acad Sci U S A

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Chloride binding site of neurotransmitter sodium symporters.

Publication Date: 2013 May 21 PMID: 23641004
Authors: Kantcheva, A. K. - Quick, M. - Shi, L. - Winther, A. M. - Stolzenberg, S. - Weinstein, H. - Javitch, J. A. - Nissen, P.
Journal: Proc Natl Acad Sci U S A

Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion in the Na1 site organize a connection between their coordinating residues and the extracellular gate of LeuT through a continuous H-bond network. The specific insights from the structures, combined with results from substrate binding studies and molecular dynamics simulations, reveal an anion-dependent occlusion mechanism for NSS and shed light on the functional role of chloride binding.

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Dnmt2-dependent methylomes lack defined DNA methylation patterns.

Publication Date: 2013 May 21 PMID: 23641003
Authors: Raddatz, G. - Guzzardo, P. M. - Olova, N. - Fantappie, M. R. - Rampp, M. - Schaefer, M. - Reik, W. - Hannon, G. J. - Lyko, F.
Journal: Proc Natl Acad Sci U S A

Several organisms have retained methyltransferase 2 (Dnmt2) as their only candidate DNA methyltransferase gene. However, information about Dnmt2-dependent methylation patterns has been limited to a few isolated loci and the results have been discussed controversially. In addition, recent studies have shown that Dnmt2 functions as a tRNA methyltransferase, which raised the possibility that Dnmt2-only genomes might be unmethylated. We have now used whole-genome bisulfite sequencing to analyze the methylomes of Dnmt2-only organisms at single-base resolution. Our results show that the genomes of Schistosoma mansoni and Drosophila melanogaster lack detectable DNA methylation patterns. Residual unconverted cytosine residues shared many attributes with bisulfite deamination artifacts and were observed at comparable levels in Dnmt2-deficient flies. Furthermore, genetically modified Dnmt2-only mouse embryonic stem cells lost the DNA methylation patterns found in wild-type cells. Our results thus uncover fundamental differences among animal methylomes and suggest that DNA methylation is dispensable for a considerable number of eukaryotic organisms.

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Dynamic gradients of an intermediate filament-like cytoskeleton are recruited by a polarity landmark during apical growth.

Publication Date: 2013 May 21 PMID: 23641002
Authors: Fuchino, K. - Bagchi, S. - Cantlay, S. - Sandblad, L. - Wu, D. - Bergman, J. - Kamali-Moghaddam, M. - Flardh, K. - Ausmees, N.
Journal: Proc Natl Acad Sci U S A

Intermediate filament (IF)-like cytoskeleton emerges as a versatile tool for cellular organization in all kingdoms of life, underscoring the importance of mechanistically understanding its diverse manifestations. We showed previously that, in Streptomyces (a bacterium with a mycelial lifestyle similar to that of filamentous fungi, including extreme cell and growth polarity), the IF protein FilP confers rigidity to the hyphae by an unknown mechanism. Here, we provide a possible explanation for the IF-like function of FilP by demonstrating its ability to self-assemble into a cis-interconnected regular network in vitro and its localization into structures consistent with a cytoskeletal network in vivo. Furthermore, we reveal that a spatially restricted interaction between FilP and DivIVA, the main component of the Streptomyces polarisome complex, leads to formation of apical gradients of FilP in hyphae undergoing active tip extension. We propose that the coupling between the mechanism driving polar growth and the assembly of an IF cytoskeleton provides each new hypha with an additional stress-bearing structure at its tip, where the nascent cell wall is inevitably more flexible and compliant while it is being assembled and matured. Our data suggest that recruitment of cytoskeleton around a cell polarity landmark is a broadly conserved strategy in tip-growing cells.

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Peptide library-based evaluation of T-cell receptor breadth detects defects in global and regulatory activation in human immunologic diseases.

Publication Date: 2013 May 14 PMID: 23637345
Authors: Barber, J. S. - Yokomizo, L. K. - Sheikh, V. - Freeman, A. F. - Garabedian, E. - van Dijk, E. - Sokolic, R. - Candotti, F. - Weng, N. P. - Sereti, I. - Milner, J. D.
Journal: Proc Natl Acad Sci U S A

The ability of T-cells to respond to foreign antigens and to appropriately regulate this response is crucial for maintaining immune homeostasis. Using combinatorial peptide libraries, we functionally measured broad T-cell reactivity and observed impaired reactivity in established models of T-cell receptor repertoire restriction and in previously unrecognized disease contexts. By concurrently analyzing T-regulatory and T-effector cells, we show strong functional correlation between these subsets in healthy individuals and, strikingly, that alterations of this balance are associated with T helper type 2 (Th2)-mediated disease in a lymphopenic setting. Finally, we demonstrate that peptide-based priming of polyclonal naive cells with relatively low concentrations skews toward Th2 differentiation. These findings provide unique insight into the pathophysiology and functional consequences of abnormal T-cell repertoires and into differentiation of human naive T-cells.

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Rational integration of noisy evidence and prior semantic expectations in sentence interpretation.

Publication Date: 2013 May 14 PMID: 23637344
Authors: Gibson, E. - Bergen, L. - Piantadosi, S. T.
Journal: Proc Natl Acad Sci U S A

Sentence processing theories typically assume that the input to our language processing mechanisms is an error-free sequence of words. However, this assumption is an oversimplification because noise is present in typical language use (for instance, due to a noisy environment, producer errors, or perceiver errors). A complete theory of human sentence comprehension therefore needs to explain how humans understand language given imperfect input. Indeed, like many cognitive systems, language processing mechanisms may even be "well designed"-in this case for the task of recovering intended meaning from noisy utterances. In particular, comprehension mechanisms may be sensitive to the types of information that an idealized statistical comprehender would be sensitive to. Here, we evaluate four predictions about such a rational (Bayesian) noisy-channel language comprehender in a sentence comprehension task: (i) semantic cues should pull sentence interpretation towards plausible meanings, especially if the wording of the more plausible meaning is close to the observed utterance in terms of the number of edits; (ii) this process should asymmetrically treat insertions and deletions due to the Bayesian "size principle"; such nonliteral interpretation of sentences should (iii) increase with the perceived noise rate of the communicative situation and (iv) decrease if semantically anomalous meanings are more likely to be communicated. These predictions are borne out, strongly suggesting that human language relies on rational statistical inference over a noisy channel.

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DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV.

Publication Date: 2013 May 14 PMID: 23637343
Authors: Zhang, H. - Ma, Z. Y. - Zeng, L. - Tanaka, K. - Zhang, C. J. - Ma, J. - Bai, G. - Wang, P. - Zhang, S. W. - Liu, Z. W. - Cai, T. - Tang, K. - Liu, R. - Shi, X. - He, X. J. - Zhu, J. K.
Journal: Proc Natl Acad Sci U S A

DNA methylation is an important epigenetic mark in many eukaryotic organisms. De novo DNA methylation in plants can be achieved by the RNA-directed DNA methylation (RdDM) pathway, where the plant-specific DNA-dependent RNA polymerase IV (Pol IV) transcribes target sequences to initiate 24-nt siRNA production and action. The putative DNA binding protein DTF1/SHH1 of Arabidopsis has been shown to associate with Pol IV and is required for 24-nt siRNA accumulation and transcriptional silencing at several RdDM target loci. However, the extent and mechanism of DTF1 function in RdDM is unclear. We show here that DTF1 is necessary for the accumulation of the majority of Pol IV-dependent 24-nt siRNAs. It is also required for a large proportion of Pol IV-dependent de novo DNA methylation. Interestingly, there is a group of RdDM target loci where 24-nt siRNA accumulation but not DNA methylation is dependent on DTF1. DTF1 interacts directly with the chromatin remodeling protein CLASSY 1 (CLSY1), and both DTF1 and CLSY1 are associated in vivo with Pol IV but not Pol V, which functions downstream in the RdDM effector complex. DTF1 and DTF2 (a DTF1-like protein) contain a SAWADEE domain, which was found to bind specifically to histone H3 containing H3K9 methylation. Taken together, our results show that DTF1 is a core component of the RdDM pathway, and suggest that DTF1 interacts with CLSY1 to assist in the recruitment of Pol IV to RdDM target loci where H3K9 methylation may be an important feature. Our results also suggest the involvement of DTF1 in an important negative feedback mechanism for DNA methylation at some RdDM target loci.

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Global view of the evolution and diversity of metazoan neuropeptide signaling.

Publication Date: 2013 May 21 PMID: 23637342
Authors: Jekely, G.
Journal: Proc Natl Acad Sci U S A

Neuropeptides are signaling molecules that commonly act via G protein-coupled receptors (GPCRs) and are generated in neurons by proneuropeptide (pNP) cleavage. Present in both cnidarians and bilaterians, neuropeptides represent an ancient and widespread mode of neuronal communication. Due to the inherent difficulties of analyzing highly diverse and repetitive pNPs, the relationships among different families are often elusive. Using similarity-based clustering and sensitive similarity searches, I obtained a global view of metazoan pNP diversity and evolution. Clustering revealed a large and diffuse network of sequences connected by significant sequence similarity encompassing one-quarter of all families. pNPs belonging to this cluster were also identified in the early-branching neuronless animal Trichoplax adhaerens. Clustering of neuropeptide GPCRs identified several orthology groups and allowed the reconstruction of the phyletic distribution of receptor families. GPCR phyletic distribution closely paralleled that of pNPs, indicating extensive conservation and long-term coevolution of receptor-ligand pairs. Receptor orthology and intermediate sequences also revealed the homology of pNPs so far considered unrelated, including allatotropin and orexin. These findings, together with the identification of deuterostome achatin and luqin and protostome opioid pNPs, extended the neuropeptide complement of the urbilaterian. Several pNPs were also identified from the hemichordate Saccoglossus kowalevskii and the cephalochordate Branchiostoma floridae, elucidating pNP evolution in deuterostomes. Receptor-ligand conservation also allowed ligand predictions for many uncharacterized GPCRs from nonmodel species. The reconstruction of the neuropeptide-signaling repertoire at deep nodes of the animal phylogeny allowed the formulation of a testable scenario of the evolution of animal neuroendocrine systems.

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Diversity, host switching and evolution of Plasmodium vivax infecting African great apes.

Publication Date: 2013 May 14 PMID: 23637341
Authors: Prugnolle, F. - Rougeron, V. - Becquart, P. - Berry, A. - Makanga, B. - Rahola, N. - Arnathau, C. - Ngoubangoye, B. - Menard, S. - Willaume, E. - Ayala, F. J. - Fontenille, D. - Ollomo, B. - Durand, P. - Paupy, C. - Renaud, F.
Journal: Proc Natl Acad Sci U S A

Plasmodium vivax is considered to be absent from Central and West Africa because of the protective effect of Duffy negativity. However, there are reports of persons returning from these areas infected with this parasite and observations suggesting the existence of transmission. Among the possible explanations for this apparent paradox, the existence of a zoonotic reservoir has been proposed. May great apes be this reservoir? We analyze the mitochondrial and nuclear genetic diversity of P. vivax parasites isolated from great apes in Africa and compare it to parasites isolated from travelers returning from these regions of Africa, as well as to human isolates distributed all over the world. We show that the P. vivax sequences from parasites of great apes form a clade genetically distinct from the parasites circulating in humans. We show that this clade's parasites can be infectious to humans by describing the case of a traveler returning from the Central African Republic infected with one of them. The relationship between this P. vivax clade in great apes and the human isolates is discussed.

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IL-15 in tumor microenvironment causes rejection of large established tumors by T cells in a noncognate T cell receptor-dependent manner.

Publication Date: 2013 May 14 PMID: 23637340
Authors: Liu, R. B. - Engels, B. - Schreiber, K. - Ciszewski, C. - Schietinger, A. - Schreiber, H. - Jabri, B.
Journal: Proc Natl Acad Sci U S A

A major challenge of cancer immunotherapy is the persistence and outgrowth of subpopulations that lose expression of the target antigen. IL-15 is a potent cytokine that can promote organ-specific autoimmunity when up-regulated on tissue cells. Here we report that T cells eradicated 2-wk-old solid tumors that expressed IL-15, eliminating antigen-negative cells. In contrast, control tumors that lacked IL-15 expression consistently relapsed. Interestingly, even tumors lacking expression of cognate antigen were rejected when expressing IL-15, indicating that rejection after adoptive T-cell transfer was independent of cognate antigen expression. Nevertheless, the T-cell receptor of the transferred T cells influenced the outcome, consistent with the notion that T-cell receptor activation and effector status determine whether IL-15 can confer lymphokine killer activity-like properties to T cells. The effect was limited to the microenvironment of tumors expressing IL-15; there were no noticeable effects on contralateral tumors lacking IL-15. Taken together, these results indicate that expression of IL-15 in the tumor microenvironment may prevent the escape of antigen loss variants and subsequent tumor recurrence by enabling T cells to eliminate cancer cells lacking cognate antigen expression in a locally restricted manner.

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The transition in southern Iberia: Insights from paleoclimatology and the Early Upper Palaeolithic.

Publication Date: 2013 Apr 30 PMID: 23633575
Authors: de la Pena, P.
Journal: Proc Natl Acad Sci U S A

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Inadequate methods and questionable conclusions in atmospheric life study.

Publication Date: 2013 Apr 30 PMID: 23633574
Authors: Smith, D. J. - Griffin, D. W.
Journal: Proc Natl Acad Sci U S A

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Quantifying primary loops in polymer gels by linear viscoelasticity.

Publication Date: 2013 Apr 30 PMID: 23633573
Authors: Stadler, F. J.
Journal: Proc Natl Acad Sci U S A

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Surface display of a massively variable lipoprotein by a Legionella diversity-generating retroelement.

Publication Date: 2013 May 14 PMID: 23633572
Authors: Arambula, D. - Wong, W. - Medhekar, B. A. - Guo, H. - Gingery, M. - Czornyj, E. - Liu, M. - Dey, S. - Ghosh, P. - Miller, J. F.
Journal: Proc Natl Acad Sci U S A

Diversity-generating retroelements (DGRs) are a unique family of retroelements that confer selective advantages to their hosts by facilitating localized DNA sequence evolution through a specialized error-prone reverse transcription process. We characterized a DGR in Legionella pneumophila, an opportunistic human pathogen that causes Legionnaires disease. The L. pneumophila DGR is found within a horizontally acquired genomic island, and it can theoretically generate 10(26) unique nucleotide sequences in its target gene, legionella determinent target A (ldtA), creating a repertoire of 10(19) distinct proteins. Expression of the L. pneumophila DGR resulted in transfer of DNA sequence information from a template repeat to a variable repeat (VR) accompanied by adenine-specific mutagenesis of progeny VRs at the 3'end of ldtA. ldtA encodes a twin-arginine translocated lipoprotein that is anchored in the outer leaflet of the outer membrane, with its C-terminal variable region surface exposed. Related DGRs were identified in L. pneumophila clinical isolates that encode unique target proteins with homologous VRs, demonstrating the adaptability of DGR components. This work characterizes a DGR that diversifies a bacterial protein and confirms the hypothesis that DGR-mediated mutagenic homing occurs through a conserved mechanism. Comparative bioinformatics predicts that surface display of massively variable proteins is a defining feature of a subset of bacterial DGRs.

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Fast neurotransmitter release regulated by the endocytic scaffold intersectin.

Publication Date: 2013 May 14 PMID: 23633571
Authors: Sakaba, T. - Kononenko, N. L. - Bacetic, J. - Pechstein, A. - Schmoranzer, J. - Yao, L. - Barth, H. - Shupliakov, O. - Kobler, O. - Aktories, K. - Haucke, V.
Journal: Proc Natl Acad Sci U S A

Sustained fast neurotransmission requires the rapid replenishment of release-ready synaptic vesicles (SVs) at presynaptic active zones. Although the machineries for exocytic fusion and for subsequent endocytic membrane retrieval have been well characterized, little is known about the mechanisms underlying the rapid recruitment of SVs to release sites. Here we show that the Down syndrome-associated endocytic scaffold protein intersectin 1 is a crucial factor for the recruitment of release-ready SVs. Genetic deletion of intersectin 1 expression or acute interference with intersectin function inhibited the replenishment of release-ready vesicles, resulting in short-term depression, without significantly affecting the rate of endocytic membrane retrieval. Acute perturbation experiments suggest that intersectin-mediated vesicle replenishment involves the association of intersectin with the fissioning enzyme dynamin and with the actin regulatory GTPase CDC42. Our data indicate a role for the endocytic scaffold intersectin in fast neurotransmitter release, which may be of prime importance for information processing in the brain.

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Juvenile hormone and its receptor, methoprene-tolerant, control the dynamics of mosquito gene expression.

Publication Date: 2013 Apr 30 PMID: 23633570
Authors: Zou, Z. - Saha, T. T. - Roy, S. - Shin, S. W. - Backman, T. W. - Girke, T. - White, K. P. - Raikhel, A. S.
Journal: Proc Natl Acad Sci U S A

Juvenile hormone III (JH) plays a key role in regulating the reproduction of female mosquitoes. Microarray time-course analysis revealed dynamic changes in gene expression during posteclosion (PE) development in the fat body of female Aedes aegypti. Hierarchical clustering identified three major gene clusters: 1,843 early-PE (EPE) genes maximally expressed at 6 h PE, 457 mid-PE (MPE) genes at 24 h PE, and 1,815 late-PE (LPE) genes at 66 h PE. The RNAi microarray screen for the JH receptor Methoprene-tolerant (Met) showed that 27% of EPE and 40% of MPE genes were up-regulated whereas 36% of LPE genes were down-regulated in the absence of this receptor. Met repression of EPE and MPE and activation of LPE genes were validated by an in vitro fat-body culture experiment using Met RNAi. Sequence motif analysis revealed the consensus for a 9-mer Met-binding motif, CACGC/TGA/GT/AG. Met-binding motif variants were overrepresented within the first 300 bases of the promoters of Met RNAi-down-regulated (LPE) genes but not in Met RNAi-up-regulated (EPE) genes. EMSAs using a combination of mutational and anti-Met antibody supershift analyses confirmed the binding properties of the Met consensus motif variants. There was a striking temporal separation of expression profiles among major functional gene groups, with carbohydrate, lipid, and xenobiotics metabolism belonging to the EPE and MPE clusters and transcription and translation to the LPE cluster. This study represents a significant advancement in the understanding of the regulation of gene expression by JH and its receptor Met during female mosquito reproduction.

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Limits to upward movement of subalpine forests in a warming climate.

Publication Date: 2013 May 14 PMID: 23633569
Authors: Donato, D. C.
Journal: Proc Natl Acad Sci U S A

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Targeted resequencing implicates the familial Mediterranean fever gene MEFV and the toll-like receptor 4 gene TLR4 in Behcet disease.

Publication Date: 2013 May 14 PMID: 23633568
Authors: Kirino, Y. - Zhou, Q. - Ishigatsubo, Y. - Mizuki, N. - Tugal-Tutkun, I. - Seyahi, E. - Ozyazgan, Y. - Ugurlu, S. - Erer, B. - Abaci, N. - Ustek, D. - Meguro, A. - Ueda, A. - Takeno, M. - Inoko, H. - Ombrello, M. J. - Satorius, C. L. - Maskeri, B. - Mullikin, J. C. - Sun, H. W. - Gutierrez-Cruz, G. - Kim, Y. - Wilson, A. F. - Kastner, D. L. - Gul, A. - Remmers, E. F.
Journal: Proc Natl Acad Sci U S A

Genome-wide association studies (GWAS) are a powerful means of identifying genes with disease-associated common variants, but they are not well-suited to detecting genes with disease-associated rare and low-frequency variants. In the current study of Behcet disease (BD), nonsynonymous variants (NSVs) identified by deep exonic resequencing of 10 genes found by GWAS (IL10, IL23R, CCR1, STAT4, KLRK1, KLRC1, KLRC2, KLRC3, KLRC4, and ERAP1) and 11 genes selected for their role in innate immunity (IL1B, IL1R1, IL1RN, NLRP3, MEFV, TNFRSF1A, PSTPIP1, CASP1, PYCARD, NOD2, and TLR4) were evaluated for BD association. A differential distribution of the rare and low-frequency NSVs of a gene in 2,461 BD cases compared with 2,458 controls indicated their collective association with disease. By stringent criteria requiring at least a single burden test with study-wide significance and a corroborating test with at least nominal significance, rare and low-frequency NSVs in one GWAS-identified gene, IL23R (P = 6.9 x 10(-5)), and one gene involved in innate immunity, TLR4 (P = 8.0 x 10(-4)), were associated with BD. In addition, damaging or rare damaging NOD2 variants were nominally significant across all three burden tests applied (P = 0.0063-0.045). Furthermore, carriage of the familial Mediterranean fever gene (MEFV) mutation Met694Val, which is known to cause recessively inherited familial Mediterranean fever, conferred BD risk in the Turkish population (OR, 2.65; P = 1.8 x 10(-12)). The disease-associated NSVs in MEFV and TLR4 implicate innate immune and bacterial sensing mechanisms in BD pathogenesis.

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Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa.

Publication Date: 2013 May 14 PMID: 23633567
Authors: Fujihara, Y. - Tokuhiro, K. - Muro, Y. - Kondoh, G. - Araki, Y. - Ikawa, M. - Okabe, M.
Journal: Proc Natl Acad Sci U S A

Formation of spermatozoa of normal shape, number, and motility is insufficient for the male siring of pups. The spermatozoa must be accompanied by sound fertilizing ability. We found that males with disrupted testis-expressed gene 101 (Tex101) produce normal-looking but fertilization-incompetent spermatozoa, which were accompanied by a deficiency of a disintegrin and metallopeptidase domain 3 (ADAM3) on sperm plasma membrane. It was also found that the existence of TEX101 on spermatozoa was regulated by angiotensin-converting enzyme (ACE). The removal of GPI-anchored protein TEX101 by ACE was essential to produce fertile spermatozoa, and the function of ACE was not depending on its well-known peptidase activity. The finding of TEX101 as a unique specific substrate for ACE may provide a potential target for the production of an awaited contraceptive medicine for men.

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Fe-carbonyl is a key player in planetary magmas.

Publication Date: 2013 May 14 PMID: 23633566
Authors: Hirschmann, M. M.
Journal: Proc Natl Acad Sci U S A

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Genomic analysis of diffuse pediatric low-grade gliomas identifies recurrent oncogenic truncating rearrangements in the transcription factor MYBL1.

Publication Date: 2013 May 14 PMID: 23633565
Authors: Ramkissoon, L. A. - Horowitz, P. M. - Craig, J. M. - Ramkissoon, S. H. - Rich, B. E. - Schumacher, S. E. - McKenna, A. - Lawrence, M. S. - Bergthold, G. - Brastianos, P. K. - Tabak, B. - Ducar, M. D. - Van Hummelen, P. - Macconaill, L. E. - Pouissant-Young, T. - Cho, Y. J. - Taha, H. - Mahmoud, M. - Bowers, D. C. - Margraf, L. - Tabori, U. - Hawkins, C. - Packer, R. J. - Hill, D. A. - Pomeroy, S. L. - Eberhart, C. G. - Dunn, I. F. - Goumnerova, L. - Getz, G. - Chan, J. A. - Santagata, S. - Hahn, W. C. - Stiles, C. D. - Ligon, A. H. - Kieran, M. W. - Beroukhim, R. - Ligon, K. L.
Journal: Proc Natl Acad Sci U S A

Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but, apart from BRAF kinase mutations or duplications in specific subclasses, few genetic driver events are known. Diffuse PLGGs comprise a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. We performed high-resolution copy-number analysis on 44 formalin-fixed, paraffin-embedded diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gain, was observed in 28% of diffuse astrocytoma grade IIs and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene v-myb avian myeloblastosis viral oncogene homolog (MYB) on 6q23.3. Whole-genome sequencing of a MYBL1-rearranged diffuse astrocytoma grade II demonstrated MYBL1 tandem duplication and few other events. Truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas.

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A new member of the 4-methylideneimidazole-5-one-containing aminomutase family from the enediyne kedarcidin biosynthetic pathway.

Publication Date: 2013 May 14 PMID: 23633564
Authors: Huang, S. X. - Lohman, J. R. - Huang, T. - Shen, B.
Journal: Proc Natl Acad Sci U S A

4-Methylideneimidazole-5-one (MIO)-containing aminomutases catalyze the conversion of l-alpha-amino acids to beta-amino acids with either an (R) or an (S) configuration. l-Phenylalanine and l-tyrosine are the only two natural substrates identified to date. The enediyne chromophore of the chromoprotein antitumor antibiotic kedarcidin (KED) harbors an (R)-2-aza-3-chloro-beta-tyrosine moiety reminiscent of the (S)-3-chloro-5-hydroxy-beta-tyrosine moiety of the C-1027 enediyne chromophore, the biosynthesis of which uncovered the first known MIO-containing aminomutase, SgcC4. Comparative analysis of the KED and C-1027 biosynthetic gene clusters inspired the proposal for (R)-2-aza-3-chloro-beta-tyrosine biosynthesis starting from 2-aza-l-tyrosine, featuring KedY4 as a putative MIO-containing aminomutase. Here we report the biochemical characterization of KedY4, confirming its proposed role in KED biosynthesis. KedY4 is an MIO-containing aminomutase that stereospecifically catalyzes the conversion of 2-aza-l-tyrosine to (R)-2-aza-beta-tyrosine, exhibiting no detectable activity toward 2-aza-l-phenylalanine or l-tyrosine as an alternative substrate. In contrast, SgcC4, which stereospecifically catalyzes the conversion of l-tyrosine to (S)-beta-tyrosine in C-1027 biosynthesis, exhibits minimal activity with 2-aza-l-tyrosine as an alternative substrate but generating (S)-2-aza-beta-tyrosine, a product with the opposite stereochemistry of KedY4. This report of KedY4 broadens the scope of known substrates for the MIO-containing aminomutase family, and comparative studies of KedY4 and SgcC4 provide an outstanding opportunity to examine how MIO-containing aminomutases control substrate specificity and product enantioselectivity.

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PNAS Plus Significance Statements.

Publication Date: 2013 Apr 30 PMID: 23633557
Authors:
Journal: Proc Natl Acad Sci U S A

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Seeking sailors to help measure phytoplankton populations.

Publication Date: 2013 Apr 30 PMID: 23633556
Authors: Horne, R.
Journal: Proc Natl Acad Sci U S A

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Solving tough problems with games.

Publication Date: 2013 Apr 30 PMID: 23633555
Authors: Schrope, M.
Journal: Proc Natl Acad Sci U S A

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Toxic RNA as a driver of disease in a common form of ALS and dementia.

Publication Date: 2013 May 7 PMID: 23630297
Authors: Orr, H. T.
Journal: Proc Natl Acad Sci U S A

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miRNA regulation of BK polyomavirus replication during early infection.

Publication Date: 2013 May 14 PMID: 23630296
Authors: Broekema, N. M. - Imperiale, M. J.
Journal: Proc Natl Acad Sci U S A

Viral microRNAs (miRNAs) play an important role during infection by posttranscriptionally regulating both host and viral gene expression. However, the function of many viral miRNAs remains poorly understood. In this study, we investigated the role of the BK polyomavirus (BKPyV) miRNA in regulating virus replication. The function of the polyomavirus miRNA was investigated in archetype BKPyV, which is the transmissible form of the virus and thought to establish a persistent infection in the host urinary tract. In agreement with previous studies, we show that the BKPyV miRNA targets early mRNAs. Importantly, we show that the miRNA plays a significant role in limiting archetype BKPyV replication in a natural host cell model of infection. This regulation is accomplished through the balance of regulatory elements located within the noncoding control region that control early gene expression and miRNA expression before genome replication. We therefore provide evidence for a unique function of the polyomavirus miRNA that may have important implications for the mechanism of viral persistence.

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Provincialization of terrestrial faunas following the end-Permian mass extinction.

Publication Date: 2013 May 14 PMID: 23630295
Authors: Sidor, C. A. - Vilhena, D. A. - Angielczyk, K. D. - Huttenlocker, A. K. - Nesbitt, S. J. - Peecook, B. R. - Steyer, J. S. - Smith, R. M. - Tsuji, L. A.
Journal: Proc Natl Acad Sci U S A

In addition to their devastating effects on global biodiversity, mass extinctions have had a long-term influence on the history of life by eliminating dominant lineages that suppressed ecological change. Here, we test whether the end-Permian mass extinction (252.3 Ma) affected the distribution of tetrapod faunas within the southern hemisphere and apply quantitative methods to analyze four components of biogeographic structure: connectedness, clustering, range size, and endemism. For all four components, we detected increased provincialism between our Permian and Triassic datasets. In southern Pangea, a more homogeneous and broadly distributed fauna in the Late Permian (Wuchiapingian, approximately 257 Ma) was replaced by a provincial and biogeographically fragmented fauna by Middle Triassic times (Anisian, approximately 242 Ma). Importantly in the Triassic, lower latitude basins in Tanzania and Zambia included dinosaur predecessors and other archosaurs unknown elsewhere. The recognition of heterogeneous tetrapod communities in the Triassic implies that the end-Permian mass extinction afforded ecologically marginalized lineages the ecospace to diversify, and that biotic controls (i.e., evolutionary incumbency) were fundamentally reset. Archosaurs, which began diversifying in the Early Triassic, were likely beneficiaries of this ecological release and remained dominant for much of the later Mesozoic.

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Regulator of G protein signaling is a crucial modulator of antidepressant drug action in depression and neuropathic pain models.

Publication Date: 2013 May 14 PMID: 23630294
Authors: Stratinaki, M. - Varidaki, A. - Mitsi, V. - Ghose, S. - Magida, J. - Dias, C. - Russo, S. J. - Vialou, V. - Caldarone, B. J. - Tamminga, C. A. - Nestler, E. J. - Zachariou, V.
Journal: Proc Natl Acad Sci U S A

Regulator of G protein signaling 4 (Rgs4) is a signal transduction protein that controls the function of monoamine, opiate, muscarinic, and other G protein-coupled receptors via interactions with Galpha subunits. Rgs4 is expressed in several brain regions involved in mood, movement, cognition, and addiction and is regulated by psychotropic drugs, stress, and corticosteroids. In this study, we use genetic mouse models and viral-mediated gene transfer to examine the role of Rgs4 in the actions of antidepressant medications. We first analyzed human postmortem brain tissue and found robust up-regulation of RGS4 expression in the nucleus accumbens (NAc) of subjects receiving standard antidepressant medications that target monoamine systems. Behavioral studies of mice lacking Rgs4, including specific knockdowns in NAc, demonstrate that Rgs4 in this brain region acts as a positive modulator of the antidepressant-like and antiallodynic-like actions of several monoamine-directed antidepressant drugs, including tricyclic antidepressants, selective serotonin reuptake inhibitors, and norepinephrine reuptake inhibitors. Studies using viral-mediated increases in Rgs4 activity in NAc further support this hypothesis. Interestingly, in prefrontal cortex, Rgs4 acts as a negative modulator of the actions of nonmonoamine-directed drugs that are purported to act as antidepressants: the N-methyl-D-aspartate glutamate receptor antagonist ketamine and the delta opioid agonist (+)-4-[(alphaR)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzy l]-N,N-diethylbenzamide. Together, these data reveal a unique modulatory role of Rgs4 in the brain region-specific actions of a wide range of antidepressant drugs and indicate that pharmacological interventions at the level of RGS4 activity may enhance the actions of such drugs used for the treatment of depression and neuropathic pain.

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A model comparison approach shows stronger support for economic models of fertility decline.

Publication Date: 2013 May 14 PMID: 23630293
Authors: Shenk, M. K. - Towner, M. C. - Kress, H. C. - Alam, N.
Journal: Proc Natl Acad Sci U S A

The demographic transition is an ongoing global phenomenon in which high fertility and mortality rates are replaced by low fertility and mortality. Despite intense interest in the causes of the transition, especially with respect to decreasing fertility rates, the underlying mechanisms motivating it are still subject to much debate. The literature is crowded with competing theories, including causal models that emphasize (i) mortality and extrinsic risk, (ii) the economic costs and benefits of investing in self and children, and (iii) the cultural transmission of low-fertility social norms. Distinguishing between models, however, requires more comprehensive, better-controlled studies than have been published to date. We use detailed demographic data from recent fieldwork to determine which models produce the most robust explanation of the rapid, recent demographic transition in rural Bangladesh. To rigorously compare models, we use an evidence-based statistical approach using model selection techniques derived from likelihood theory. This approach allows us to quantify the relative evidence the data give to alternative models, even when model predictions are not mutually exclusive. Results indicate that fertility, measured as either total fertility or surviving children, is best explained by models emphasizing economic factors and related motivations for parental investment. Our results also suggest important synergies between models, implicating multiple causal pathways in the rapidity and degree of recent demographic transitions.

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Genetic deletion of Rnd3 results in aqueductal stenosis leading to hydrocephalus through up-regulation of Notch signaling.

Publication Date: 2013 May 14 PMID: 23630292
Authors: Lin, X. - Liu, B. - Yang, X. - Yue, X. - Diao, L. - Wang, J. - Chang, J.
Journal: Proc Natl Acad Sci U S A

Rho family guanosine triphosphatase (GTPase) 3 (Rnd3), a member of the small Rho GTPase family, is involved in the regulation of cell actin cytoskeleton dynamics, cell migration, and proliferation through the Rho kinase-dependent signaling pathway. We report a role of Rnd3 in the pathogenesis of hydrocephalus disorder. Mice with Rnd3 genetic deletion developed severe obstructive hydrocephalus with enlargement of the lateral and third ventricles, but not of the fourth ventricles. The cerebral aqueducts in Rnd3-null mice were partially or completely blocked by the overgrowth of ependymal epithelia. We examined the molecular mechanism contributing to this Rnd3-deficiency-mediated hydrocephalus and found that Rnd3 is a regulator of Notch signaling. Rnd3 deficiency, through either gene deletion or siRNA knockdown, resulted in a decrease in Notch intracellular domain (NICD) protein degradation. However, there was no correlated change in mRNA change, which in turn led to an increase in NICD protein levels. Immunoprecipitation analysis demonstrated that Rnd3 and NICD physically interacted, and that down-regulation of Rnd3 attenuated NICD protein ubiquitination. This eventually enhanced Notch signaling activity and promoted aberrant growth of aqueduct ependymal cells, resulting in aqueduct stenosis and the development of congenital hydrocephalus. Inhibition of Notch activity rescued the hydrocephalus disorder in the mutant animals.

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Blood meal-induced changes to antennal transcriptome profiles reveal shifts in odor sensitivities in Anopheles gambiae.

Publication Date: 2013 May 14 PMID: 23630291
Authors: Rinker, D. C. - Pitts, R. J. - Zhou, X. - Suh, E. - Rokas, A. - Zwiebel, L. J.
Journal: Proc Natl Acad Sci U S A

Olfactory-driven behaviors are central to the lifecycle of the malaria vector mosquito Anopheles gambiae and are initiated by peripheral signaling in the antenna and other olfactory tissues. To continue gaining insight into the relationship between gene expression and olfaction, we have performed cohort comparisons of antennal transcript abundances at five time points after a blood meal, a key event in both reproduction and disease transmission cycles. We found that more than 5,000 transcripts displayed significant abundance differences, many of which were correlated by cluster analysis. Within the chemosensory gene families, we observed a general reduction in the level of chemosensory gene transcripts, although a subset of odorant receptors (AgOrs) was modestly enhanced in post-blood-fed samples. Integration of AgOr transcript abundance data with previously characterized AgOr excitatory odorant response profiles revealed potential changes in antennal odorant receptivity that coincided with the shift from host-seeking to oviposition behaviors in blood-fed female mosquitoes. Behavioral testing of ovipositing females to odorants highlighted by this synthetic analysis identified two unique, unitary oviposition cues for An. gambiae, 2-propylphenol and 4-methylcyclohexanol. We posit that modest, yet cumulative, alterations of AgOr transcript levels modulate peripheral odor coding resulting in biologically relevant behavioral effects. Moreover, these results demonstrate that highly quantitative, RNAseq transcript abundance data can be successfully integrated with functional data to generate testable hypotheses.

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In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras-effector interaction.

Publication Date: 2013 May 14 PMID: 23630290
Authors: Shima, F. - Yoshikawa, Y. - Ye, M. - Araki, M. - Matsumoto, S. - Liao, J. - Hu, L. - Sugimoto, T. - Ijiri, Y. - Takeda, A. - Nishiyama, Y. - Sato, C. - Muraoka, S. - Tamura, A. - Osoda, T. - Tsuda, K. - Miyakawa, T. - Fukunishi, H. - Shimada, J. - Kumasaka, T. - Yamamoto, M. - Kataoka, T.
Journal: Proc Natl Acad Sci U S A

Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of RasGTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-RasGTP carrying an H-Ras-type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-RasGTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H-ras(G12V)-transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras(G12V) gene by oral administration. The NMR structure of a complex of the compound with H-RasGTP(T35S), exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple RasGTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target RasGTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.

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Subjective costs drive overly patient foraging strategies in rats on an intertemporal foraging task.

Publication Date: 2013 May 14 PMID: 23630289
Authors: Wikenheiser, A. M. - Stephens, D. W. - Redish, A. D.
Journal: Proc Natl Acad Sci U S A

Laboratory studies of decision making often take the form of two-alternative, forced-choice paradigms. In natural settings, however, many decision problems arise as stay/go choices. We designed a foraging task to test intertemporal decision making in rats via stay/go decisions. Subjects did not follow the rate-maximizing strategy of choosing only food items associated with short delays. Instead, rats were often willing to wait for surprisingly long periods, and consequently earned a lower rate of food intake than they might have by ignoring long-delay options. We tested whether foraging theory or delay discounting models predicted the behavior we observed but found that these models could not account for the strategies subjects selected. Subjects' behavior was well accounted for by a model that incorporated a cost for rejecting potential food items. Interestingly, subjects' cost sensitivity was proportional to environmental richness. These findings are at odds with traditional normative accounts of decision making but are consistent with retrospective considerations having a deleterious influence on decisions (as in the "sunk-cost" effect). More broadly, these findings highlight the utility of complementing existing assays of decision making with tasks that mimic more natural decision topologies.

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Obesity-driven synaptic remodeling affects endocannabinoid control of orexinergic neurons.

Publication Date: 2013 Apr 29 PMID: 23630288
Authors: Cristino, L. - Busetto, G. - Imperatore, R. - Ferrandino, I. - Palomba, L. - Silvestri, C. - Petrosino, S. - Orlando, P. - Bentivoglio, M. - Mackie, K. - Di Marzo, V.
Journal: Proc Natl Acad Sci U S A

Acute or chronic alterations in energy status alter the balance between excitatory and inhibitory synaptic transmission and associated synaptic plasticity to allow for the adaptation of energy metabolism to new homeostatic requirements. The impact of such changes on endocannabinoid and cannabinoid receptor type 1 (CB1)-mediated modulation of synaptic transmission and strength is not known, despite the fact that this signaling system is an important target for the development of new drugs against obesity. We investigated whether CB1-expressing excitatory vs. inhibitory inputs to orexin-A-containing neurons in the lateral hypothalamus are altered in obesity and how this modifies endocannabinoid control of these neurons. In lean mice, these inputs are mostly excitatory. By confocal and ultrastructural microscopic analyses, we observed that in leptin-knockout (ob/ob) obese mice, and in mice with diet-induced obesity, orexinergic neurons receive predominantly inhibitory CB1-expressing inputs and overexpress the biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol, which retrogradely inhibits synaptic transmission at CB1-expressing axon terminals. Patch-clamp recordings also showed increased CB1-sensitive inhibitory innervation of orexinergic neurons in ob/ob mice. These alterations are reversed by leptin administration, partly through activation of the mammalian target of rapamycin pathway in neuropeptide-Y-ergic neurons of the arcuate nucleus, and are accompanied by CB1-mediated enhancement of orexinergic innervation of target brain areas. We propose that enhanced inhibitory control of orexin-A neurons, and their CB1-mediated disinhibition, are a consequence of leptin signaling impairment in the arcuate nucleus. We also provide initial evidence of the participation of this phenomenon in hyperphagia and hormonal dysregulation in obesity.

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Evidence of a liquid-liquid phase transition in hot dense hydrogen.

Publication Date: 2013 May 14 PMID: 23630287
Authors: Dzyabura, V. - Zaghoo, M. - Silvera, I. F.
Journal: Proc Natl Acad Sci U S A

We use pulsed-laser heating of hydrogen at static pressures in the megabar pressure region to search for the plasma phase transition to liquid atomic metallic hydrogen. We heat our samples substantially above the melting line and observe a plateau in a temperature vs. laser power curve that otherwise increases with power. This anomaly in the heating curve appears correlated with theoretical predictions for the plasma phase transition.

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Neural predictors of individual differences in response to math tutoring in primary-grade school children.

Publication Date: 2013 May 14 PMID: 23630286
Authors: Supekar, K. - Swigart, A. G. - Tenison, C. - Jolles, D. D. - Rosenberg-Lee, M. - Fuchs, L. - Menon, V.
Journal: Proc Natl Acad Sci U S A

Now, more than ever, the ability to acquire mathematical skills efficiently is critical for academic and professional success, yet little is known about the behavioral and neural mechanisms that drive some children to acquire these skills faster than others. Here we investigate the behavioral and neural predictors of individual differences in arithmetic skill acquisition in response to 8-wk of one-to-one math tutoring. Twenty-four children in grade 3 (ages 8-9 y), a critical period for acquisition of basic mathematical skills, underwent structural and resting-state functional MRI scans pretutoring. A significant shift in arithmetic problem-solving strategies from counting to fact retrieval was observed with tutoring. Notably, the speed and accuracy of arithmetic problem solving increased with tutoring, with some children improving significantly more than others. Next, we examined whether pretutoring behavioral and brain measures could predict individual differences in arithmetic performance improvements with tutoring. No behavioral measures, including intelligence quotient, working memory, or mathematical abilities, predicted performance improvements. In contrast, pretutoring hippocampal volume predicted performance improvements. Furthermore, pretutoring intrinsic functional connectivity of the hippocampus with dorsolateral and ventrolateral prefrontal cortices and the basal ganglia also predicted performance improvements. Our findings provide evidence that individual differences in morphometry and connectivity of brain regions associated with learning and memory, and not regions typically involved in arithmetic processing, are strong predictors of responsiveness to math tutoring in children. More generally, our study suggests that quantitative measures of brain structure and intrinsic brain organization can provide a more sensitive marker of skill acquisition than behavioral measures.

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Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3.

Publication Date: 2013 May 14 PMID: 23630285
Authors: Demir, F. - Horntrich, C. - Blachutzik, J. O. - Scherzer, S. - Reinders, Y. - Kierszniowska, S. - Schulze, W. X. - Harms, G. S. - Hedrich, R. - Geiger, D. - Kreuzer, I.
Journal: Proc Natl Acad Sci U S A

The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin Arabidopsis thaliana remorin 1.3 (AtRem 1.3). After transient coexpression, CPK21 interacted with SLAH3 [slow anion channel 1 (SLAC1) homolog 3] and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9 [regulatory components of ABA receptor 1/PYR1 (pyrabactin resistance 1)-like protein 9]. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21.

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Intermediate intrinsic diversity enhances neural population coding.

Publication Date: 2013 May 14 PMID: 23630284
Authors: Tripathy, S. J. - Padmanabhan, K. - Gerkin, R. C. - Urban, N. N.
Journal: Proc Natl Acad Sci U S A

Cell-to-cell variability in molecular, genetic, and physiological features is increasingly recognized as a critical feature of complex biological systems, including the brain. Although such variability has potential advantages in robustness and reliability, how and why biological circuits assemble heterogeneous cells into functional groups is poorly understood. Here, we develop analytic approaches toward answering how neuron-level variation in intrinsic biophysical properties of olfactory bulb mitral cells influences population coding of fluctuating stimuli. We capture the intrinsic diversity of recorded populations of neurons through a statistical approach based on generalized linear models. These models are flexible enough to predict the diverse responses of individual neurons yet provide a common reference frame for comparing one neuron to the next. We then use Bayesian stimulus decoding to ask how effectively different populations of mitral cells, varying in their diversity, encode a common stimulus. We show that a key advantage provided by physiological levels of intrinsic diversity is more efficient and more robust encoding of stimuli by the population as a whole. However, we find that the populations that best encode stimulus features are not simply the most heterogeneous, but those that balance diversity with the benefits of neural similarity.

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Lipid phosphatases identified by screening a mouse phosphatase shRNA library regulate T-cell differentiation and Protein kinase B AKT signaling.

Publication Date: 2013 May 14 PMID: 23630283
Authors: Guo, L. - Martens, C. - Bruno, D. - Porcella, S. F. - Yamane, H. - Caucheteux, S. M. - Zhu, J. - Paul, W. E.
Journal: Proc Natl Acad Sci U S A

Screening a complete mouse phosphatase lentiviral shRNA library using high-throughput sequencing revealed several phosphatases that regulate CD4 T-cell differentiation. We concentrated on two lipid phosphatases, the myotubularin-related protein (MTMR)9 and -7. Silencing MTMR9 by shRNA or siRNA resulted in enhanced T-helper (Th)1 differentiation and increased Th1 protein kinase B (PKB)/AKT phosphorylation while silencing MTMR7 caused increased Th2 and Th17 differentiation and increased AKT phosphorylation in these cells. Irradiated mice reconstituted with MTMR9 shRNA-transduced bone marrow cells had an elevated proportion of T-box transcription factor T-bet expressors among their CD4 T cells. After adoptive transfer of naive cells from such reconstituted mice, immunization resulted in a greater proportion of T-box transcription factor T-bet-expressing cells. Thus, myotubularin-related proteins have a role in controlling in vitro and in vivo Th-cell differentiation, possibly through regulation of phosphatidylinositol [3,4,5]-trisphosphate activity.

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Central role of liver in anticancer and radioprotective activities of Toll-like receptor 5 agonist.

Publication Date: 2013 May 14 PMID: 23630282
Authors: Burdelya, L. G. - Brackett, C. M. - Kojouharov, B. - Gitlin, I. I. - Leonova, K. I. - Gleiberman, A. S. - Aygun-Sunar, S. - Veith, J. - Johnson, C. - Haderski, G. J. - Stanhope-Baker, P. - Allamaneni, S. - Skitzki, J. - Zeng, M. - Martsen, E. - Medvedev, A. - Scheblyakov, D. - Artemicheva, N. M. - Logunov, D. Y. - Gintsburg, A. L. - Naroditsky, B. S. - Makarov, S. S. - Gudkov, A. V.
Journal: Proc Natl Acad Sci U S A

Vertebrate Toll-like receptor 5 (TLR5) recognizes bacterial flagellin proteins and activates innate immune responses to motile bacteria. In addition, activation of TLR5 signaling can inhibit growth of TLR5-expressing tumors and protect normal tissues from radiation and ischemia-reperfusion injuries. To understand the mechanisms behind these phenomena at the organismal level, we assessed nuclear factor kappa B (NF-kappaB) activation (indicative of TLR5 signaling) in tissues and cells of mice treated with CBLB502, a pharmacologically optimized flagellin derivative. This identified the liver and gastrointestinal tract as primary CBLB502 target organs. In particular, liver hepatocytes were the main cell type directly and specifically responding to systemic administration of CBLB502 but not to that of the TLR4 agonist LPS. To assess CBLB502 impact on other pathways, we created multireporter mice with hepatocytes transduced in vivo with reporters for 46 inducible transcription factor families and found that along with NF-kappaB, CBLB502 strongly activated STAT3-, phenobarbital-responsive enhancer module (PREM), and activator protein 1 (AP-1-) -driven pathways. Livers of CBLB502-treated mice displayed induction of numerous immunomodulatory factors and massive recruitment of various types of immune cells. This led to inhibition of growth of liver metastases of multiple tumors regardless of their TLR5 status. The changed liver microenvironment was not, however, hepatotoxic, because CBLB502 induced resistance to Fas-mediated apoptosis in normal liver cells. Temporary occlusion of liver blood circulation prevented CBLB502 from protecting hematopoietic progenitors in lethally irradiated mice, indicating involvement of a factor secreted by responding liver cells. These results define the liver as the key mediator of TLR5-dependent effects in vivo and suggest clinical applications for TLR5 agonists as hepatoprotective and antimetastatic agents.

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Aptamer to ErbB-2/HER2 enhances degradation of the target and inhibits tumorigenic growth.

Publication Date: 2013 May 14 PMID: 23630281
Authors: Mahlknecht, G. - Maron, R. - Mancini, M. - Schechter, B. - Sela, M. - Yarden, Y.
Journal: Proc Natl Acad Sci U S A

Aptamers, oligonucleotides able to avidly bind cellular targets, are emerging as promising therapeutic agents, analogous to monoclonal antibodies. We selected from a DNA library an aptamer specifically recognizing human epidermal growth factor receptor 2 (ErbB-2/HER2), a receptor tyrosine kinase, which is overexpressed in a variety of human cancers, including breast and gastric tumors. Treatment of human gastric cancer cells with a trimeric version (42 nucleotides) of the selected aptamer (14 nucleotides) resulted in reduced cell growth in vitro, but a monomeric version was ineffective. Likewise, when treated with the trimeric aptamer, animals bearing tumor xenografts of human gastric origin reflected reduced rates of tumor growth. The antitumor effect of the aptamer was nearly twofold stronger than that of a monoclonal anti-ErbB-2/HER2 antibody. Consistent with aptamer-induced intracellular degradation of ErbB-2/HER2, incubation of gastric cancer cells with the trimeric aptamer promoted translocation of ErbB-2/HER2 from the cell surface to cytoplasmic puncta. This translocation was associated with a lysosomal hydrolase-dependent clearance of the ErbB-2/HER2 protein from cell extracts. We conclude that targeting ErbB-2/HER2 with DNA aptamers might retard the tumorigenic growth of gastric cancer by means of accelerating lysosomal degradation of the oncoprotein. This work exemplifies the potential pharmacological utility of aptamers directed at cell surface proteins, and it highlights an endocytosis-mediated mechanism of tumor inhibition.

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Escalation of polymerization in a thermal gradient.

Publication Date: 2013 May 14 PMID: 23630280
Authors: Mast, C. B. - Schink, S. - Gerland, U. - Braun, D.
Journal: Proc Natl Acad Sci U S A

For the emergence of early life, the formation of biopolymers such as RNA is essential. However, the addition of nucleotide monomers to existing oligonucleotides requires millimolar concentrations. Even in such optimistic settings, no polymerization of RNA longer than about 20 bases could be demonstrated. How then could self-replicating ribozymes appear, for which recent experiments suggest a minimal length of 200 nt? Here, we demonstrate a mechanism to bridge this gap: the escalated polymerization of nucleotides by a spatially confined thermal gradient. The gradient accumulates monomers by thermophoresis and convection while retaining longer polymers exponentially better. Polymerization and accumulation become mutually self-enhancing and result in a hyperexponential escalation of polymer length. We describe this escalation theoretically under the conservative assumption of reversible polymerization. Taking into account the separately measured thermophoretic properties of RNA, we extrapolate the results for primordial RNA polymerization inside a temperature gradient in pores or fissures of rocks. With a dilute, nanomolar concentration of monomers the model predicts that a pore length of 5 cm and a temperature difference of 10 K suffice to polymerize 200-mers of RNA in micromolar concentrations. The probability to generate these long RNAs is raised by a factor of >10(600) compared with polymerization in a physical equilibrium. We experimentally validate the theory with the reversible polymerization of DNA blocks in a laser-driven thermal trap. The results confirm that a thermal gradient can significantly enlarge the available sequence space for the emergence of catalytically active polymers.

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AMPA receptor exchange underlies transient memory destabilization on retrieval.

Publication Date: 2013 May 14 PMID: 23630279
Authors: Hong, I. - Kim, J. - Kim, J. - Lee, S. - Ko, H. G. - Nader, K. - Kaang, B. K. - Tsien, R. W. - Choi, S.
Journal: Proc Natl Acad Sci U S A

A consolidated memory can be transiently destabilized by memory retrieval, after which memories are reconsolidated within a few hours; however, the molecular substrates underlying this destabilization process remain essentially unknown. Here we show that at lateral amygdala synapses, fear memory consolidation correlates with increased surface expression of calcium-impermeable AMPA receptors (CI-AMPARs), which are known to be more stable at the synapse, whereas memory retrieval induces an abrupt exchange of CI-AMPARs to calcium-permeable AMPARs (CP-AMPARs), which are known to be less stable at the synapse. We found that blockade of either CI-AMPAR endocytosis or NMDA receptor activity during memory retrieval, both of which blocked the exchange to CP-AMPARs, prevented memory destabilization, indicating that this transient exchange of AMPARs may underlie the transformation of a stable memory into an unstable memory. These newly inserted CP-AMPARs gradually exchanged back to CI-AMPARs within hours, which coincided with the course of reconsolidation. Furthermore, blocking the activity of these newly inserted CP-AMPARs after retrieval impaired reconsolidation, suggesting that they serve as synaptic "tags" that support synapse-specific reconsolidation. Taken together, our results reveal unexpected physiological roles of CI-AMPARs and CP-AMPARs in transforming a consolidated memory into an unstable memory and subsequently guiding reconsolidation.

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An essential role for gamma-herpesvirus latency-associated nuclear antigen homolog in an acute lymphoproliferative disease of cattle.

Publication Date: 2013 May 21 PMID: 23630278
Authors: Palmeira, L. - Sorel, O. - Van Campe, W. - Boudry, C. - Roels, S. - Myster, F. - Reschner, A. - Coulie, P. G. - Kerkhofs, P. - Vanderplasschen, A. - Dewals, B. G.
Journal: Proc Natl Acad Sci U S A

Wildebeests carry asymptomatically alcelaphine herpesvirus 1 (AlHV-1), a gamma-herpesvirus inducing malignant catarrhal fever (MCF) to several ruminant species (including cattle). This acute and lethal lymphoproliferative disease occurs after a prolonged asymptomatic incubation period after transmission. Our recent findings with the rabbit model indicated that AlHV-1 infection is not productive during MCF. Here, we investigated whether latency establishment could explain this apparent absence of productive infection and sought to determine its role in MCF pathogenesis. First, whole-genome cellular and viral gene expression analyses were performed in lymph nodes of MCF-developing calves. Whereas a severe disruption in cellular genes was observed, only 10% of the entire AlHV-1 genome was expressed, contrasting with the 45% observed during productive infection in vitro. In vivo, the expressed viral genes included the latency-associated nuclear antigen homolog ORF73 but none of the regions known to be essential for productive infection. Next, genomic conformation analyses revealed that AlHV-1 was essentially episomal, further suggesting that MCF might be the consequence of a latent infection rather than abortive lytic infection. This hypothesis was further supported by the high frequencies of infected CD8(+) T cells during MCF using immunodetection of ORF73 protein and single-cell RT-PCR approaches. Finally, the role of latency-associated ORF73 was addressed. A lack of ORF73 did not impair initial virus replication in vivo, but it rendered AlHV-1 unable to induce MCF and persist in vivo and conferred protection against a lethal challenge with a WT virus. Together, these findings suggest that a latent infection is essential for MCF induction.

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Self-cleaning of superhydrophobic surfaces by self-propelled jumping condensate.

Publication Date: 2013 May 14 PMID: 23630277
Authors: Wisdom, K. M. - Watson, J. A. - Qu, X. - Liu, F. - Watson, G. S. - Chen, C. H.
Journal: Proc Natl Acad Sci U S A

The self-cleaning function of superhydrophobic surfaces is conventionally attributed to the removal of contaminating particles by impacting or rolling water droplets, which implies the action of external forces such as gravity. Here, we demonstrate a unique self-cleaning mechanism whereby the contaminated superhydrophobic surface is exposed to condensing water vapor, and the contaminants are autonomously removed by the self-propelled jumping motion of the resulting liquid condensate, which partially covers or fully encloses the contaminating particles. The jumping motion off the superhydrophobic surface is powered by the surface energy released upon coalescence of the condensed water phase around the contaminants. The jumping-condensate mechanism is shown to spontaneously clean superhydrophobic cicada wings, where the contaminating particles cannot be removed by gravity, wing vibration, or wind flow. Our findings offer insights for the development of self-cleaning materials.

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Loss of function of Ribonuclease T2, an ancient and phylogenetically conserved RNase, plays a crucial role in ovarian tumorigenesis.

Publication Date: 2013 May 14 PMID: 23630276
Authors: Acquati, F. - Lualdi, M. - Bertilaccio, S. - Monti, L. - Turconi, G. - Fabbri, M. - Grimaldi, A. - Anselmo, A. - Inforzato, A. - Collotta, A. - Cimetti, L. - Riva, C. - Gribaldo, L. - Ghia, P. - Taramelli, R.
Journal: Proc Natl Acad Sci U S A

In recent years, the role played by the stromal microenvironment has been given growing attention in order to achieve a full understanding of cancer initiation and progression. Because cancer is a tissue-based disease, the integrity of tissue architecture is a major constraint toward cancer growth. Indeed, a large contribution of the natural resistance to cancer stems from stromal microenvironment components, the dysregulation of which can facilitate cancer occurrence. For instance, recent experimental evidence has highlighted the involvement of stromal cells in ovarian carcinogenesis, as epitomized by ovarian xenografts obtained by a double KO of the murine Dicer and Pten genes. Likewise, we reported the role of an ancient extracellular RNase, called Ribonuclease T2 (RNASET2), within the ovarian stromal microenvironment. Indeed, hyperexpression of RNASET2 is able to control tumorigenesis by recruiting macrophages (mostly of the anticancer M1 subtype) at the tumor sites. We present biological data obtained by RNASET2 silencing in the poorly tumorigenetic and highly RNASET2-expressing human OVCAR3 cell line. RNASET2 knockdown was shown to stimulate in vivo tumor growth early after microinjection of OVCAR3 cells in nude mice. Moreover, we have investigated by molecular profiling the in vivo expression signature of human and mouse cell xenografts and disclosed the activation of pathways related to activation of the innate immune response and modulation of ECM components. Finally, we provide evidence for a role of RNASET2 in triggering an in vitro chemotactic response in macrophages. These results further highlight the critical role played by the microenvironment in RNASET2-mediated ovarian tumor suppression, which could eventually contribute to better clarify the pathogenesis of this disease.

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Steroidogenic enzyme Cyp11a1 regulates Type 2 CD8+ T cell skewing in allergic lung disease.

Publication Date: 2013 May 14 PMID: 23630275
Authors: Jia, Y. - Domenico, J. - Takeda, K. - Han, J. - Wang, M. - Armstrong, M. - Reisdorph, N. - O'Connor, B. P. - Lucas, J. J. - Gelfand, E. W.
Journal: Proc Natl Acad Sci U S A

Allergic asthma is a heterogeneous inflammatory disorder of the airways characterized by chronic airway inflammation and airway hyperresponsiveness. Numbers of CD8(+)IL-13(+) T cells are increased in asthmatics and during the development of experimental asthma in mice. In an atopic environment rich in IL-4, these CD8(+) T cells mediate asthmatic responses, but the mechanisms regulating the conversion of CD8(+) effector T cells from IFN-gamma- to pathogenic IL-13-producing effector cells that contribute to an asthma phenotype have not been defined. Here, we show that cholesterol side-chain cleavage P450 enzyme, Cyp11a1, is a key regulator of CD8(+) T-cell conversion. Expression of the gene, protein, and enzymatic activity of Cyp11a1 were markedly increased in CD8(+) T cells differentiated in the presence of IL-2 plus IL-4 compared with cells differentiated in IL-2 alone. Inhibition of Cyp11a1 enzymatic activity with aminoglutethimide or reduction in the expression of Cyp11a1 using short hairpin RNA prevented the IL-4-induced conversion of IFN-gamma- to IL-13-producing cells without affecting expression of the lineage-specific transcription factors T-box expressed in T cells (T-bet) or GATA binding protein 3 (GATA3). Adoptive transfer of aminoglutethimide-treated CD8(+) T cells into sensitized and challenged CD8-deficient recipients failed to restore airway hyperresponsiveness and inflammation. We demonstrate that Cyp11a1 controls the phenotypic conversion of CD8(+) T cells from IFN-gamma to IL-13 production, linking steroidogenesis in CD8(+) T cells, a nonclassical steroidogenic tissue, to a proallergic differentiation pathway.

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Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state.

Publication Date: 2013 Apr 29 PMID: 23630274
Authors: Fagan, C. E. - Dunkle, J. A. - Maehigashi, T. - Dang, M. N. - Devaraj, A. - Miles, S. J. - Qin, D. - Fredrick, K. - Dunham, C. M.
Journal: Proc Natl Acad Sci U S A

After four decades of research aimed at understanding tRNA selection on the ribosome, the mechanism by which ribosomal ambiguity (ram) mutations promote miscoding remains unclear. Here, we present two X-ray crystal structures of the Thermus thermophilus 70S ribosome containing 16S rRNA ram mutations, G347U and G299A. Each of these mutations causes miscoding in vivo and stimulates elongation factor thermo unstable (EF-Tu)-dependent GTP hydrolysis in vitro. Mutation G299A is located near the interface of ribosomal proteins S4 and S5 on the solvent side of the subunit, whereas G347U is located 77 A distant, at intersubunit bridge B8, close to where EF-Tu engages the ribosome. Despite these disparate locations, both mutations induce almost identical structural rearrangements that disrupt the B8 bridge-namely, the interaction of h8/h14 with L14 and L19. This conformation most closely resembles that seen upon EF-TuGTPaminoacyl-tRNA binding to the 70S ribosome. These data provide evidence that disruption and/or distortion of B8 is an important aspect of GTPase activation. We propose that, by destabilizing B8, G299A and G347U reduce the energetic cost of attaining the GTPase-activated state and thereby decrease the stringency of decoding. This previously unappreciated role for B8 in controlling the decoding process may hold relevance for many other ribosomal mutations known to influence translational fidelity.

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Single-molecule DNA repair in live bacteria.

Publication Date: 2013 May 14 PMID: 23630273
Authors: Uphoff, S. - Reyes-Lamothe, R. - Garza de Leon, F. - Sherratt, D. J. - Kapanidis, A. N.
Journal: Proc Natl Acad Sci U S A

Cellular DNA damage is reversed by balanced repair pathways that avoid accumulation of toxic intermediates. Despite their importance, the organization of DNA repair pathways and the function of repair enzymes in vivo have remained unclear because of the inability to directly observe individual reactions in living cells. Here, we used photoactivation, localization, and tracking in live Escherichia coli to directly visualize single fluorescent labeled DNA polymerase I (Pol) and ligase (Lig) molecules searching for DNA gaps and nicks, performing transient reactions, and releasing their products. Our general approach provides enzymatic rates and copy numbers, substrate-search times, diffusion characteristics, and the spatial distribution of reaction sites, at the single-cell level, all in one measurement. Single repair events last 2.1 s (Pol) and 2.5 s (Lig), respectively. Pol and Lig activities increased fivefold over the basal level within minutes of DNA methylation damage; their rates were limited by upstream base excision repair pathway steps. Pol and Lig spent >80% of their time searching for free substrates, thereby minimizing both the number and lifetime of toxic repair intermediates. We integrated these single-molecule observations to generate a quantitative, systems-level description of a model repair pathway in vivo.

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Childhood maltreatment is associated with distinct genomic and epigenetic profiles in posttraumatic stress disorder.

Publication Date: 2013 May 14 PMID: 23630272
Authors: Mehta, D. - Klengel, T. - Conneely, K. N. - Smith, A. K. - Altmann, A. - Pace, T. W. - Rex-Haffner, M. - Loeschner, A. - Gonik, M. - Mercer, K. B. - Bradley, B. - Muller-Myhsok, B. - Ressler, K. J. - Binder, E. B.
Journal: Proc Natl Acad Sci U S A

Childhood maltreatment is likely to influence fundamental biological processes and engrave long-lasting epigenetic marks, leading to adverse health outcomes in adulthood. We aimed to elucidate the impact of different early environment on disease-related genome-wide gene expression and DNA methylation in peripheral blood cells in patients with posttraumatic stress disorder (PTSD). Compared with the same trauma-exposed controls (n = 108), gene-expression profiles of PTSD patients with similar clinical symptoms and matched adult trauma exposure but different childhood adverse events (n = 32 and 29) were almost completely nonoverlapping (98%). These differences on the level of individual transcripts were paralleled by the enrichment of several distinct biological networks between the groups. Moreover, these gene-expression changes were accompanied and likely mediated by changes in DNA methylation in the same loci to a much larger proportion in the childhood abuse (69%) vs. the non-child abuse-only group (34%). This study is unique in providing genome-wide evidence of distinct biological modifications in PTSD in the presence or absence of exposure to childhood abuse. The findings that nonoverlapping biological pathways seem to be affected in the two PTSD groups and that changes in DNA methylation appear to have a much greater impact in the childhood-abuse group might reflect differences in the pathophysiology of PTSD, in dependence of exposure to childhood maltreatment. These results contribute to a better understanding of the extent of influence of differences in trauma exposure on pathophysiological processes in stress-related psychiatric disorders and may have implications for personalized medicine.

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Used planet: A global history.

Publication Date: 2013 May 14 PMID: 23630271
Authors: Ellis, E. C. - Kaplan, J. O. - Fuller, D. Q. - Vavrus, S. - Klein Goldewijk, K. - Verburg, P. H.
Journal: Proc Natl Acad Sci U S A

Human use of land has transformed ecosystem pattern and process across most of the terrestrial biosphere, a global change often described as historically recent and potentially catastrophic for both humanity and the biosphere. Interdisciplinary paleoecological, archaeological, and historical studies challenge this view, indicating that land use has been extensive and sustained for millennia in some regions and that recent trends may represent as much a recovery as an acceleration. Here we synthesize recent scientific evidence and theory on the emergence, history, and future of land use as a process transforming the Earth System and use this to explain why relatively small human populations likely caused widespread and profound ecological changes more than 3,000 y ago, whereas the largest and wealthiest human populations in history are using less arable land per person every decade. Contrasting two spatially explicit global reconstructions of land-use history shows that reconstructions incorporating adaptive changes in land-use systems over time, including land-use intensification, offer a more spatially detailed and plausible assessment of our planet's history, with a biosphere and perhaps even climate long ago affected by humans. Although land-use processes are now shifting rapidly from historical patterns in both type and scale, integrative global land-use models that incorporate dynamic adaptations in human-environment relationships help to advance our understanding of both past and future land-use changes, including their sustainability and potential global effects.

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A tribute to C. Everett Koop.

Publication Date: 2013 Apr 30 PMID: 23630270
Authors: Kessler, D. A. - Nesbit, J. A. - Westmoreland, T. M. - Albright, M. B.
Journal: Proc Natl Acad Sci U S A

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Ash from the Toba supereruption in Lake Malawi shows no volcanic winter in East Africa at 75 ka.

Publication Date: 2013 May 14 PMID: 23630269
Authors: Lane, C. S. - Chorn, B. T. - Johnson, T. C.
Journal: Proc Natl Acad Sci U S A

The most explosive volcanic event of the Quaternary was the eruption of Mt. Toba, Sumatra, 75,000 y ago, which produced voluminous ash deposits found across much of the Indian Ocean, Indian Peninsula, and South China Sea. A major climatic downturn observed within the Greenland ice cores has been attributed to the cooling effects of the ash and aerosols ejected during the eruption of the Youngest Toba Tuff (YTT). These events coincided roughly with a hypothesized human genetic bottleneck, when the number of our species in Africa may have been reduced to near extinction. Some have speculated that the demise of early modern humans at that time was due in part to a dramatic climate shift triggered by the supereruption. Others have argued that environmental conditions would not have been so severe to have such an impact on our ancestors, and furthermore, that modern humans may have already expanded beyond Africa by this time. We report an observation of the YTT in Africa, recovered as a cryptotephra layer in Lake Malawi sediments, >7,000 km west of the source volcano. The YTT isochron provides an accurate and precise age estimate for the Lake Malawi paleoclimate record, which revises the chronology of past climatic events in East Africa. The YTT in Lake Malawi is not accompanied by a major change in sediment composition or evidence for substantial temperature change, implying that the eruption did not significantly impact the climate of East Africa and was not the cause of a human genetic bottleneck at that time.

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Synaptopodin regulates denervation-induced homeostatic synaptic plasticity.

Publication Date: 2013 May 14 PMID: 23630268
Authors: Vlachos, A. - Ikenberg, B. - Lenz, M. - Becker, D. - Reifenberg, K. - Bas-Orth, C. - Deller, T.
Journal: Proc Natl Acad Sci U S A

Synaptopodin (SP) is a marker and essential component of the spine apparatus (SA), an enigmatic cellular organelle composed of stacked smooth endoplasmic reticulum that has been linked to synaptic plasticity. However, SP/SA-mediated synaptic plasticity remains incompletely understood. To study the role of SP/SA in homeostatic synaptic plasticity we here used denervation-induced synaptic scaling of mouse dentate granule cells as a model system. This form of plasticity is of considerable interest in the context of neurological diseases that are associated with the loss of neurons and subsequent denervation of connected brain regions. In entorhino-hippocampal slice cultures prepared from SP-deficient mice, which lack the SA, a compensatory increase in excitatory synaptic strength was not observed following partial deafferentation. In line with this finding, prolonged blockade of sodium channels with tetrodotoxin induced homeostatic synaptic scaling in wild-type, but not SP-deficient, slice cultures. By crossing SP-deficient mice with a newly generated transgenic mouse strain that expresses GFP-tagged SP under the control of the Thy1.2 promoter, the ability of dentate granule cells to form the SA and to homeostatically strengthen excitatory synapses was rescued. Interestingly, homeostatic synaptic strengthening was accompanied by a compensatory increase in SP cluster size/stability and SA stack number, suggesting that activity-dependent SP/SA remodeling could be part of a negative feedback mechanism that aims at adjusting the strength of excitatory synapses to persisting changes in network activity. Thus, our results disclose an important role for SP/SA in homeostatic synaptic plasticity.

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Mechanism of somatic hypermutation at the WA motif by human DNA polymerase eta.

Publication Date: 2013 May 14 PMID: 23630267
Authors: Zhao, Y. - Gregory, M. T. - Biertumpfel, C. - Hua, Y. J. - Hanaoka, F. - Yang, W.
Journal: Proc Natl Acad Sci U S A

Somatic hypermutation is programmed base substitutions in the variable regions of Ig genes for high-affinity antibody generation. Two motifs, RGYW and WA (R, purine; Y, pyrimidine; W, A or T), have been found to be somatic hypermutation hotspots. Overwhelming evidence suggests that DNA polymerase eta (Pol eta) is responsible for converting the WA motif to WG by misincorporating dGTP opposite the templating T. To elucidate the molecular mechanism, crystal structures and kinetics of human Pol eta substituting dGTP for dATP in four sequence contexts, TA, AA, GA, and CA, have been determined and compared. The T:dGTP wobble base pair is stabilized by Gln-38 and Arg-61, two uniquely conserved residues among Pol eta. Weak base paring of the W (T:A or A:T) at the primer end and their distinct interactions with Pol eta lead to misincorporation of G in the WA motif. Between two WA motifs, our kinetic and structural data indicate that A-to-G mutation occurs more readily in the TA context than AA. Finally, Pol eta can extend the T:G mispair efficiently to complete the mutagenesis.

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Political ideology affects energy-efficiency attitudes and choices.

Publication Date: 2013 Apr 29 PMID: 23630266
Authors: Gromet, D. M. - Kunreuther, H. - Larrick, R. P.
Journal: Proc Natl Acad Sci U S A

This research demonstrates how promoting the environment can negatively affect adoption of energy efficiency in the United States because of the political polarization surrounding environmental issues. Study 1 demonstrated that more politically conservative individuals were less in favor of investment in energy-efficient technology than were those who were more politically liberal. This finding was driven primarily by the lessened psychological value that more conservative individuals placed on reducing carbon emissions. Study 2 showed that this difference has consequences: In a real-choice context, more conservative individuals were less likely to purchase a more expensive energy-efficient light bulb when it was labeled with an environmental message than when it was unlabeled. These results highlight the importance of taking into account psychological value-based considerations in the individual adoption of energy-efficient technology in the United States and beyond.

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Dynamics of internal pore opening in KV channels probed by a fluorescent unnatural amino acid.

Publication Date: 2013 May 14 PMID: 23630265
Authors: Kalstrup, T. - Blunck, R.
Journal: Proc Natl Acad Sci U S A

Atomic-scale models on the gating mechanism of voltage-gated potassium channels (Kv) are based on linear interpolations between static structures of their initial and final state derived from crystallography and molecular dynamics simulations, and, thus, lack dynamic structural information. The lack of information on dynamics and intermediate states makes it difficult to associate the structural with the dynamic functional data obtained with electrophysiology. Although voltage-clamp fluorometry fills this gap, it is limited to sites extracellularly accessible, when the key region for gating is located at the cytosolic side of the channels. Here, we solved this problem by performing voltage-clamp fluorometry with a fluorescent unnatural amino acid. By using an orthogonal tRNA-synthetase pair, the fluorescent unnatural amino acid was incorporated in the Shaker voltage-gated potassium channel at key regions that were previously inaccessible. Thus, we defined which parts act independently and which parts act cooperatively and found pore opening to occur in two sequential transitions.

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Glycolytic strategy as a tradeoff between energy yield and protein cost.

Publication Date: 2013 Apr 29 PMID: 23630264
Authors: Flamholz, A. - Noor, E. - Bar-Even, A. - Liebermeister, W. - Milo, R.
Journal: Proc Natl Acad Sci U S A

Contrary to the textbook portrayal of glycolysis as a single pathway conserved across all domains of life, not all sugar-consuming organisms use the canonical Embden-Meyerhoff-Parnass (EMP) glycolytic pathway. Prokaryotic glucose metabolism is particularly diverse, including several alternative glycolytic pathways, the most common of which is the Entner-Doudoroff (ED) pathway. The prevalence of the ED pathway is puzzling as it produces only one ATP per glucose-half as much as the EMP pathway. We argue that the diversity of prokaryotic glucose metabolism may reflect a tradeoff between a pathway's energy (ATP) yield and the amount of enzymatic protein required to catalyze pathway flux. We introduce methods for analyzing pathways in terms of thermodynamics and kinetics and show that the ED pathway is expected to require several-fold less enzymatic protein to achieve the same glucose conversion rate as the EMP pathway. Through genomic analysis, we further show that prokaryotes use different glycolytic pathways depending on their energy supply. Specifically, energy-deprived anaerobes overwhelmingly rely upon the higher ATP yield of the EMP pathway, whereas the ED pathway is common among facultative anaerobes and even more common among aerobes. In addition to demonstrating how protein costs can explain the use of alternative metabolic strategies, this study illustrates a direct connection between an organism's environment and the thermodynamic and biochemical properties of the metabolic pathways it employs.

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Consumers mediate the effects of experimental ocean acidification and warming on primary producers.

Publication Date: 2013 May 21 PMID: 23630263
Authors: Alsterberg, C. - Eklof, J. S. - Gamfeldt, L. - Havenhand, J. N. - Sundback, K.
Journal: Proc Natl Acad Sci U S A

It is well known that ocean acidification can have profound impacts on marine organisms. However, we know little about the direct and indirect effects of ocean acidification and also how these effects interact with other features of environmental change such as warming and declining consumer pressure. In this study, we tested whether the presence of consumers (invertebrate mesograzers) influenced the interactive effects of ocean acidification and warming on benthic microalgae in a seagrass community mesocosm experiment. Net effects of acidification and warming on benthic microalgal biomass and production, as assessed by analysis of variance, were relatively weak regardless of grazer presence. However, partitioning these net effects into direct and indirect effects using structural equation modeling revealed several strong relationships. In the absence of grazers, benthic microalgae were negatively and indirectly affected by sediment-associated microalgal grazers and macroalgal shading, but directly and positively affected by acidification and warming. Combining indirect and direct effects yielded no or weak net effects. In the presence of grazers, almost all direct and indirect climate effects were nonsignificant. Our analyses highlight that (i) indirect effects of climate change may be at least as strong as direct effects, (ii) grazers are crucial in mediating these effects, and (iii) effects of ocean acidification may be apparent only through indirect effects and in combination with other variables (e.g., warming). These findings highlight the importance of experimental designs and statistical analyses that allow us to separate and quantify the direct and indirect effects of multiple climate variables on natural communities.

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Activation of heme biosynthesis by a small molecule that is toxic to fermenting Staphylococcus aureus.

Publication Date: 2013 May 14 PMID: 23630262
Authors: Mike, L. A. - Dutter, B. F. - Stauff, D. L. - Moore, J. L. - Vitko, N. P. - Aranmolate, O. - Kehl-Fie, T. E. - Sullivan, S. - Reid, P. R. - Dubois, J. L. - Richardson, A. R. - Caprioli, R. M. - Sulikowski, G. A. - Skaar, E. P.
Journal: Proc Natl Acad Sci U S A

Staphylococcus aureus is a significant infectious threat to global public health. Acquisition or synthesis of heme is required for S. aureus to capture energy through respiration, but an excess of this critical cofactor is toxic to bacteria. S. aureus employs the heme sensor system (HssRS) to overcome heme toxicity; however, the mechanism of heme sensing is not defined. Here, we describe the identification of a small molecule activator of HssRS that induces endogenous heme biosynthesis by perturbing central metabolism. This molecule is toxic to fermenting S. aureus, including clinically relevant small colony variants. The utility of targeting fermenting bacteria is exemplified by the fact that this compound prevents the emergence of antibiotic resistance, enhances phagocyte killing, and reduces S. aureus pathogenesis. Not only is this small molecule a powerful tool for studying bacterial heme biosynthesis and central metabolism; it also establishes targeting of fermentation as a viable antibacterial strategy.

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Structures of the phage Sf6 large terminase provide new insights into DNA translocation and cleavage.

Publication Date: 2013 May 14 PMID: 23630261
Authors: Zhao, H. - Christensen, T. E. - Kamau, Y. N. - Tang, L.
Journal: Proc Natl Acad Sci U S A

Many DNA viruses use powerful molecular motors to cleave concatemeric viral DNA into genome-length units and package them into preformed procapsid powered by ATP hydrolysis. Here we report the structures of the DNA-packaging motor gp2 of bacteriophage Sf6, which reveal a unique clade of RecA-like ATPase domain and an RNase H-like nuclease domain tethered by a regulatory linker domain, exhibiting a strikingly distinct domain arrangement. The gp2 structures complexed with nucleotides reveal, at the atomic detail, the catalytic center embraced by the ATPase domain and the linker domain. The gp2 nuclease activity is modulated by the ATPase domain and is stimulated by ATP. An extended DNA-binding surface is formed by the linker domain and the nuclease domain. These results suggest a unique mechanism for translation of chemical reaction into physical motion of DNA and provide insights into coordination of DNA translocation and cleavage in a viral DNA-packaging motor, which may be achieved via linker-domain-mediated interdomain communication driven by ATP hydrolysis.

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Controlling electron transfer at the microbe-mineral interface.

Publication Date: 2013 May 7 PMID: 23630260
Authors: Richardson, D. J. - Butt, J. N. - Clarke, T. A.
Journal: Proc Natl Acad Sci U S A

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Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars.

Publication Date: 2013 May 14 PMID: 23630259
Authors: Cavanagh, C. R. - Chao, S. - Wang, S. - Huang, B. E. - Stephen, S. - Kiani, S. - Forrest, K. - Saintenac, C. - Brown-Guedira, G. L. - Akhunova, A. - See, D. - Bai, G. - Pumphrey, M. - Tomar, L. - Wong, D. - Kong, S. - Reynolds, M. - da Silva, M. L. - Bockelman, H. - Talbert, L. - Anderson, J. A. - Dreisigacker, S. - Baenziger, S. - Carter, A. - Korzun, V. - Morrell, P. L. - Dubcovsky, J. - Morell, M. K. - Sorrells, M. E. - Hayden, M. J. - Akhunov, E.
Journal: Proc Natl Acad Sci U S A

Domesticated crops experience strong human-mediated selection aimed at developing high-yielding varieties adapted to local conditions. To detect regions of the wheat genome subject to selection during improvement, we developed a high-throughput array to interrogate 9,000 gene-associated single-nucleotide polymorphisms (SNP) in a worldwide sample of 2,994 accessions of hexaploid wheat including landraces and modern cultivars. Using a SNP-based diversity map we characterized the impact of crop improvement on genomic and geographic patterns of genetic diversity. We found evidence of a small population bottleneck and extensive use of ancestral variation often traceable to founders of cultivars from diverse geographic regions. Analyzing genetic differentiation among populations and the extent of haplotype sharing, we identified allelic variants subjected to selection during improvement. Selective sweeps were found around genes involved in the regulation of flowering time and phenology. An introgression of a wild relative-derived gene conferring resistance to a fungal pathogen was detected by haplotype-based analysis. Comparing selective sweeps identified in different populations, we show that selection likely acts on distinct targets or multiple functionally equivalent alleles in different portions of the geographic range of wheat. The majority of the selected alleles were present at low frequency in local populations, suggesting either weak selection pressure or temporal variation in the targets of directional selection during breeding probably associated with changing agricultural practices or environmental conditions. The developed SNP chip and map of genetic variation provide a resource for advancing wheat breeding and supporting future population genomic and genome-wide association studies in wheat.

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Self-assembled, aptamer-tethered DNA nanotrains for targeted transport of molecular drugs in cancer theranostics.

Publication Date: 2013 May 14 PMID: 23630258
Authors: Zhu, G. - Zheng, J. - Song, E. - Donovan, M. - Zhang, K. - Liu, C. - Tan, W.
Journal: Proc Natl Acad Sci U S A

Nanotechnology has allowed the construction of various nanostructures for applications, including biomedicine. However, a simple target-specific, economical, and biocompatible drug delivery platform with high maximum tolerated doses is still in demand. Here, we report aptamer-tethered DNA nanotrains (aptNTrs) as carriers for targeted drug transport in cancer therapy. Long aptNTrs were self-assembled from only two short DNA upon initiation by modified aptamers, which worked like locomotives guiding nanotrains toward target cancer cells. Meanwhile, tandem "boxcars" served as carriers with high payload capacity of drugs that were transported to target cells and induced selective cytotoxicity. aptNTrs enhanced maximum tolerated dose in nontarget cells. Potent antitumor efficacy and reduced side effects of drugs delivered by biocompatible aptNTrs were demonstrated in a mouse xenograft tumor model. Moreover, fluorophores on nanotrains and drug fluorescence dequenching upon release allowed intracellular signaling of nanotrains and drugs. These results make aptNTrs a promising targeted drug transport platform for cancer theranostics.

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Nanoscale analysis of pyritized microfossils reveals differential heterotrophic consumption in the ~1.9-Ga Gunflint chert.

Publication Date: 2013 May 14 PMID: 23630257
Authors: Wacey, D. - McLoughlin, N. - Kilburn, M. R. - Saunders, M. - Cliff, J. B. - Kong, C. - Barley, M. E. - Brasier, M. D.
Journal: Proc Natl Acad Sci U S A

The 1.88-Ga Gunflint biota is one of the most famous Precambrian microfossil lagerstatten and provides a key record of the biosphere at a time of changing oceanic redox structure and chemistry. Here, we report on pyritized replicas of the iconic autotrophic Gunflintia-Huroniospora microfossil assemblage from the Schreiber Locality, Canada, that help capture a view through multiple trophic levels in a Paleoproterozoic ecosystem. Nanoscale analysis of pyritic Gunflintia (sheaths) and Huroniospora (cysts) reveals differing relic carbon and nitrogen distributions caused by contrasting spectra of decay and pyritization between taxa, reflecting in part their primary organic compositions. In situ sulfur isotope measurements from individual microfossils (delta(34)SV-CDT +6.7 per thousand to +21.5 per thousand) show that pyritization was mediated by sulfate-reducing microbes within sediment pore waters whose sulfate ion concentrations rapidly became depleted, owing to occlusion of pore space by coeval silicification. Three-dimensional nanotomography reveals additional pyritized biomaterial, including hollow, cellular epibionts and extracellular polymeric substances, showing a preference for attachment to Gunflintia over Huroniospora and interpreted as components of a saprophytic heterotrophic, decomposing community. This work also extends the record of remarkable biological preservation in pyrite back to the Paleoproterozoic and provides criteria to assess the authenticity of even older pyritized microstructures that may represent some of the earliest evidence for life on our planet.

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Cofactor-dependent specificity of a DEAD-box protein.

Publication Date: 2013 Apr 29 PMID: 23630256
Authors: Young, C. L. - Khoshnevis, S. - Karbstein, K.
Journal: Proc Natl Acad Sci U S A

DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.

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Honey constituents up-regulate detoxification and immunity genes in the western honey bee Apis mellifera.

Publication Date: 2013 Apr 29 PMID: 23630255
Authors: Mao, W. - Schuler, M. A. - Berenbaum, M. R.
Journal: Proc Natl Acad Sci U S A

As a managed pollinator, the honey bee Apis mellifera is critical to the American agricultural enterprise. Recent colony losses have thus raised concerns; possible explanations for bee decline include nutritional deficiencies and exposures to pesticides and pathogens. We determined that constituents found in honey, including p-coumaric acid, pinocembrin, and pinobanksin 5-methyl ether, specifically induce detoxification genes. These inducers are primarily found not in nectar but in pollen in the case of p-coumaric acid (a monomer of sporopollenin, the principal constituent of pollen cell walls) and propolis, a resinous material gathered and processed by bees to line wax cells. RNA-seq analysis (massively parallel RNA sequencing) revealed that p-coumaric acid specifically up-regulates all classes of detoxification genes as well as select antimicrobial peptide genes. This up-regulation has functional significance in that that adding p-coumaric acid to a diet of sucrose increases midgut metabolism of coumaphos, a widely used in-hive acaricide, by approximately 60%. As a major component of pollen grains, p-coumaric acid is ubiquitous in the natural diet of honey bees and may function as a nutraceutical regulating immune and detoxification processes. The widespread apicultural use of honey substitutes, including high-fructose corn syrup, may thus compromise the ability of honey bees to cope with pesticides and pathogens and contribute to colony losses.

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Gene silencing in adipose tissue macrophages regulates whole-body metabolism in obese mice.

Publication Date: 2013 May 14 PMID: 23630254
Authors: Aouadi, M. - Tencerova, M. - Vangala, P. - Yawe, J. C. - Nicoloro, S. M. - Amano, S. U. - Cohen, J. L. - Czech, M. P.
Journal: Proc Natl Acad Sci U S A

Adipose tissue (AT) inflammation and infiltration by macrophages is associated with insulin resistance and type 2 diabetes in obese humans, offering a potential target for therapeutics. However, whether AT macrophages (ATMs) directly contribute to systemic glucose intolerance has not been determined. The reason is the lack of methods to ablate inflammatory genes expressed in macrophages specifically localized within AT depots, leaving macrophages in other tissues unaffected. Here we report that i.p. administration of siRNA encapsulated by glucan shells in obese mice selectively silences genes in epididymal ATMs, whereas macrophages within lung, spleen, kidney, heart, skeletal muscle, subcutaneous (SubQ) adipose, and liver are not targeted. Such administration of GeRPs to silence the inflammatory cytokines TNF-alpha or osteopontin in epididymal ATMs of obese mice caused significant improvement in glucose tolerance. These data are consistent with the hypothesis that cytokines produced by ATMs can exacerbate whole-body glucose intolerance.

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Quantification of cellular penetrative forces using lab-on-a-chip technology and finite element modeling.

Publication Date: 2013 May 14 PMID: 23630253
Authors: Sanati Nezhad, A. - Naghavi, M. - Packirisamy, M. - Bhat, R. - Geitmann, A.
Journal: Proc Natl Acad Sci U S A

Tip-growing cells have the unique property of invading living tissues and abiotic growth matrices. To do so, they exert significant penetrative forces. In plant and fungal cells, these forces are generated by the hydrostatic turgor pressure. Using the TipChip, a microfluidic lab-on-a-chip device developed for tip-growing cells, we tested the ability to exert penetrative forces generated in pollen tubes, the fastest-growing plant cells. The tubes were guided to grow through microscopic gaps made of elastic polydimethylsiloxane material. Based on the deformation of the gaps, the force exerted by the elongating tubes to permit passage was determined using finite element methods. The data revealed that increasing mechanical impedance was met by the pollen tubes through modulation of the cell wall compliance and, thus, a change in the force acting on the obstacle. Tubes that successfully passed a narrow gap frequently burst, raising questions about the sperm discharge mechanism in the flowering plants.

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Female mice lack adult germ-line stem cells but sustain oogenesis using stable primordial follicles.

Publication Date: 2013 May 21 PMID: 23630252
Authors: Lei, L. - Spradling, A. C.
Journal: Proc Natl Acad Sci U S A

Whether or not mammalian females generate new oocytes during adulthood from germ-line stem cells to sustain the ovarian follicle pool has recently generated controversy. We used a sensitive lineage-labeling system to determine whether stem cells are needed in female adult mice to compensate for follicular losses and to directly identify active germ-line stem cells. Primordial follicles generated during fetal life are highly stable, with a half-life during adulthood of 10 mo, and thus are sufficient to sustain adult oogenesis without a source of renewal. Moreover, in normal mice or following germ-cell depletion with Busulfan, only stable, single oocytes are lineage-labeled, rather than cell clusters indicative of new oocyte formation. Even one germ-line stem cell division per 2 wk would have been detected by our method, based on the kinetics of fetal follicle formation. Thus, adult female mice neither require nor contain active germ-line stem cells or produce new oocytes in vivo.

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Structure-kinetic relationship study of CDK8/CycC specific compounds.

Publication Date: 2013 May 14 PMID: 23630251
Authors: Schneider, E. V. - Bottcher, J. - Huber, R. - Maskos, K. - Neumann, L.
Journal: Proc Natl Acad Sci U S A

In contrast with the very well explored concept of structure-activity relationship, similar studies are missing for the dependency between binding kinetics and compound structure of a protein ligand complex, the structure-kinetic relationship. Here, we present a structure-kinetic relationship study of the cyclin-dependent kinase 8 (CDK8)/cyclin C (CycC) complex. The scaffold moiety of the compounds is anchored in the kinase deep pocket and extended with diverse functional groups toward the hinge region and the front pocket. These variations can cause the compounds to change from fast to slow binding kinetics, resulting in an improved residence time. The flip of the DFG motif ("DMG" in CDK8) to the inactive DFG-out conformation appears to have relatively little influence on the velocity of binding. Hydrogen bonding with the kinase hinge region contributes to the residence time but has less impact than hydrophobic complementarities within the kinase front pocket.

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Metallothioneins negatively regulate IL-27-induced type 1 regulatory T-cell differentiation.

Publication Date: 2013 May 7 PMID: 23630250
Authors: Wu, C. - Pot, C. - Apetoh, L. - Thalhamer, T. - Zhu, B. - Murugaiyan, G. - Xiao, S. - Lee, Y. - Rangachari, M. - Yosef, N. - Kuchroo, V. K.
Journal: Proc Natl Acad Sci U S A

IL-27-induced type 1 regulatory T (Tr1) cells suppress autoimmunity by producing IL-10. Signal transducer and activator of transcription (STAT) 1 and STAT3 have been described as key transcription factors that promote IL-10 secretion from Tr1 cells induced by IL-27. However, the molecular pathways for negatively regulating Tr1 cell differentiation remain elusive. Here, we show that IL-27 induces metallothioneins (MTs) that in turn prevent Tr1 cell development. MT expression leads to the reduction of STAT1 and STAT3 phosphorylation under Tr1 differentiation condition, resulting in impaired IL-10 production. Accordingly, Tr1 cells derived from MT-deficient mice showed an increased ability to produce IL-10 and potently suppress experimental autoimmune encephalomyelitis upon adoptive transfer. Moreover, activation of STAT1 and/or STAT3 can overcome the suppression of IL-10 by MTs, indicating a dynamic balance between STATs and MTs in regulating IL-10 during Tr1 cell differentiation.

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Differential hERG ion channel activity of ultrasmall gold nanoparticles.

Publication Date: 2013 May 14 PMID: 23630249
Authors: Leifert, A. - Pan, Y. - Kinkeldey, A. - Schiefer, F. - Setzler, J. - Scheel, O. - Lichtenbeld, H. - Schmid, G. - Wenzel, W. - Jahnen-Dechent, W. - Simon, U.
Journal: Proc Natl Acad Sci U S A

Understanding the mechanism of toxicity of nanomaterials remains a challenge with respect to both mechanisms involved and product regulation. Here we show toxicity of ultrasmall gold nanoparticles (AuNPs). Depending on the ligand chemistry, 1.4-nm-diameter AuNPs failed electrophysiology-based safety testing using human embryonic kidney cell line 293 cells expressing human ether-a-go-go-Related gene (hERG), a Food and Drug Administration-established drug safety test. In patch-clamp experiments, phosphine-stabilized AuNPs irreversibly blocked hERG channels, whereas thiol-stabilized AuNPs of similar size had no effect in vitro, and neither particle blocked the channel in vivo. We conclude that safety regulations may need to be reevaluated and adapted to reflect the fact that the binding modality of surface functional groups becomes a relevant parameter for the design of nanoscale bioactive compounds.

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Engineering recombinant reoviruses with tandem repeats and a tetravirus 2A-like element for exogenous polypeptide expression.

Publication Date: 2013 May 14 PMID: 23630248
Authors: Demidenko, A. A. - Blattman, J. N. - Blattman, N. N. - Greenberg, P. D. - Nibert, M. L.
Journal: Proc Natl Acad Sci U S A

We tested a strategy for engineering recombinant mammalian reoviruses (rMRVs) to express exogenous polypeptides. One important feature is that these rMRVs are designed to propagate autonomously and can therefore be tested in animals as potential vaccine vectors. The strategy has been applied so far to three of the 10 MRV genome segments: S3, M1, and L1. To engineer the modified segments, a 5' or 3' region of the essential, long ORF in each was duplicated, and then exogenous sequences were inserted between the repeats. The inner repeat and exogenous insert were positioned in frame with the native protein-encoding sequences but were separated from them by an in-frame "2A-like" sequence element that specifies a cotranslational "stop/continue" event releasing the exogenous polypeptide from the essential MRV protein. This design preserves a terminal region of the MRV genome segment with essential activities in RNA packaging, assortment, replication, transcription, and/or translation and alters the encoded MRV protein to a limited degree. Recovery of rMRVs with longer inserts was made more efficient by wobble-mutagenizing both the inner repeat and the exogenous insert, which possibly helped via respective reductions in homologous recombination and RNA structure. Immunogenicity of a 300-aa portion of the simian immunodeficiency virus Gag protein expressed in mice by an L1-modified rMRV was confirmed by detection of Gag-specific T-cell responses. The engineering strategy was further used for mapping the minimal 5'-terminal region essential to MRV genome segment S3.

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Latency of Epstein-Barr virus is disrupted by gain-of-function mutant cellular AP-1 proteins that preferentially bind methylated DNA.

Publication Date: 2013 May 14 PMID: 23625009
Authors: Yu, K. P. - Heston, L. - Park, R. - Ding, Z. - Wang'ondu, R. - Delecluse, H. J. - Miller, G.
Journal: Proc Natl Acad Sci U S A

ZEBReplication Activator (ZEBRA), a viral basic zipper protein that initiates the Epstein-Barr viral lytic cycle, binds to DNA and activates transcription through heptamer ZEBRA response elements (ZREs) related to AP-1 sites. A component of the biologic action of ZEBRA is attributable to binding methylated CpGs in ZREs present in the promoters of viral lytic cycle genes. Residue S186 of ZEBRA, Z(S186), which is absolutely required for disruption of latency, participates in the recognition of methylated DNA. We find that mutant cellular AP-1 proteins, Jun(A266S) and Fos(A151S), with alanine-to-serine substitutions homologous to Z(S186), exhibit altered DNA-binding affinity and preferentially bind methylated ZREs. These mutant AP-1 proteins acquire functions of ZEBRA; they activate expression of many viral early lytic cycle gene transcripts in cells harboring latent EBV but are selectively defective in activating expression of some viral proteins and are unable to promote viral DNA replication. Transcriptional activation by mutant c-Jun and c-Fos that have acquired the capacity to bind methylated CpG challenges the paradigm that DNA methylation represses gene expression.

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Nogo-A is a negative regulator of CNS angiogenesis.

Publication Date: 2013 May 21 PMID: 23625008
Authors: Walchli, T. - Pernet, V. - Weinmann, O. - Shiu, J. Y. - Guzik-Kornacka, A. - Decrey, G. - Yuksel, D. - Schneider, H. - Vogel, J. - Ingber, D. E. - Vogel, V. - Frei, K. - Schwab, M. E.
Journal: Proc Natl Acad Sci U S A

Nogo-A is an important axonal growth inhibitor in the adult and developing CNS. In vitro, Nogo-A has been shown to inhibit migration and cell spreading of neuronal and nonneuronal cell types. Here, we studied in vivo and in vitro effects of Nogo-A on vascular endothelial cells during angiogenesis of the early postnatal brain and retina in which Nogo-A is expressed by many types of neurons. Genetic ablation or virus-mediated knock down of Nogo-A or neutralization of Nogo-A with an antibody caused a marked increase in the blood vessel density in vivo. In culture, Nogo-A inhibited spreading, migration, and sprouting of primary brain microvascular endothelial cells (MVECs) in a dose-dependent manner and induced the retraction of MVEC lamellipodia and filopodia. Mechanistically, we show that only the Nogo-A-specific Delta 20 domain exerts inhibitory effects on MVECs, but the Nogo-66 fragment, an inhibitory domain common to Nogo-A, -B, and -C, does not. Furthermore, the action of Nogo-A Delta 20 on MVECs required the intracellular activation of the Ras homolog gene family, member A (Rho-A)-associated, coiled-coil containing protein kinase (ROCK)-Myosin II pathway. The inhibitory effects of early postnatal brain membranes or cultured neurons on MVECs were relieved significantly by anti-Nogo-A antibodies. These findings identify Nogo-A as an important negative regulator of developmental angiogenesis in the CNS. They may have important implications in CNS pathologies involving angiogenesis such as stroke, brain tumors, and retinopathies.

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On Ramanujan's definition of mock theta function.

Publication Date: 2013 May 7 PMID: 23625007
Authors: Rhoades, R. C.
Journal: Proc Natl Acad Sci U S A

In his famous "deathbed" letter, Ramanujan "defined" the notion of a mock theta function and offered some examples of functions he believed satisfied his definition. Very recently, Griffin et al. established for the first time that Ramanujan's mock theta functions actually satisfy his own definition. On the other hand, Zwegers' 2002 doctoral thesis [Zwegers S (2002) Mock theta functions. PhD thesis (Univ Utrecht, Utrecht, The Netherlands)] showed that all of Ramanujan's examples are holomorphic parts of harmonic Maass forms. This has led to an alternate definition of a mock theta function. This paper shows that Ramanujan's definition of mock theta function is not equivalent to the modern definition.

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The oceanic cadmium cycle: Biological mistake or utilization?

Publication Date: 2013 May 21 PMID: 23620523
Authors: Morel, F. M.
Journal: Proc Natl Acad Sci U S A

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Misplaced helix slows down ultrafast pressure-jump protein folding.

Publication Date: 2013 May 14 PMID: 23620522
Authors: Prigozhin, M. B. - Liu, Y. - Wirth, A. J. - Kapoor, S. - Winter, R. - Schulten, K. - Gruebele, M.
Journal: Proc Natl Acad Sci U S A

Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein lambda*YA, the Y22W/Q33Y/G46,48A mutant of lambda-repressor fragment 6-85, from 3 mus to 5 ms after a 1,200-bar P-drop. In addition to a microsecond phase, we observe a slower 1.4-ms phase during refolding to the native state. Unlike temperature denaturation, pressure denaturation produces a highly reversible helix-coil-rich state. This difference highlights the importance of the denatured initial condition in folding experiments and leads us to assign a compact nonnative helical trap as the reason for slower P-jump-induced refolding. To complement the experiments, we performed over 50 mus of all-atom molecular dynamics P-drop refolding simulations with four different force fields. Two of the force fields yield compact nonnative states with misplaced alpha-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of lambda*YA contains mainly residual helix and little beta-sheet; following a fast P-drop, at least some lambda*YA forms misplaced helical structure within microseconds. We hypothesize that nonnative helix at helix-turn interfaces traps the protein in compact nonnative conformations. These traps delay the folding of at least some of the population for 1.4 ms en route to the native state. Based on molecular dynamics, we predict specific mutations at the helix-turn interfaces that should speed up refolding from the pressure-denatured state, if this hypothesis is correct.

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Profile of Susan S. Golden.

Publication Date: 2013 Apr 25 PMID: 23620521
Authors: Gupta, S.
Journal: Proc Natl Acad Sci U S A

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Viscosity of alpha-pinene secondary organic material and implications for particle growth and reactivity.

Publication Date: 2013 May 14 PMID: 23620520
Authors: Renbaum-Wolff, L. - Grayson, J. W. - Bateman, A. P. - Kuwata, M. - Sellier, M. - Murray, B. J. - Shilling, J. E. - Martin, S. T. - Bertram, A. K.
Journal: Proc Natl Acad Sci U S A

Particles composed of secondary organic material (SOM) are abundant in the lower troposphere. The viscosity of these particles is a fundamental property that is presently poorly quantified yet required for accurate modeling of their formation, growth, evaporation, and environmental impacts. Using two unique techniques, namely a "bead-mobility" technique and a "poke-flow" technique, in conjunction with simulations of fluid flow, the viscosity of the water-soluble component of SOM produced by alpha-pinene ozonolysis is quantified for 20- to 50-mum particles at 293-295 K. The viscosity is comparable to that of honey at 90% relative humidity (RH), similar to that of peanut butter at 70% RH, and at least as viscous as bitumen at
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Bringing the ocean into the laboratory to probe the chemical complexity of sea spray aerosol.

Publication Date: 2013 May 7 PMID: 23620519
Authors: Prather, K. A. - Bertram, T. H. - Grassian, V. H. - Deane, G. B. - Stokes, M. D. - Demott, P. J. - Aluwihare, L. I. - Palenik, B. P. - Azam, F. - Seinfeld, J. H. - Moffet, R. C. - Molina, M. J. - Cappa, C. D. - Geiger, F. M. - Roberts, G. C. - Russell, L. M. - Ault, A. P. - Baltrusaitis, J. - Collins, D. B. - Corrigan, C. E. - Cuadra-Rodriguez, L. A. - Ebben, C. J. - Forestieri, S. D. - Guasco, T. L. - Hersey, S. P. - Kim, M. J. - Lambert, W. F. - Modini, R. L. - Mui, W. - Pedler, B. E. - Ruppel, M. J. - Ryder, O. S. - Schoepp, N. G. - Sullivan, R. C. - Zhao, D.
Journal: Proc Natl Acad Sci U S A

The production, size, and chemical composition of sea spray aerosol (SSA) particles strongly depend on seawater chemistry, which is controlled by physical, chemical, and biological processes. Despite decades of studies in marine environments, a direct relationship has yet to be established between ocean biology and the physicochemical properties of SSA. The ability to establish such relationships is hindered by the fact that SSA measurements are typically dominated by overwhelming background aerosol concentrations even in remote marine environments. Herein, we describe a newly developed approach for reproducing the chemical complexity of SSA in a laboratory setting, comprising a unique ocean-atmosphere facility equipped with actual breaking waves. A mesocosm experiment was performed in natural seawater, using controlled phytoplankton and heterotrophic bacteria concentrations, which showed SSA size and chemical mixing state are acutely sensitive to the aerosol production mechanism, as well as to the type of biological species present. The largest reduction in the hygroscopicity of SSA occurred as heterotrophic bacteria concentrations increased, whereas phytoplankton and chlorophyll-a concentrations decreased, directly corresponding to a change in mixing state in the smallest (60-180 nm) size range. Using this newly developed approach to generate realistic SSA, systematic studies can now be performed to advance our fundamental understanding of the impact of ocean biology on SSA chemical mixing state, heterogeneous reactivity, and the resulting climate-relevant properties.

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Modulation of the endoplasmic reticulum-mitochondria interface in Alzheimer's disease and related models.

Publication Date: 2013 May 7 PMID: 23620518
Authors: Hedskog, L. - Pinho, C. M. - Filadi, R. - Ronnback, A. - Hertwig, L. - Wiehager, B. - Larssen, P. - Gellhaar, S. - Sandebring, A. - Westerlund, M. - Graff, C. - Winblad, B. - Galter, D. - Behbahani, H. - Pizzo, P. - Glaser, E. - Ankarcrona, M.
Journal: Proc Natl Acad Sci U S A

It is well-established that subcompartments of endoplasmic reticulum (ER) are in physical contact with the mitochondria. These lipid raft-like regions of ER are referred to as mitochondria-associated ER membranes (MAMs), and they play an important role in, for example, lipid synthesis, calcium homeostasis, and apoptotic signaling. Perturbation of MAM function has previously been suggested in Alzheimer's disease (AD) as shown in fibroblasts from AD patients and a neuroblastoma cell line containing familial presenilin-2 AD mutation. The effect of AD pathogenesis on the ER-mitochondria interplay in the brain has so far remained unknown. Here, we studied ER-mitochondria contacts in human AD brain and related AD mouse and neuronal cell models. We found uniform distribution of MAM in neurons. Phosphofurin acidic cluster sorting protein-2 and sigma1 receptor, two MAM-associated proteins, were shown to be essential for neuronal survival, because siRNA knockdown resulted in degeneration. Up-regulated MAM-associated proteins were found in the AD brain and amyloid precursor protein (APP)Swe/Lon mouse model, in which up-regulation was observed before the appearance of plaques. By studying an ER-mitochondria bridging complex, inositol-1,4,5-triphosphate receptor-voltage-dependent anion channel, we revealed that nanomolar concentrations of amyloid beta-peptide increased inositol-1,4,5-triphosphate receptor and voltage-dependent anion channel protein expression and elevated the number of ER-mitochondria contact points and mitochondrial calcium concentrations. Our data suggest an important role of ER-mitochondria contacts and cross-talk in AD pathology.

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Human amnesia and the medial temporal lobe illuminated by neuropsychological and neurohistological findings for patient E.P.

Publication Date: 2013 May 21 PMID: 23620517
Authors: Insausti, R. - Annese, J. - Amaral, D. G. - Squire, L. R.
Journal: Proc Natl Acad Sci U S A

We present neurohistological information for a case of bilateral, symmetrical damage to the medial temporal lobe and well-documented memory impairment. E.P. developed profound memory impairment at age 70 y and then was studied for 14 y He had no capacity for learning facts and events and had retrograde amnesia covering several decades. He also had a modest impairment of semantic knowledge. Neurohistological analysis revealed bilaterally symmetrical lesions of the medial temporal lobe that eliminated the temporal pole, the amygdala, the entorhinal cortex, the hippocampus, the perirhinal cortex, and rostral parahippocampal cortex. The lesion also extended laterally to involve the fusiform gyrus substantially. Last, the superior, inferior, and middle temporal gyri were atrophic, and subjacent white matter was gliotic. Several considerations indicate that E.P.'s severe memory impairment was caused by his medial temporal lesions, whereas his impaired semantic knowledge was caused by lateral temporal damage. His lateral temporal damage also may have contributed to his extensive retrograde amnesia. The findings illuminate the anatomical relationship between memory, perception, and semantic knowledge.

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Characterization of pathogenic human monoclonal autoantibodies against GM-CSF.

Publication Date: 2013 May 7 PMID: 23620516
Authors: Wang, Y. - Thomson, C. A. - Allan, L. L. - Jackson, L. M. - Olson, M. - Hercus, T. R. - Nero, T. L. - Turner, A. - Parker, M. W. - Lopez, A. L. - Waddell, T. K. - Anderson, G. P. - Hamilton, J. A. - Schrader, J. W.
Journal: Proc Natl Acad Sci U S A

The origin of pathogenic autoantibodies remains unknown. Idiopathic pulmonary alveolar proteinosis is caused by autoantibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF). We generated 19 monoclonal autoantibodies against GM-CSF from six patients with idiopathic pulmonary alveolar proteinosis. The autoantibodies used multiple V genes, excluding preferred V-gene use as an etiology, and targeted at least four nonoverlapping epitopes on GM-CSF, suggesting that GM-CSF is driving the autoantibodies and not a B-cell epitope on a pathogen cross-reacting with GM-CSF. The number of somatic mutations in the autoantibodies suggests that the memory B cells have been helped by T cells and re-entered germinal centers. All autoantibodies neutralized GM-CSF bioactivity, with general correlations to affinity and off-rate. The binding of certain autoantibodies was changed by point mutations in GM-CSF that reduced binding to the GM-CSF receptor. Those monoclonal autoantibodies that potently neutralize GM-CSF may be useful in treating inflammatory disease, such as rheumatoid arthritis and multiple sclerosis, cancer, and pain.

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Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2.

Publication Date: 2013 May 7 PMID: 23620515
Authors: Knutson, S. K. - Warholic, N. M. - Wigle, T. J. - Klaus, C. R. - Allain, C. J. - Raimondi, A. - Porter Scott, M. - Chesworth, R. - Moyer, M. P. - Copeland, R. A. - Richon, V. M. - Pollock, R. M. - Kuntz, K. W. - Keilhack, H.
Journal: Proc Natl Acad Sci U S A

Inactivation of the switch/sucrose nonfermentable complex component SMARCB1 is extremely prevalent in pediatric malignant rhabdoid tumors (MRTs) or atypical teratoid rhabdoid tumors. This alteration is hypothesized to confer oncogenic dependency on EZH2 in these cancers. We report the discovery of a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity, (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-py ran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide). The compound induces apoptosis and differentiation specifically in SMARCB1-deleted MRT cells. Treatment of xenograft-bearing mice with (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-py ran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide) leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation. These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers.

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Deciphering the rules of ceRNA networks.

Publication Date: 2013 Apr 30 PMID: 23620514
Authors: Cesana, M. - Daley, G. Q.
Journal: Proc Natl Acad Sci U S A

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ApoE influences amyloid-beta (Abeta) clearance despite minimal apoE/Abeta association in physiological conditions.

Publication Date: 2013 May 7 PMID: 23620513
Authors: Verghese, P. B. - Castellano, J. M. - Garai, K. - Wang, Y. - Jiang, H. - Shah, A. - Bu, G. - Frieden, C. - Holtzman, D. M.
Journal: Proc Natl Acad Sci U S A

Apolipoprotein E gene (APOE) alleles may shift the onset of Alzheimer's disease (AD) through apoE protein isoforms changing the probability of amyloid-beta (Abeta) accumulation. It has been proposed that differential physical interactions of apoE isoforms with soluble Abeta (sAbeta) in brain fluids influence the metabolism of Abeta, providing a mechanism to account for how APOE influences AD risk. In contrast, we provide clear evidence that apoE and sAbeta interactions occur minimally in solution and in the cerebrospinal fluid of human subjects, producing apoE3 and apoE4 isoforms as assessed by multiple biochemical and analytical techniques. Despite minimal extracellular interactions with sAbeta in fluid, we find that apoE isoforms regulate the metabolism of sAbeta by astrocytes and in the interstitial fluid of mice that received apoE infusions during brain Abeta microdialysis. We find that a significant portion of apoE and sAbeta compete for the low-density lipoprotein receptor-related protein 1 (LRP1)-dependent cellular uptake pathway in astrocytes, providing a mechanism to account for apoE's regulation of sAbeta metabolism despite minimal evidence of direct interactions in extracellular fluids. We propose that apoE influences sAbeta metabolism not through direct binding to sAbeta in solution but through its actions with other interacting receptors/transporters and cell surfaces. These results provide an alternative frame work for the mechanistic explanations on how apoE isoforms influence the risk of AD pathogenesis.

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p63-expressing cells are the stem cells of developing prostate, bladder, and colorectal epithelia.

Publication Date: 2013 May 14 PMID: 23620512
Authors: Pignon, J. C. - Grisanzio, C. - Geng, Y. - Song, J. - Shivdasani, R. A. - Signoretti, S.
Journal: Proc Natl Acad Sci U S A

The tumor protein p63 (p63), and more specifically the NH2-terminal truncated (DeltaN) p63 isoform, is a marker of basal epithelial cells and is required for normal development of several epithelial tissues, including the bladder and prostate glands. Although p63-expressing cells are proposed to be the stem cells of the developing prostate epithelium and bladder urothelium, cell lineages in these endoderm-derived epithelia remain highly controversial, and rigorous lineage tracing studies are warranted. Here, we generated knock-in mice expressing Cre recombinase (Cre) under the control of the endogenous DeltaNp63 promoter. Heterozygote DeltaNp63(+/Cre) mice were phenotypically normal and fertile. Cre-mediated recombination in DeltaNp63(+/Cre);ROSA26(EYFP) reporter mice faithfully recapitulated the pattern of DeltaNp63 expression and were useful for genetic lineage tracing of DeltaNp63-expressing cells of the caudal endoderm in vivo. We found that DeltaNp63-positive cells of the urogenital sinus generated all epithelial lineages of the prostate and bladder, indicating that these cells represent the stem/progenitor cells of those epithelia during development. We also observed DeltaNp63 expression in caudal gut endoderm and the contribution of DeltaNp63-positive cells to the stem/progenitor compartment of adult colorectal epithelium. Because p63 is a master regulator of stratified epithelial development, this finding provides a unique developmental insight into the cell of origin of squamous cell metaplasia and squamous cell carcinoma of the colon.

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Hippo-Foxa2 signaling pathway plays a role in peripheral lung maturation and surfactant homeostasis.

Publication Date: 2013 May 7 PMID: 23620511
Authors: Chung, C. - Kim, T. - Kim, M. - Kim, M. - Song, H. - Kim, T. S. - Seo, E. - Lee, S. H. - Kim, H. - Kim, S. K. - Yoo, G. - Lee, D. H. - Hwang, D. S. - Kinashi, T. - Kim, J. M. - Lim, D. S.
Journal: Proc Natl Acad Sci U S A

Respiratory distress syndrome (RDS), which is induced by insufficient production of surfactant, is the leading cause of mortality in preterm babies. Although several transcription factors are known to be involved in surfactant protein expression, the molecular mechanisms and signaling pathways upstream of these transcription factors have remained elusive. Here, using mammalian Hippo kinases (Mst1/2, mammalian sterile 20-like kinase 1/2) conditional knockout mice, we demonstrate that Mst1/2 kinases are critical for orchestration of transcription factors involved in surfactant protein homeostasis and prevention of RDS. Mice lacking Mst1/2 in the respiratory epithelium exhibited perinatal mortality with respiratory failure and their lungs contained fewer type I pneumocytes and more immature type II pneumocytes lacking microvilli, lamellar bodies, and surfactant protein expression, pointing to peripheral lung immaturity and RDS. In contrast to previous findings of YAP (Yes-associated protein)-mediated canonical Hippo signaling in the liver and intestine, loss of Mst1/2 kinases induced the defects in pneumocyte differentiation independently of YAP hyperactivity. We instead found that Mst1/2 kinases stabilized and phosphorylated the transcription factor Foxa2 (forkhead box A2), which regulates pneumocyte maturation and surfactant protein expression. Taken together, our results suggest that the mammalian Hippo kinases play crucial roles in surfactant homeostasis and coordination of peripheral lung differentiation through regulation of Foxa2 rather than of YAP.

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E4orf4 induces PP2A- and Src-dependent cell death in Drosophila melanogaster and at the same time inhibits classic apoptosis pathways.

Publication Date: 2013 May 7 PMID: 23613593
Authors: Pechkovsky, A. - Lahav, M. - Bitman, E. - Salzberg, A. - Kleinberger, T.
Journal: Proc Natl Acad Sci U S A

The adenovirus E4orf4 protein regulates the progression of viral infection, and when expressed alone in mammalian tissue culture cells it induces protein phosphatase 2A (PP2A)-B55- and Src-dependent cell death, which is more efficient in oncogene-transformed cells than in normal cells. This form of cell death is caspase-independent, although it interacts with classic caspase-dependent apoptosis. PP2A-B55-dependent E4orf4-induced toxicity is highly conserved in evolution from yeast to mammalian cells. In this work we investigated E4orf4-induced cell death in a whole multicellular organism, Drosophila melanogaster. We show that E4orf4 induced low levels of cell killing, caused by both caspase-dependent and -independent mechanisms. Drosophila PP2A-B55 (twins/abnormal anaphase resolution) and Src64B contributed additively to this form of cell death. Our results provide insight into E4orf4-induced cell death, demonstrating that in parallel to activating caspase-dependent apoptosis, E4orf4 also inhibited this form of cell death induced by the proapoptotic genes reaper, head involution defective, and grim. The combination of both induction and inhibition of caspase-dependent cell death resulted in low levels of tissue damage that may explain the inefficient cell killing induced by E4orf4 in normal cells in tissue culture. Furthermore, E4orf4 inhibited JNK-dependent cell killing as well. However, JNK inhibition did not impede E4orf4-induced toxicity and even enhanced it, indicating that E4orf4-induced cell killing is a distinctive form of cell death that differs from both JNK- and Rpr/Hid/Grim-induced forms of cell death.

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Transporter-mediated biofuel secretion.

Publication Date: 2013 May 7 PMID: 23613592
Authors: Doshi, R. - Nguyen, T. - Chang, G.
Journal: Proc Natl Acad Sci U S A

Engineering microorganisms to produce biofuels is currently among the most promising strategies in renewable energy. However, harvesting these organisms for extracting biofuels is energy- and cost-intensive, limiting the commercial feasibility of large-scale production. Here, we demonstrate the use of a class of transport proteins of pharmacological interest to circumvent the need to harvest biomass during biofuel production. We show that membrane-embedded transporters, better known to efflux lipids and drugs, can be used to mediate the secretion of intracellularly synthesized model isoprenoid biofuel compounds to the extracellular milieu. Transporter-mediated biofuel secretion sustainably maintained an approximate three- to fivefold boost in biofuel production in our Escherichia coli test system. Because the transporters used in this study belong to the ubiquitous ATP-binding cassette protein family, we propose their use as "plug-and-play" biofuel-secreting systems in a variety of bacteria, cyanobacteria, diatoms, yeast, and algae used for biofuel production. This investigation showcases the potential of expressing desired membrane transport proteins in cell factories to achieve the export or import of substances of economic, environmental, or therapeutic importance.

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Ancestral amphibian v2rs are expressed in the main olfactory epithelium.

Publication Date: 2013 May 7 PMID: 23613591
Authors: Syed, A. S. - Sansone, A. - Nadler, W. - Manzini, I. - Korsching, S. I.
Journal: Proc Natl Acad Sci U S A

Mammalian olfactory receptor families are segregated into different olfactory organs, with type 2 vomeronasal receptor (v2r) genes expressed in a basal layer of the vomeronasal epithelium. In contrast, teleost fish v2r genes are intermingled with all other olfactory receptor genes in a single sensory surface. We report here that, strikingly different from both lineages, the v2r gene family of the amphibian Xenopus laevis is expressed in the main olfactory as well as the vomeronasal epithelium. Interestingly, late diverging v2r genes are expressed exclusively in the vomeronasal epithelium, whereas "ancestral" v2r genes, including the single member of v2r family C, are restricted to the main olfactory epithelium. Moreover, within the main olfactory epithelium, v2r genes are expressed in a basal zone, partially overlapping, but clearly distinct from an apical zone of olfactory marker protein and odorant receptor-expressing cells. These zones are also apparent in the spatial distribution of odor responses, enabling a tentative assignment of odor responses to olfactory receptor gene families. Responses to alcohols, aldehydes, and ketones show an apical localization, consistent with being mediated by odorant receptors, whereas amino acid responses overlap extensively with the basal v2r-expressing zone. The unique bimodal v2r expression pattern in main and accessory olfactory system of amphibians presents an excellent opportunity to study the transition of v2r gene expression during evolution of higher vertebrates.

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The c-ring stoichiometry of ATP synthase is adapted to cell physiological requirements of alkaliphilic Bacillus pseudofirmus OF4.

Publication Date: 2013 May 7 PMID: 23613590
Authors: Preiss, L. - Klyszejko, A. L. - Hicks, D. B. - Liu, J. - Fackelmayer, O. J. - Yildiz, O. - Krulwich, T. A. - Meier, T.
Journal: Proc Natl Acad Sci U S A

The c-rings of ATP synthases consist of individual c-subunits, all of which harbor a conserved motif of repetitive glycine residues (GxGxGxG) important for tight transmembrane alpha-helix packing. The c-ring stoichiometry determines the number of ions transferred during enzyme operation and has a direct impact on the ion-to-ATP ratio, a cornerstone parameter of cell bioenergetics. In the extreme alkaliphile Bacillus pseudofirmus OF4, the glycine motif is replaced by AxAxAxA. We performed a structural study on two mutants with alanine-to-glycine changes using atomic force microscopy and X-ray crystallography, and found that mutants form smaller c12 rings compared with the WT c13. The molar growth yields of B. pseudofirmus OF4 cells on malate further revealed that the c12 mutants have a considerably reduced capacity to grow on limiting malate at high pH. Our results demonstrate that the mutant ATP synthases with either c12 or c13 can support ATP synthesis, and also underscore the critical importance of an alanine motif with c13 ring stoichiometry for optimal growth at pH >10. The data indicate a direct connection between the precisely adapted ATP synthase c-ring stoichiometry and its ion-to-ATP ratio on cell physiology, and also demonstrate the bioenergetic challenges and evolutionary adaptation strategies of extremophiles.

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Mechanism for the endocytosis of spherical nucleic acid nanoparticle conjugates.

Publication Date: 2013 May 7 PMID: 23613589
Authors: Choi, C. H. - Hao, L. - Narayan, S. P. - Auyeung, E. - Mirkin, C. A.
Journal: Proc Natl Acad Sci U S A

Intracellular delivery of nucleic acids as gene regulation agents typically requires the use of cationic carriers or viral vectors, yet issues related to cellular toxicity or immune responses hamper their attractiveness as therapeutic candidates. The discovery that spherical nucleic acids (SNAs), polyanionic structures comprised of densely packed, highly oriented oligonucleotides covalently attached to the surface of nanoparticles, can effectively enter more than 50 different cell types presents a potential strategy for overcoming the limitations of conventional transfection agents. Unfortunately, little is known about the mechanism of endocytosis of SNAs, including the pathway of entry and specific proteins involved. Here, we demonstrate that the rapid cellular uptake kinetics and intracellular transport of SNAs stem from the arrangement of oligonucleotides into a 3D architecture, which supports their targeting of class A scavenger receptors and endocytosis via a lipid-raft-dependent, caveolae-mediated pathway. These results reinforce the notion that SNAs can serve as therapeutic payloads and targeting structures to engage biological pathways not readily accessible with linear oligonucleotides.

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Serum microRNA-122 level correlates with virologic responses to pegylated interferon therapy in chronic hepatitis C.

Publication Date: 2013 May 7 PMID: 23613588
Authors: Su, T. H. - Liu, C. H. - Liu, C. J. - Chen, C. L. - Ting, T. T. - Tseng, T. C. - Chen, P. J. - Kao, J. H. - Chen, D. S.
Journal: Proc Natl Acad Sci U S A

MicroRNA-122 (miR-122) facilitates hepatitis C virus replication in vitro. Serum miR-122 has been implicated as a biomarker for various liver diseases; however, its role in chronic hepatitis C remains unclear. To address this issue, 126 patients with chronic hepatitis C who completed pegylated IFN plus ribavirin therapy with sustained virologic response (SVR) or nonresponse (NR) were retrospectively included, and their pretreatment clinical profiles and treatment responses were collected. Serum miR-122 was quantified before and during treatment. Another 51 patients in SVR and NR groups were prospectively enrolled for validation. Serum miR-122 was found to be a surrogate for hepatic miR-122 and positively correlated with hepatic necroinflammation. Patients who showed complete early virologic response and SVR had significantly higher pretreatment serum miR-122 levels than those with NR (P = 0.001 and P = 0.008, respectively), especially in subgroups of patients with hepatitis C virus genotype 2 and IL-28B rs8099917 TT genotype. Patients with IL-28B TT genotype had significantly better treatment responses and higher pretreatment serum miR-122 level than those with GT or GG genotypes. Univariate analysis showed that pretreatment body mass index, gamma-glutamyl transpeptidase, triglyceride, IL-28B TT genotype, and serum miR-122 are predictors for SVR. Multivariate analysis specifically in IL-28B TT genotype demonstrated that pretreatment serum miR-122 independently predicted SVR. The validation cohort confirmed a significantly greater pretreatment serum miR-122 level in patients with SVR compared with NR (P = 0.025). In conclusion, serum miR-122 may serve as a surrogate of hepatic miR-122, and a higher pretreatment serum miR-122 level can help predict virologic responses to pegylated IFN plus ribavirin therapy.

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Structural analysis of Stc1 provides insights into the coupling of RNAi and chromatin modification.

Publication Date: 2013 May 21 PMID: 23613586
Authors: He, C. - Pillai, S. S. - Taglini, F. - Li, F. - Ruan, K. - Zhang, J. - Wu, J. - Shi, Y. - Bayne, E. H.
Journal: Proc Natl Acad Sci U S A

Noncoding RNAs can modulate gene expression by directing modifications to histones that alter chromatin structure. In fission yeast, siRNAs produced via the RNAi pathway direct modifications associated with heterochromatin formation. siRNAs associate with the RNAi effector protein Argonaute 1 (Ago1), targeting the Ago1-containing RNA-induced transcriptional silencing (RITS) complex to homologous nascent transcripts. This promotes recruitment of the Clr4 complex (CLRC), which mediates methylation of histone H3 on lysine 9 (H3K9me) in cognate chromatin. A key question is how the RNAi and chromatin modification machineries are connected. Stc1 is a small protein recently shown to associate with both Ago1 and CLRC and to play a pivotal role in mediating the RNAi-dependent recruitment of CLRC to chromatin. To understand its mode of action, we have performed a detailed structural and functional analysis of the Stc1 protein. Our analyses reveal that the conserved N-terminal region of Stc1 represents an unusual tandem zinc finger domain, with similarities to common LIM domains but distinguished by a lack of preferred relative orientation of the two zinc fingers. We demonstrate that this tandem zinc finger domain is involved in binding Ago1, whereas the nonconserved C-terminal region mediates association with CLRC. These findings elucidate the molecular basis for the coupling of RNAi to chromatin modification in fission yeast.

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Assessment of radiative feedback in climate models using satellite observations of annual flux variation.

Publication Date: 2013 May 7 PMID: 23613585
Authors: Tsushima, Y. - Manabe, S.
Journal: Proc Natl Acad Sci U S A

In the climate system, two types of radiative feedback are in operation. The feedback of the first kind involves the radiative damping of the vertically uniform temperature perturbation of the troposphere and Earth's surface that approximately follows the Stefan-Boltzmann law of blackbody radiation. The second kind involves the change in the vertical lapse rate of temperature, water vapor, and clouds in the troposphere and albedo of the Earth's surface. Using satellite observations of the annual variation of the outgoing flux of longwave radiation and that of reflected solar radiation at the top of the atmosphere, this study estimates the so-called "gain factor," which characterizes the strength of radiative feedback of the second kind that operates on the annually varying, global-scale perturbation of temperature at the Earth's surface. The gain factor is computed not only for all sky but also for clear sky. The gain factor of so-called "cloud radiative forcing" is then computed as the difference between the two. The gain factors thus obtained are compared with those obtained from 35 models that were used for the fourth and fifth Intergovernmental Panel on Climate Change assessment. Here, we show that the gain factors obtained from satellite observations of cloud radiative forcing are effective for identifying systematic biases of the feedback processes that control the sensitivity of simulated climate, providing useful information for validating and improving a climate model.

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Smoke-derived karrikin perception by the alpha/beta-hydrolase KAI2 from Arabidopsis.

Publication Date: 2013 May 14 PMID: 23613584
Authors: Guo, Y. - Zheng, Z. - La Clair, J. J. - Chory, J. - Noel, J. P.
Journal: Proc Natl Acad Sci U S A

Genetic studies in Arabidopsis implicate an alpha/beta-hydrolase, KARRIKIN-INSENSITIVE 2 (KAI2) as a receptor for karrikins, germination-promoting butenolide small molecules found in the smoke of burned plants. However, direct biochemical evidence for the interaction between KAI2 and karrikin and for the mechanism of downstream signaling by a KAI2-karrikin complex remain elusive. We report crystallographic analyses and ligand-binding experiments for KAI2 recognition of karrikins. The karrikin-1 (KAR1) ligand sits in the opening to the active site abutting a helical domain insert but distal from the canonical catalytic triad (Ser95-His246-Asp217) of alpha/beta-hydrolases, consistent with the lack of detectable hydrolytic activity by purified KAI2. The closest approach of KAR1 to Ser95-His246-Asp217 is 3.8 A from His246. Six aromatic side chains, including His246, encapsulate KAR1 through geometrically defined aromatic-aromatic interactions. KAR1 binding induces a conformational change in KAI2 at the active site entrance. A crevice of hydrophobic residues linking the polar edge of KAR1 and the helical domain insert suggests that KAI2-KAR1 creates a contiguous interface for binding signaling partners in a ligand-dependent manner.

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Organic-matter loading determines regime shifts and alternative states in an aquatic ecosystem.

Publication Date: 2013 May 7 PMID: 23613583
Authors: Sirota, J. - Baiser, B. - Gotelli, N. J. - Ellison, A. M.
Journal: Proc Natl Acad Sci U S A

Slow changes in underlying state variables can lead to "tipping points," rapid transitions between alternative states ("regime shifts") in a wide range of complex systems. Tipping points and regime shifts routinely are documented retrospectively in long time series of observational data. Experimental induction of tipping points and regime shifts is rare, but could lead to new methods for detecting impending tipping points and forestalling regime shifts. By using controlled additions of detrital organic matter (dried, ground arthropod prey), we experimentally induced a shift from aerobic to anaerobic states in a miniature aquatic ecosystem: the self-contained pools that form in leaves of the carnivorous northern pitcher plant, Sarracenia purpurea. In unfed controls, the concentration of dissolved oxygen ([O2]) in all replicates exhibited regular diurnal cycles associated with daytime photosynthesis and nocturnal plant respiration. In low prey-addition treatments, the regular diurnal cycles of [O2] were disrupted, but a regime shift was not detected. In high prey-addition treatments, the variance of the [O2] time series increased until the system tipped from an aerobic to an anaerobic state. In these treatments, replicate [O2] time series predictably crossed a tipping point at approximately 45 h as [O2] was decoupled from diurnal cycles of photosynthesis and respiration. Increasing organic-matter loading led to predictable changes in [O2] dynamics, with high loading consistently driving the system past a well-defined tipping point. The Sarracenia microecosystem functions as a tractable experimental system in which to explore the forecasting and management of tipping points and alternative regimes.

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Control of electrolyte balance through ubiquitination.

Publication Date: 2013 May 7 PMID: 23613582
Authors: Herrera, M. - Coffman, T. M.
Journal: Proc Natl Acad Sci U S A

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Salicylic acid interferes with clathrin-mediated endocytic protein trafficking.

Publication Date: 2013 May 7 PMID: 23613581
Authors: Du, Y. - Tejos, R. - Beck, M. - Himschoot, E. - Li, H. - Robatzek, S. - Vanneste, S. - Friml, J.
Journal: Proc Natl Acad Sci U S A

Removal of cargos from the cell surface via endocytosis is an efficient mechanism to regulate activities of plasma membrane (PM)-resident proteins, such as receptors or transporters. Salicylic acid (SA) is an important plant hormone that is traditionally associated with pathogen defense. Here, we describe an unanticipated effect of SA on subcellular endocytic cycling of proteins. Both exogenous treatments and endogenously enhanced SA levels repressed endocytosis of different PM proteins. The SA effect on endocytosis did not involve transcription or known components of the SA signaling pathway for transcriptional regulation. SA likely targets an endocytic mechanism that involves the coat protein clathrin, because SA interfered with the clathrin incidence at the PM and clathrin-deficient mutants were less sensitive to the impact of SA on the auxin distribution and root bending during the gravitropic response. By contrast, SA did not affect the ligand-induced endocytosis of the FLAGELLIN SENSING2 (FLS2) receptor during pathogen responses. Our data suggest that the established SA impact on transcription in plant immunity and the nontranscriptional effect of SA on clathrin-mediated endocytosis are independent mechanisms by which SA regulates distinct aspects of plant physiology.

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Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm.

Publication Date: 2013 May 7 PMID: 23613580
Authors: Rodrigues, J. A. - Ruan, R. - Nishimura, T. - Sharma, M. K. - Sharma, R. - Ronald, P. C. - Fischer, R. L. - Zilberman, D.
Journal: Proc Natl Acad Sci U S A

Arabidopsis thaliana endosperm, a transient tissue that nourishes the embryo, exhibits extensive localized DNA demethylation on maternally inherited chromosomes. Demethylation mediates parent-of-origin-specific (imprinted) gene expression but is apparently unnecessary for the extensive accumulation of maternally biased small RNA (sRNA) molecules detected in seeds. Endosperm DNA in the distantly related monocots rice and maize is likewise locally hypomethylated, but whether this hypomethylation is generally parent-of-origin specific is unknown. Imprinted expression of sRNA also remains uninvestigated in monocot seeds. Here, we report high-coverage sequencing of the Kitaake rice cultivar that enabled us to show that localized hypomethylation in rice endosperm occurs solely on the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana. However, unlike in A. thaliana, rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small class II (cut-and-paste) transposable elements, those in endosperm are more uniformly derived, including sequences from other transposon classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that localized hypomethylation of maternal endosperm DNA is conserved in flowering plants.

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Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator.

Publication Date: 2013 May 7 PMID: 23613579
Authors: Zalachoras, I. - Houtman, R. - Atucha, E. - Devos, R. - Tijssen, A. M. - Hu, P. - Lockey, P. M. - Datson, N. A. - Belanoff, J. K. - Lucassen, P. J. - Joels, M. - de Kloet, E. R. - Roozendaal, B. - Hunt, H. - Meijer, O. C.
Journal: Proc Natl Acad Sci U S A

Glucocorticoid receptor (GR) antagonism may be of considerable therapeutic value in stress-related psychopathology such as depression. However, blockade of all GR-dependent processes in the brain will lead to unnecessary and even counteractive effects, such as elevated endogenous cortisol levels. Selective GR modulators are ligands that can act both as agonist and as antagonist and may be used to separate beneficial from harmful treatment effects. We have discovered that the high-affinity GR ligand C108297 is a selective modulator in the rat brain. We first demonstrate that C108297 induces a unique interaction profile between GR and its downstream effector molecules, the nuclear receptor coregulators, compared with the full agonist dexamethasone and the antagonist RU486 (mifepristone). C108297 displays partial agonistic activity for the suppression of hypothalamic corticotropin-releasing hormone (CRH) gene expression and potently enhances GR-dependent memory consolidation of training on an inhibitory avoidance task. In contrast, it lacks agonistic effects on the expression of CRH in the central amygdala and antagonizes GR-mediated reduction in hippocampal neurogenesis after chronic corticosterone exposure. Importantly, the compound does not lead to disinhibition of the hypothalamus-pituitary-adrenal axis. Thus, C108297 represents a class of ligands that has the potential to more selectively abrogate pathogenic GR-dependent processes in the brain, while retaining beneficial aspects of GR signaling.

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Dnr1 mutations cause neurodegeneration in Drosophila by activating the innate immune response in the brain.

Publication Date: 2013 May 7 PMID: 23613578
Authors: Cao, Y. - Chtarbanova, S. - Petersen, A. J. - Ganetzky, B.
Journal: Proc Natl Acad Sci U S A

A growing body of evidence in humans implicates chronic activation of the innate immune response in the brain as a major cause of neuropathology in various neurodegenerative conditions, although the mechanisms remain unclear. In an unbiased genetic screen for mutants exhibiting neurodegeneration in Drosophila, we have recovered a mutation of dnr1 (defense repressor 1), a negative regulator of the Imd (immune deficiency) innate immune-response pathway. dnr1 mutants exhibit shortened lifespan and progressive, age-dependent neuropathology associated with activation of the Imd pathway and elevated expression of AMP (antimicrobial peptide) genes. To test the hypothesis that overactivation of innate immune-response pathways in the brain is responsible for neurodegeneration, we demonstrated that direct bacterial infection in the brain of wild-type flies also triggers neurodegeneration. In both cases, neurodegeneration is dependent on the NF-kappaB transcription factor, Relish. Moreover, we found that neural overexpression of individual AMP genes is sufficient to cause neurodegeneration. These results provide a mechanistic link between innate immune responses and neurodegeneration and may have important implications for the role of neuroinflammation in human neurodegenerative diseases as well.

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The molecular basis for Mucosal-Associated Invariant T cell recognition of MR1 proteins.

Publication Date: 2013 May 7 PMID: 23613577
Authors: Lopez-Sagaseta, J. - Dulberger, C. L. - Crooks, J. E. - Parks, C. D. - Luoma, A. M. - McFedries, A. - Van Rhijn, I. - Saghatelian, A. - Adams, E. J.
Journal: Proc Natl Acad Sci U S A

Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved alphabeta T-cell lineage that express a semi-invariant T-cell receptor (TCR) restricted to the MHC related-1 (MR1) protein. MAIT cells are dependent upon MR1 expression and exposure to microbes for their development and stimulation, yet these cells can exhibit microbial-independent stimulation when responding to MR1 from different species. We have used this microbial-independent, cross-species reactivity of MAIT cells to define the molecular basis of MAIT-TCR/MR1 engagement and present here a 2.85 A complex structure of a human MAIT-TCR bound to bovine MR1. The MR1 binding groove is similar in backbone structure to classical peptide-presenting MHC class I molecules (MHCp), yet is partially occluded by large aromatic residues that form cavities suitable for small ligand presentation. The docking of the MAIT-TCR on MR1 is perpendicular to the MR1 surface and straddles the MR1 alpha1 and alpha2 helices, similar to classical alphabeta TCR engagement of MHCp. However, the MAIT-TCR contacts are dominated by the alpha-chain, focused on the MR1 alpha2 helix. TCR beta-chain contacts are mostly through the variable CDR3beta loop that is positioned proximal to the CDR3alpha loop directly over the MR1 open groove. The elucidation of the MAIT TCR/MR1 complex structure explains how the semi-invariant MAIT-TCR engages the nonpolymorphic MR1 protein, and sheds light onto ligand discrimination by this cell type. Importantly, this structure also provides a critical link in our understanding of the evolution of alphabeta T-cell recognition of MHC and MHC-like ligands.

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Neuropeptides control life-phase transitions.

Publication Date: 2013 May 14 PMID: 23613576
Authors: Schoofs, L. - Beets, I.
Journal: Proc Natl Acad Sci U S A

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Autocrine signaling based selection of combinatorial antibodies that transdifferentiate human stem cells.

Publication Date: 2013 May 14 PMID: 23613575
Authors: Xie, J. - Zhang, H. - Yea, K. - Lerner, R. A.
Journal: Proc Natl Acad Sci U S A

We report here the generation of antibody agonists from intracellular combinatorial libraries that transdifferentiate human stem cells. Antibodies that are agonists for the granulocyte colony stimulating factor receptor were selected from intracellular libraries on the basis of their ability to activate signaling pathways in reporter cells. We used a specialized "near neighbor" approach in which the entire antibody library and its target receptor are cointegrated into the plasma membranes of a population of reporter cells. This format favors unusual interactions between receptors and their protein ligands and ensures that the antibody acts in an autocrine manner on the cells that produce it. Unlike the natural granulocyte-colony stimulating factor that activates cells to differentiate along a predetermined pathway, the isolated agonist antibodies transdifferentiated human myeloid lineage CD34+ bone marrow cells into neural progenitors. This transdifferentiation by agonist antibodies is different from more commonly used methods because initiation is agenetic. Antibodies that act at the plasma membrane may have therapeutic potential as agents that transdifferentiate autologous cells.

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Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation.

Publication Date: 2013 May 21 PMID: 23610442
Authors: Jurado, K. A. - Wang, H. - Slaughter, A. - Feng, L. - Kessl, J. J. - Koh, Y. - Wang, W. - Ballandras-Colas, A. - Patel, P. A. - Fuchs, J. R. - Kvaratskhelia, M. - Engelman, A.
Journal: Proc Natl Acad Sci U S A

Integration is essential for HIV-1 replication, and the viral integrase (IN) protein is an important therapeutic target. Allosteric IN inhibitors (ALLINIs) that engage the IN dimer interface at the binding site for the host protein lens epithelium-derived growth factor (LEDGF)/transcriptional coactivator p75 are an emerging class of small molecule antagonists. Consistent with the inhibition of a multivalent drug target, ALLINIs display steep antiviral dose-response curves ex vivo. ALLINIs multimerize IN protein and concordantly block its assembly with viral DNA in vitro, indicating that the disruption of two integration-associated functions, IN catalysis and the IN-LEDGF/p75 interaction, determines the multimode mechanism of ALLINI action. We now demonstrate that ALLINI potency is unexpectedly accounted for during the late phase of HIV-1 replication. The compounds promote virion IN multimerization and, reminiscent of class II IN mutations, block the formation of the electron-dense viral core and inhibit reverse transcription and integration in subsequently infected target cells. Mature virions are recalcitrant to ALLINI treatment, and compound potency during virus production is independent of the level of LEDGF/p75 expression. We conclude that cooperative multimerization of IN by ALLINIs together with the inability for LEDGF/p75 to effectively engage the virus during its egress from cells underscores the multimodal mechanism of ALLINI action. Our results highlight the versatile nature of allosteric inhibitors to primarily inhibit viral replication at a step that is distinct from the catalytic requirement for the target enzyme. The vulnerability of IN to small molecules during the late phase of HIV-1 replication unveils a pharmacological Achilles' heel for exploitation in clinical ALLINI development.

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Single-molecule analysis of combinatorial epigenomic states in normal and tumor cells.

Publication Date: 2013 May 7 PMID: 23610441
Authors: Murphy, P. J. - Cipriany, B. R. - Wallin, C. B. - Ju, C. Y. - Szeto, K. - Hagarman, J. A. - Benitez, J. J. - Craighead, H. G. - Soloway, P. D.
Journal: Proc Natl Acad Sci U S A

Proper placement of epigenetic marks on DNA and histones is fundamental to normal development, and perturbations contribute to a variety of disease states. Combinations of marks act together to control gene expression; therefore, detecting their colocalization is important, but because of technical challenges, such measurements are rarely reported. Instead, measurements of epigenetic marks are typically performed one at a time in a population of cells, and their colocalization is inferred by association. Here, we describe a single-molecule analytical approach that can perform direct detection of multiple epigenetic marks simultaneously and use it to identify mechanisms coordinating placement of three gene silencing marks, trimethylated histone H3 lysine 9, lysine 27 (H3K9me3, H3K27me3), and cytosine methylation (mC), in the normal and cancer genome. We show that H3K9me3 and mC are present together on individual chromatin fragments in mouse embryonic stem cells and that half of the H3K9me3 marks require mC for their placement. In contrast, mC and H3K27me3 coincidence is rare, and in fact, mC antagonizes H3K27me3 in both embryonic stem cells and primary mouse fibroblasts, indicating this antagonism is shared among primary cells. However, upon immortalization or tumorigenic transformation of mouse fibroblasts, mC is required for complete H3K27me3 placement. Importantly, in human promyelocytic cells, H3K27me3 is also dependent on mC. Because aberrant placement of gene silencing marks at tumor suppressor genes contributes to tumor progression, the improper dependency of H3K27me3 by mC in immortalized cells is likely to be fundamental to cancer. Our platform can enable other studies involving coordination of epigenetic marks and leverage efforts to discover disease biomarkers and epigenome-modifying drugs.

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Control of cell proliferation, endoreduplication, cell size, and cell death by the retinoblastoma-related pathway in maize endosperm.

Publication Date: 2013 May 7 PMID: 23610440
Authors: Sabelli, P. A. - Liu, Y. - Dante, R. A. - Lizarraga, L. E. - Nguyen, H. N. - Brown, S. W. - Klingler, J. P. - Yu, J. - Labrant, E. - Layton, T. M. - Feldman, M. - Larkins, B. A.
Journal: Proc Natl Acad Sci U S A

The endosperm of cereal grains is one of the most valuable products of modern agriculture. Cereal endosperm development comprises different phases characterized by mitotic cell proliferation, endoreduplication, the accumulation of storage compounds, and programmed cell death. Although manipulation of these processes could maximize grain yield, how they are regulated and integrated is poorly understood. We show that the Retinoblastoma-related (RBR) pathway controls key aspects of endosperm development in maize. Down-regulation of RBR1 by RNAi resulted in up-regulation of RBR3-type genes, as well as the MINICHROMOSOME MAINTENANCE 2-7 gene family and PROLIFERATING CELL NUCLEAR ANTIGEN, which encode essential DNA replication factors. Both the mitotic and endoreduplication cell cycles were stimulated. Developing transgenic endosperm contained 42-58% more cells and approximately 70% more DNA than wild type, whereas there was a reduction in cell and nuclear sizes. In addition, cell death was enhanced. The DNA content of mature endosperm increased 43% upon RBR1 down-regulation, whereas storage protein content and kernel weight were essentially not affected. Down-regulation of both RBR1 and CYCLIN DEPENDENT KINASE A (CDKA);1 indicated that CDKA;1 is epistatic to RBR1 and controls endoreduplication through an RBR1-dependent pathway. However, the repressive activity of RBR1 on downstream targets was independent from CDKA;1, suggesting diversification of RBR1 activities. Furthermore, RBR1 negatively regulated CDK activity, suggesting the presence of a feedback loop. These results indicate that the RBR1 pathway plays a major role in regulation of different processes during maize endosperm development and suggest the presence of tissue/organ-level regulation of endosperm/seed homeostasis.

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Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells.

Publication Date: 2013 May 7 PMID: 23610439
Authors: Truong, L. N. - Li, Y. - Shi, L. Z. - Hwang, P. Y. - He, J. - Wang, H. - Razavian, N. - Berns, M. W. - Wu, X.
Journal: Proc Natl Acad Sci U S A

Microhomology-mediated end joining (MMEJ) is a major pathway for Ku-independent alternative nonhomologous end joining, which contributes to chromosomal translocations and telomere fusions, but the underlying mechanism of MMEJ in mammalian cells is not well understood. In this study, we demonstrated that, distinct from Ku-dependent classical nonhomologous end joining, MMEJ-even with very limited end resection-requires cyclin-dependent kinase activities and increases significantly when cells enter S phase. We also showed that MMEJ shares the initial end resection step with homologous recombination (HR) by requiring meiotic recombination 11 homolog A (Mre11) nuclease activity, which is needed for subsequent recruitment of Bloom syndrome protein (BLM) and exonuclease 1 (Exo1) to DNA double-strand breaks (DSBs) to promote extended end resection and HR. MMEJ does not require S139-phosphorylated histone H2AX (gamma-H2AX), suggesting that initial end resection likely occurs at DSB ends. Using a MMEJ and HR competition repair substrate, we demonstrated that MMEJ with short end resection is used in mammalian cells at the level of 10-20% of HR when both HR and nonhomologous end joining are available. Furthermore, MMEJ is used to repair DSBs generated at collapsed replication forks. These studies suggest that MMEJ not only is a backup repair pathway in mammalian cells, but also has important physiological roles in repairing DSBs to maintain cell viability, especially under genomic stress.

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Mapping remodeling of thalamocortical projections in the living reeler mouse brain by diffusion tractography.

Publication Date: 2013 May 7 PMID: 23610438
Authors: Harsan, L. A. - David, C. - Reisert, M. - Schnell, S. - Hennig, J. - von Elverfeldt, D. - Staiger, J. F.
Journal: Proc Natl Acad Sci U S A

A major challenge in neuroscience is to accurately decipher in vivo the entire brain circuitry (connectome) at a microscopic level. Currently, the only methodology providing a global noninvasive window into structural brain connectivity is diffusion tractography. The extent to which the reconstructed pathways reflect realistic neuronal networks depends, however, on data acquisition and postprocessing factors. Through a unique combination of approaches, we designed and evaluated herein a framework for reliable fiber tracking and mapping of the living mouse brain connectome. One important wiring scheme, connecting gray matter regions and passing fiber-crossing areas, was closely examined: the lemniscal thalamocortical (TC) pathway. We quantitatively validated the TC projections inferred from in vivo tractography with correlative histological axonal tracing in the same wild-type and reeler mutant mice. We demonstrated noninvasively that changes in patterning of the cortical sheet, such as highly disorganized cortical lamination in reeler, led to spectacular compensatory remodeling of the TC pathway.

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Cigarette smoke (CS) and nicotine delay neutrophil spontaneous death via suppressing production of diphosphoinositol pentakisphosphate.

Publication Date: 2013 May 7 PMID: 23610437
Authors: Xu, Y. - Li, H. - Bajrami, B. - Kwak, H. - Cao, S. - Liu, P. - Zhou, J. - Zhou, Y. - Zhu, H. - Ye, K. - Luo, H. R.
Journal: Proc Natl Acad Sci U S A

Diphosphoinositol pentakisphosphate (InsP7), a higher inositol phosphate containing energetic pyrophosphate bonds, is beginning to emerge as a key cellular signaling molecule. However, the various physiological and pathological processes that involve InsP7 are not completely understood. Here we report that cigarette smoke (CS) extract and nicotine reduce InsP7 levels in aging neutrophils. This subsequently leads to suppression of Akt deactivation, a causal mediator of neutrophil spontaneous death, and delayed neutrophil death. The effect of CS extract and nicotine on neutrophil death can be suppressed by either directly inhibiting the PtdIns(3,4,5)P3/Akt pathway, or increasing InsP7 levels via overexpression of InsP6K1, an inositol hexakisphosphate (InsP6) kinase responsible for InsP7 production in neutrophils. Delayed neutrophil death contributes to the pathogenesis of CS-induced chronic obstructive pulmonary disease. Therefore, disruption of InsP6K1 augments CS-induced neutrophil accumulation and lung damage. Taken together, these results suggest that CS and nicotine delay neutrophil spontaneous death by suppressing InsP7 production and consequently blocking Akt deactivation in aging neutrophils. Modifying neutrophil death via this pathway provides a strategy and therapeutic target for the treatment of tobacco-induced chronic obstructive pulmonary disease.

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Genomic rearrangements and the evolution of clusters of locally adaptive loci.

Publication Date: 2013 May 7 PMID: 23610436
Authors: Yeaman, S.
Journal: Proc Natl Acad Sci U S A

Numerous studies of ecological genetics have found that alleles contributing to local adaptation sometimes cluster together, forming "genomic islands of divergence." Divergence hitchhiking theory posits that these clusters evolve by the preferential establishment of tightly linked locally adapted mutations, because such linkage reduces the rate that recombination breaks up locally favorable combinations of alleles. Here, I use calculations based on previously developed analytical models of divergence hitchhiking to show that very few clustered mutations should be expected in a single bout of adaptation, relative to the number of unlinked mutations, suggesting that divergence hitchhiking theory alone may often be insufficient to explain empirical observations. Using individual-based simulations that allow for the transposition of a single genetic locus from one position on a chromosome to another, I then show that tight clustering of the loci involved in local adaptation tends to evolve on biologically realistic time scales. These results suggest that genomic rearrangements may often be an important component of local adaptation and the evolution of genomic islands of divergence. More generally, these results suggest that genomic architecture and functional neighborhoods of genes may be actively shaped by natural selection in heterogeneous environments. Because small-scale changes in gene order are relatively common in some taxa, comparative genomic studies could be coupled with studies of adaptation to explore how commonly such rearrangements are involved in local adaptation.

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Characterizing deformability and surface friction of cancer cells.

Publication Date: 2013 May 7 PMID: 23610435
Authors: Byun, S. - Son, S. - Amodei, D. - Cermak, N. - Shaw, J. - Kang, J. H. - Hecht, V. C. - Winslow, M. M. - Jacks, T. - Mallick, P. - Manalis, S. R.
Journal: Proc Natl Acad Sci U S A

Metastasis requires the penetration of cancer cells through tight spaces, which is mediated by the physical properties of the cells as well as their interactions with the confined environment. Various microfluidic approaches have been devised to mimic traversal in vitro by measuring the time required for cells to pass through a constriction. Although a cell's passage time is expected to depend on its deformability, measurements from existing approaches are confounded by a cell's size and its frictional properties with the channel wall. Here, we introduce a device that enables the precise measurement of (i) the size of a single cell, given by its buoyant mass, (ii) the velocity of the cell entering a constricted microchannel (entry velocity), and (iii) the velocity of the cell as it transits through the constriction (transit velocity). Changing the deformability of the cell by perturbing its cytoskeleton primarily alters the entry velocity, whereas changing the surface friction by immobilizing positive charges on the constriction's walls primarily alters the transit velocity, indicating that these parameters can give insight into the factors affecting the passage of each cell. When accounting for cell buoyant mass, we find that cells possessing higher metastatic potential exhibit faster entry velocities than cells with lower metastatic potential. We additionally find that some cell types with higher metastatic potential exhibit greater than expected changes in transit velocities, suggesting that not only the increased deformability but reduced friction may be a factor in enabling invasive cancer cells to efficiently squeeze through tight spaces.

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Rapid cell death is preceded by amyloid plaque-mediated oxidative stress.

Publication Date: 2013 May 7 PMID: 23610434
Authors: Xie, H. - Hou, S. - Jiang, J. - Sekutowicz, M. - Kelly, J. - Bacskai, B. J.
Journal: Proc Natl Acad Sci U S A

Neuronal loss is the ultimate outcome in a variety of neurodegenerative diseases and central nerve system disorders. Understanding the sequelae of events that leads to cell death would provide insight into neuroprotective approaches. We imaged neurons in the living brain of a mouse model of Alzheimer's disease that overexpresses mutant human amyloid precursor protein and presenilin 1 and followed the death of individual neurons in real time. This mouse model exhibited limited neurodegeneration and atrophy, but we were able to identify a small fraction of vulnerable cells that would not have been detectable by using standard approaches. By exploiting a genetically encoded reporter of oxidative stress, we identified susceptible neurons by their increased redox potential. The oxidative stress was most dramatic in neurites near plaques, propagated to cell bodies, and preceded activation of caspases that led to cell death within 24 h. Thus, local oxidative stress surrounding plaques contributes to long-range toxicity and selective neuronal death in Alzheimer's disease.

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Long tethers provide high-force coupling of the Dam1 ring to shortening microtubules.

Publication Date: 2013 May 7 PMID: 23610433
Authors: Volkov, V. A. - Zaytsev, A. V. - Gudimchuk, N. - Grissom, P. M. - Gintsburg, A. L. - Ataullakhanov, F. I. - McIntosh, J. R. - Grishchuk, E. L.
Journal: Proc Natl Acad Sci U S A

Microtubule kinetochore attachments are essential for accurate mitosis, but how these force-generating connections move chromosomes remains poorly understood. Processive motion at shortening microtubule ends can be reconstituted in vitro using microbeads conjugated to the budding yeast kinetochore protein Dam1, which forms microtubule-encircling rings. Here, we report that, when Dam1 is linked to a bead cargo by elongated protein tethers, the maximum force transmitted from a disassembling microtubule increases sixfold compared with a short tether. We interpret this significant improvement with a theory that considers the geometry and mechanics of the microtubule-ring-bead system. Our results show the importance of fibrillar links in tethering microtubule ends to cargo: fibrils enable the cargo to align coaxially with the microtubule, thereby increasing the stability of attachment and the mechanical work that it can do. The force-transducing characteristics of fibril-tethered Dam1 are similar to the analogous properties of purified yeast kinetochores, suggesting that a tethered Dam1 ring comprises the main force-bearing unit of the native attachment.

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Soluble IL7Ralpha potentiates IL-7 bioactivity and promotes autoimmunity.

Publication Date: 2013 May 7 PMID: 23610432
Authors: Lundstrom, W. - Highfill, S. - Walsh, S. T. - Beq, S. - Morse, E. - Kockum, I. - Alfredsson, L. - Olsson, T. - Hillert, J. - Mackall, C. L.
Journal: Proc Natl Acad Sci U S A

Human soluble interleukin-7 receptor (sIL7R)alpha circulates in high molar excess compared with IL-7, but its biology remains unclear. We demonstrate that sIL7Ralpha has moderate affinity for IL-7 but does not bind thymic stromal lymphopoietin. Functionally, sIL7Ralpha competes with cell-associated IL-7 receptor to diminish excessive IL-7 consumption and, thus, enhances the bioactivity of IL-7 when the cytokine is limited, as it is presumed to be in vivo. IL-7 signaling in the presence of sIL7Ralpha also diminishes expression of CD95 and suppressor of cytokine signaling 1, both regulatory molecules. Murine models confirm diminished consumption of IL-7 in the presence of sIL7Ralpha and also demonstrate a potentiating effect of sIL7Ralpha on IL-7-mediated homeostatic expansion and experimental autoimmune encephalomyelitis exacerbation. In multiple sclerosis and several other autoimmune diseases, IL7R genotype influences susceptibility. We measured increased sIL7Ralpha levels, as well as increased IL-7 levels, in multiple sclerosis patients with the predisposing IL7R genotype, consistent with diminished IL-7 consumption in vivo. This work demonstrates that sIL7Ralpha potentiates IL-7 bioactivity and provides a basis to explain the increased risk of autoimmunity observed in individuals with genotype-induced elevations of sIL7Ralpha.

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Different 3D domain-swapped oligomeric cyanovirin-N structures suggest trapped folding intermediates.

Publication Date: 2013 May 7 PMID: 23610431
Authors: Koharudin, L. M. - Liu, L. - Gronenborn, A. M.
Journal: Proc Natl Acad Sci U S A

Although it has long been established that the amino acid sequence encodes the fold of a protein, how individual proteins arrive at their final conformation is still difficult to predict, especially for oligomeric structures. Here, we present a comprehensive characterization of oligomeric species of cyanovirin-N that all are formed by a polypeptide chain with the identical amino acid sequence. Structures of the oligomers were determined by X-ray crystallography, and each one exhibits 3D domain swapping. One unique 3D domain-swapped structure is observed for the trimer, while for both dimer and tetramer, two different 3D domain-swapped structures were obtained. In addition to the previously identified hinge-loop region of the 3D domain-swapped dimer, which resides between strands beta5 and beta6 in the middle of the polypeptide sequence, another hinge-loop region is observed between strands beta7 and beta8 in the structures. Plasticity in these two regions allows for variability in dihedral angles and concomitant differences in chain conformation that results in the differently 3D domain-swapped multimers. Based on all of the different structures, we propose possible folding pathways for this protein. Altogether, our results illuminate the amazing ability of cyanovirin-N to proceed down different folding paths and provide general insights into oligomer formation via 3D domain swapping.

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Conserved glycolipid termini in capsular polysaccharides synthesized by ATP-binding cassette transporter-dependent pathways in Gram-negative pathogens.

Publication Date: 2013 May 7 PMID: 23610430
Authors: Willis, L. M. - Stupak, J. - Richards, M. R. - Lowary, T. L. - Li, J. - Whitfield, C.
Journal: Proc Natl Acad Sci U S A

Bacterial capsules are surface layers made of long-chain polysaccharides. They are anchored to the outer membrane of many Gram-negative bacteria, including pathogens such as Escherichia coli, Neisseria meningitidis, Haemophilus influenzae, and Pasteurella multocida. Capsules protect pathogens from host defenses including complement-mediated killing and phagocytosis and therefore represent a major virulence factor. Capsular polysaccharides are synthesized by enzymes located in the inner (cytoplasmic) membrane and are then translocated to the cell surface. Whereas the enzymes that synthesize the polysaccharides have been studied in detail, the structure and biosynthesis of the anchoring elements have not been definitively resolved. Here we determine the structure of the glycolipid attached to the reducing terminus of the polysialic acid capsular polysaccharides from E. coli K1 and N. meningitidis group B and the heparosan-like capsular polysaccharide from E. coli K5. All possess the same unique glycolipid terminus consisting of a lyso-phosphatidylglycerol moiety with a beta-linked poly-(3-deoxy-d-manno-oct-2-ulosonic acid) (poly-Kdo) linker attached to the reducing terminus of the capsular polysaccharide.

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Interdomain lateral gene transfer of an essential ferrochelatase gene in human parasitic nematodes.

Publication Date: 2013 May 7 PMID: 23610429
Authors: Wu, B. - Novelli, J. - Jiang, D. - Dailey, H. A. - Landmann, F. - Ford, L. - Taylor, M. J. - Carlow, C. K. - Kumar, S. - Foster, J. M. - Slatko, B. E.
Journal: Proc Natl Acad Sci U S A

Lateral gene transfer events between bacteria and animals highlight an avenue for evolutionary genomic loss/gain of function. Herein, we report functional lateral gene transfer in animal parasitic nematodes. Members of the Nematoda are heme auxotrophs, lacking the ability to synthesize heme; however, the human filarial parasite Brugia malayi has acquired a bacterial gene encoding ferrochelatase (BmFeCH), the terminal step in heme biosynthesis. BmFeCH, encoded by a 9-exon gene, is a mitochondrial-targeted, functional ferrochelatase based on enzyme assays, complementation, and inhibitor studies. Homologs have been identified in several filariae and a nonfilarial nematode. RNAi and ex vivo inhibitor experiments indicate that BmFeCH is essential for viability, validating it as a potential target for filariasis control.

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Phase transition in the economically modeled growth of a cellular nervous system.

Publication Date: 2013 May 7 PMID: 23610428
Authors: Nicosia, V. - Vertes, P. E. - Schafer, W. R. - Latora, V. - Bullmore, E. T.
Journal: Proc Natl Acad Sci U S A

Spatially embedded complex networks, such as nervous systems, the Internet, and transportation networks, generally have nontrivial topological patterns of connections combined with nearly minimal wiring costs. However, the growth rules shaping these economical tradeoffs between cost and topology are not well understood. Here, we study the cellular nervous system of the nematode worm Caenorhabditis elegans, together with information on the birth times of neurons and on their spatial locations. We find that the growth of this network undergoes a transition from an accelerated to a constant increase in the number of links (synaptic connections) as a function of the number of nodes (neurons). The time of this phase transition coincides closely with the observed moment of hatching, when development switches metamorphically from oval to larval stages. We use graph analysis and generative modeling to show that the transition between different growth regimes, as well as its coincidence with the moment of hatching, may be explained by a dynamic economical model that incorporates a tradeoff between topology and cost that is continuously negotiated and renegotiated over developmental time. As the body of the animal progressively elongates, the cost of longer-distance connections is increasingly penalized. This growth process regenerates many aspects of the adult nervous system's organization, including the neuronal membership of anatomically predefined ganglia. We expect that similar economical principles may be found in the development of other biological or manmade spatially embedded complex systems.

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Bats are a major natural reservoir for hepaciviruses and pegiviruses.

Publication Date: 2013 May 14 PMID: 23610427
Authors: Quan, P. L. - Firth, C. - Conte, J. M. - Williams, S. H. - Zambrana-Torrelio, C. M. - Anthony, S. J. - Ellison, J. A. - Gilbert, A. T. - Kuzmin, I. V. - Niezgoda, M. - Osinubi, M. O. - Recuenco, S. - Markotter, W. - Breiman, R. F. - Kalemba, L. - Malekani, J. - Lindblade, K. A. - Rostal, M. K. - Ojeda-Flores, R. - Suzan, G. - Davis, L. B. - Blau, D. M. - Ogunkoya, A. B. - Alvarez Castillo, D. A. - Moran, D. - Ngam, S. - Akaibe, D. - Agwanda, B. - Briese, T. - Epstein, J. H. - Daszak, P. - Rupprecht, C. E. - Holmes, E. C. - Lipkin, W. I.
Journal: Proc Natl Acad Sci U S A

Although there are over 1,150 bat species worldwide, the diversity of viruses harbored by bats has only recently come into focus as a result of expanded wildlife surveillance. Such surveys are of importance in determining the potential for novel viruses to emerge in humans, and for optimal management of bats and their habitats. To enhance our knowledge of the viral diversity present in bats, we initially surveyed 415 sera from African and Central American bats. Unbiased high-throughput sequencing revealed the presence of a highly diverse group of bat-derived viruses related to hepaciviruses and pegiviruses within the family Flaviridae. Subsequent PCR screening of 1,258 bat specimens collected worldwide indicated the presence of these viruses also in North America and Asia. A total of 83 bat-derived viruses were identified, representing an infection rate of nearly 5%. Evolutionary analyses revealed that all known hepaciviruses and pegiviruses, including those previously documented in humans and other primates, fall within the phylogenetic diversity of the bat-derived viruses described here. The prevalence, unprecedented viral biodiversity, phylogenetic divergence, and worldwide distribution of the bat-derived viruses suggest that bats are a major and ancient natural reservoir for both hepaciviruses and pegiviruses and provide insights into the evolutionary history of hepatitis C virus and the human GB viruses.

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Disorder guides protein function.

Publication Date: 2013 Apr 30 PMID: 23610426
Authors: Whitford, P. C.
Journal: Proc Natl Acad Sci U S A

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Terrestrial cooling in Northern Europe during the Eocene-Oligocene transition.

Publication Date: 2013 May 7 PMID: 23610424
Authors: Hren, M. T. - Sheldon, N. D. - Grimes, S. T. - Collinson, M. E. - Hooker, J. J. - Bugler, M. - Lohmann, K. C.
Journal: Proc Natl Acad Sci U S A

Geochemical and modeling studies suggest that the transition from the "greenhouse" state of the Late Eocene to the "icehouse" conditions of the Oligocene 34-33.5 Ma was triggered by a reduction of atmospheric pCO2 that enabled the rapid buildup of a permanent ice sheet on the Antarctic continent. Marine records show that the drop in pCO2 during this interval was accompanied by a significant decline in high-latitude sea surface and deep ocean temperature and enhanced seasonality in middle and high latitudes. However, terrestrial records of this climate transition show heterogeneous responses to changing pCO2 and ocean temperatures, with some records showing a significant time lag in the temperature response to declining pCO2. We measured the Delta47 of aragonite shells of the freshwater gastropod Viviparus lentus from the Solent Group, Hampshire Basin, United Kingdom, to reconstruct terrestrial temperature and hydrologic change in the North Atlantic region during the Eocene-Oligocene transition. Our data show a decrease in growing-season surface water temperatures ( approximately 10 degrees C) during the Eocene-Oligocene transition, corresponding to an average decrease in mean annual air temperature of approximately 4-6 degrees C from the Late Eocene to Early Oligocene. The magnitude of cooling is similar to observed decreases in North Atlantic sea surface temperature over this interval and occurs during major glacial expansion. This suggests a close linkage between atmospheric carbon dioxide concentrations, Northern Hemisphere temperature, and expansion of the Antarctic ice sheets.

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Geometric control of vascular networks to enhance engineered tissue integration and function.

Publication Date: 2013 May 7 PMID: 23610423
Authors: Baranski, J. D. - Chaturvedi, R. R. - Stevens, K. R. - Eyckmans, J. - Carvalho, B. - Solorzano, R. D. - Yang, M. T. - Miller, J. S. - Bhatia, S. N. - Chen, C. S.
Journal: Proc Natl Acad Sci U S A

Tissue vascularization and integration with host circulation remains a key barrier to the translation of engineered tissues into clinically relevant therapies. Here, we used a microtissue molding approach to demonstrate that constructs containing highly aligned "cords" of endothelial cells triggered the formation of new capillaries along the length of the patterned cords. These vessels became perfused with host blood as early as 3 d post implantation and became progressively more mature through 28 d. Immunohistochemical analysis showed that the neovessels were composed of human and mouse endothelial cells and exhibited a mature phenotype, as indicated by the presence of alpha-smooth muscle actin-positive pericytes. Implantation of cords with a prescribed geometry demonstrated that they provided a template that defined the neovascular architecture in vivo. To explore the utility of this geometric control, we implanted primary rat and human hepatocyte constructs containing randomly organized endothelial networks vs. ordered cords. We found substantially enhanced hepatic survival and function in the constructs containing ordered cords following transplantation in mice. These findings demonstrate the importance of multicellular architecture in tissue integration and function, and our approach provides a unique strategy to engineer vascular architecture.

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Nontoxic radioactive Listeriaat is a highly effective therapy against metastatic pancreatic cancer.

Publication Date: 2013 May 21 PMID: 23610422
Authors: Quispe-Tintaya, W. - Chandra, D. - Jahangir, A. - Harris, M. - Casadevall, A. - Dadachova, E. - Gravekamp, C.
Journal: Proc Natl Acad Sci U S A

No significant improvement in therapy of pancreatic cancer has been reported over the last 25 y, underscoring the urgent need for new alternative therapies. Here, we coupled a radioisotope, (188)Rhenium, to an attenuated (at) live Listeria monocytogenes (Listeria(at)) using Listeria-binding antibodies, thus creating a unique radioactive Listeria(at) (RL). We then demonstrated in a highly metastatic pancreatic mouse tumor model (Panc-02) that RL delivered radioactivity to the metastases and less abundantly to primary tumors in vivo, without harming normal cells. This result was possible because Listeria(at) was efficiently cleared by the immune system in normal tissues but not in the heavily immune-suppressed microenvironment of metastases and primary tumor. Multiple treatments with low doses of the RL resulted in a dramatic decrease in the number of metastases ( approximately 90%) compared with control groups in the Panc-02 model. This is the first report of using live attenuated bacteria delivering a highly radioactive payload to the metastases, resulting in killing tumor cells in vivo without harming normal cells. The nontoxic RL treatment is attractive for clinical development as a therapy to prevent pancreatic cancer recurrence and metastases.

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Holo-TFIID controls the magnitude of a transcription burst and fine-tuning of transcription.

Publication Date: 2013 May 7 PMID: 23610421
Authors: Pennington, K. L. - Marr, S. K. - Chirn, G. W. - Marr, M. T. 2nd
Journal: Proc Natl Acad Sci U S A

Transcription factor (TF)IID is a central player in activated transcription initiation. Recent evidence suggests that the role and composition of TFIID are more diverse than previously understood. To investigate the effects of changing the composition of TFIID in a simple system, we depleted TATA box-binding protein-associated factor (TAF)1 from Drosophila cells and determined the consequences on metal-induced transcription at an inducible gene, metallothionein B. We observe a marked increase in the levels of both the mature message and pre-mRNA in TAF1-depleted cells. Under conditions of continued metal exposure, we show that TAF1 depletion increases the magnitude of the initial transcription burst but has no effect on the timing of that burst. We also show that TAF1 depletion causes delay in the shutoff of transcription upon removal of the stimulus. Thus, TAFs are involved in both establishing an upper limit of transcription during induction and efficiently turning the gene off once the inducer is removed. Using genome-wide nascent sequencing, we identify hundreds of genes that are controlled in a similar manner, indicating that the findings at this inducible gene are likely generalizable to a large set of promoters. There is a long-standing appreciation for the importance of the spatial and temporal control of transcription. Here we uncover an important third dimension of control: the magnitude of the response. Our results show that the magnitude of the transcriptional response to the same signaling event, even at the same promoter, can vary greatly depending on the composition of the TFIID complex in the cell.

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Accumulation of the FACT complex, as well as histone H3.3, serves as a target marker for somatic hypermutation.

Publication Date: 2013 May 7 PMID: 23610419
Authors: Aida, M. - Hamad, N. - Stanlie, A. - Begum, N. A. - Honjo, T.
Journal: Proc Natl Acad Sci U S A

Somatic hypermutation (SHM) requires not only the expression of activation-induced cytidine deaminase, but also transcription in the target regions. However, how transcription guides activation-induced cytidine deaminase in targeting SHM to the Ig genes is not fully understood. Here, we found that the "facilitates chromatin transcription" (FACT) complex promotes SHM by RNAi screening of transcription elongation factors. Furthermore, FACT and histone H3.3, a hallmark of transcription-coupled histone turnover, are enriched at the V(D)J region, 5' flanking sequence of the Smu switch region and the light chain Jkappa 5 segment region in the Ig loci. The regions with the most abundant deposition of FACT and H3.3 were also the most efficient targets of SHM. These results demonstrate the importance of histone-exchanging dynamics at the chromatin of SHM targets, especially in Ig genes.

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Pre-Miocene birth of the Yangtze River.

Publication Date: 2013 May 7 PMID: 23610418
Authors: Zheng, H. - Clift, P. D. - Wang, P. - Tada, R. - Jia, J. - He, M. - Jourdan, F.
Journal: Proc Natl Acad Sci U S A

The development of fluvial systems in East Asia is closely linked to the evolving topography following India-Eurasia collision. Despite this, the age of the Yangtze River system has been strongly debated, with estimates ranging from 40 to 45 Ma, to a more recent initiation around 2 Ma. Here, we present (40)Ar/(39)Ar ages from basalts interbedded with fluvial sediments from the lower reaches of the Yangtze together with detrital zircon U-Pb ages from sand grains within these sediments. We show that a river containing sediments indistinguishable from the modern river was established before approximately 23 Ma. We argue that the connection through the Three Gorges must postdate 36.5 Ma because of evaporite and lacustrine sedimentation in the Jianghan Basin before that time. We propose that the present Yangtze River system formed in response to regional extension throughout eastern China, synchronous with the start of strike-slip tectonism and surface uplift in eastern Tibet and fed by strengthened rains caused by the newly intensified summer monsoon.

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Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis.

Publication Date: 2013 May 7 PMID: 23610417
Authors: Gantt, R. W. - Peltier-Pain, P. - Singh, S. - Zhou, M. - Thorson, J. S.
Journal: Proc Natl Acad Sci U S A

We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in kcat/Km of >400-fold and >15-fold for formation of NDP-glucoses and UDP-sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP-sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.

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PCNA is efficiently loaded on the DNA recombination intermediate to modulate polymerase delta, eta, and zeta activities.

Publication Date: 2013 May 7 PMID: 23610416
Authors: Li, J. - Holzschu, D. L. - Sugiyama, T.
Journal: Proc Natl Acad Sci U S A

Proliferating cell nuclear antigen (PCNA) is required for DNA homologous recombination (HR), but its exact role is unclear. Here, we investigated the loading of PCNA onto a synthetic D-loop (DL) intermediate of HR and the functional interactions of PCNA with Rad51 recombinase and DNA polymerase (Pol) delta, Pol eta, and Pol zeta. PCNA was loaded onto the synthetic DL as efficiently as it was loaded onto a primed DNA substrate. Efficient PCNA loading requires Replication Protein A, which is associated with the displaced ssDNA loop and provides a binding site for the clamp-loader Replication Factor C. Loaded PCNA greatly stimulates DNA synthesis by Pol delta within the DL but does not affect primer recognition by Pol delta. This suggests that the essential role of PCNA in HR is not recruitment of Pol delta to the DL but stimulation of Pol delta to displace a DNA strand during DL extension. Both Pol eta and Pol zeta extended the DL more efficiently than Pol delta in the absence of PCNA, but little or no stimulation was observed in the presence of PCNA. Finally, Rad51 inhibited both the loading of PCNA onto the DL and the extension of the DL by Pol delta and Pol eta. However, preloaded PCNA on the DL counteracts the Rad51-mediated inhibition of the DL extension. This suggests that the inhibition of postinvasion DNA synthesis by Rad51 occurs mostly at the step of PCNA loading.

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Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli.

Publication Date: 2013 May 7 PMID: 23610415
Authors: Howard, T. P. - Middelhaufe, S. - Moore, K. - Edner, C. - Kolak, D. M. - Taylor, G. N. - Parker, D. A. - Lee, R. - Smirnoff, N. - Aves, S. J. - Love, J.
Journal: Proc Natl Acad Sci U S A

Biofuels are the most immediate, practical solution for mitigating dependence on fossil hydrocarbons, but current biofuels (alcohols and biodiesels) require significant downstream processing and are not fully compatible with modern, mass-market internal combustion engines. Rather, the ideal biofuels are structurally and chemically identical to the fossil fuels they seek to replace (i.e., aliphatic n- and iso-alkanes and -alkenes of various chain lengths). Here we report on production of such petroleum-replica hydrocarbons in Escherichia coli. The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (Cn) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane Cn and expression of the branched-chain alpha-keto acid dehydrogenase complex and beta-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.

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A monocular contribution to stimulus rivalry.

Publication Date: 2013 May 21 PMID: 23610414
Authors: Brascamp, J. - Sohn, H. - Lee, S. H. - Blake, R.
Journal: Proc Natl Acad Sci U S A

When corresponding areas of the two eyes view dissimilar images, stable perception gives way to visual competition wherein perceptual awareness alternates between those images. Moreover, a given image can remain visually dominant for several seconds at a time even when the competing images are swapped between the eyes multiple times each second. This perceptual stability across eye swaps has led to the widespread belief that this unique form of visual competition, dubbed stimulus rivalry, is governed by eye-independent neural processes at a purely binocular stage of cortical processing. We tested this idea by investigating the influence of stimulus rivalry on the buildup of the threshold elevation aftereffect, a form of contrast adaptation thought to transpire at early cortical stages that include eye-specific neural activity. Weaker threshold elevation aftereffects were observed when the adapting image was engaged in stimulus rivalry than when it was not, indicating diminished buildup of adaptation during stimulus-rivalry suppression. We then confirmed that this reduction occurred, in part, at eye-specific neural stages by showing that suppression of an image at a given moment specifically diminished adaptation associated with the eye viewing the image at that moment. Considered together, these results imply that eye-specific neural events at early cortical processing stages contribute to stimulus rivalry. We have developed a computational model of stimulus rivalry that successfully implements this idea.

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Castor is required for Hedgehog-dependent cell-fate specification and follicle stem cell maintenance in Drosophila oogenesis.

Publication Date: 2013 May 7 PMID: 23610413
Authors: Chang, Y. C. - Jang, A. C. - Lin, C. H. - Montell, D. J.
Journal: Proc Natl Acad Sci U S A

Asymmetric division of stem cells results in both self-renewal and differentiation of daughters. Understanding the molecules and mechanisms that govern differentiation of specific cell types from adult tissue stem cells is a major challenge in developmental biology and regenerative medicine. Drosophila follicle stem cells (FSCs) represent an excellent model system to study adult stem cell behavior; however, the earliest stages of follicle cell differentiation remain largely mysterious. Here we identify Castor (Cas) as a nuclear protein that is expressed in FSCs and early follicle cell precursors and then is restricted to differentiated polar and stalk cells once egg chambers form. Cas is required for FSC maintenance and polar and stalk cell fate specification. Eyes absent (Eya) is excluded from polar and stalk cells and represses their fate by inhibiting Cas expression. Hedgehog signaling is essential to repress Eya to allow Cas expression in polar and stalk cells. Finally, we show that the complementary patterns of Cas and Eya reveal the gradual differentiation of polar and stalk precursor cells at the earliest stages of their development. Our studies provide a marker for cell fates in this model and insight into the molecular and cellular mechanisms by which FSC progeny diverge into distinct fates.

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Transient formation of water-conducting states in membrane transporters.

Publication Date: 2013 May 7 PMID: 23610412
Authors: Li, J. - Shaikh, S. A. - Enkavi, G. - Wen, P. C. - Huang, Z. - Tajkhorshid, E.
Journal: Proc Natl Acad Sci U S A

Membrane transporters rely on highly coordinated structural transitions between major conformational states for their function, to prevent simultaneous access of the substrate binding site to both sides of the membrane-a mode of operation known as the alternating access model. Although this mechanism successfully accounts for the efficient exchange of the primary substrate across the membrane, accruing evidence on significant water transport and even uncoupled ion transport mediated by transporters has challenged the concept of perfect mechanical coupling and coordination of the gating mechanism in transporters, which might be expected from the alternating access model. Here, we present a large set of extended equilibrium molecular dynamics simulations performed on several classes of membrane transporters in different conformational states, to test the presence of the phenomenon in diverse transporter classes and to investigate the underlying molecular mechanism of water transport through membrane transporters. The simulations reveal spontaneous formation of transient water-conducting (channel-like) states allowing passive water diffusion through the lumen of the transporters. These channel-like states are permeable to water but occluded to substrate, thereby not hindering the uphill transport of the primary substrate, i.e., the alternating access model remains applicable to the substrate. The rise of such water-conducting states during the large-scale structural transitions of the transporter protein is indicative of imperfections in the coordinated closing and opening motions of the cytoplasmic and extracellular gates. We propose that the observed water-conducting states likely represent a universal phenomenon in membrane transporters, which is consistent with their reliance on large-scale motion for function.

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Renal intercalated cells are rather energized by a proton than a sodium pump.

Publication Date: 2013 May 7 PMID: 23610411
Authors: Chambrey, R. - Kurth, I. - Peti-Peterdi, J. - Houillier, P. - Purkerson, J. M. - Leviel, F. - Hentschke, M. - Zdebik, A. A. - Schwartz, G. J. - Hubner, C. A. - Eladari, D.
Journal: Proc Natl Acad Sci U S A

The Na(+) concentration of the intracellular milieu is very low compared with the extracellular medium. Transport of Na(+) along this gradient is used to fuel secondary transport of many solutes, and thus plays a major role for most cell functions including the control of cell volume and resting membrane potential. Because of a continuous leak, Na(+) has to be permanently removed from the intracellular milieu, a process that is thought to be exclusively mediated by the Na(+)/K(+)-ATPase in animal cells. Here, we show that intercalated cells of the mouse kidney are an exception to this general rule. By an approach combining two-photon imaging of isolated renal tubules, physiological studies, and genetically engineered animals, we demonstrate that inhibition of the H(+) vacuolar-type ATPase (V-ATPase) caused drastic cell swelling and depolarization, and also inhibited the NaCl absorption pathway that we recently discovered in intercalated cells. In contrast, pharmacological blockade of the Na(+)/K(+)-ATPase had no effects. Basolateral NaCl exit from beta-intercalated cells was independent of the Na(+)/K(+)-ATPase but critically relied on the presence of the basolateral ion transporter anion exchanger 4. We conclude that not all animal cells critically rely on the sodium pump as the unique bioenergizer, but can be replaced by the H(+) V-ATPase in renal intercalated cells. This concept is likely to apply to other animal cell types characterized by plasma membrane expression of the H(+) V-ATPase.

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Structures of complexes comprised of Fischerella transcription factor HetR with Anabaena DNA targets.

Publication Date: 2013 May 7 PMID: 23610410
Authors: Kim, Y. - Ye, Z. - Joachimiak, G. - Videau, P. - Young, J. - Hurd, K. - Callahan, S. M. - Gornicki, P. - Zhao, J. - Haselkorn, R. - Joachimiak, A.
Journal: Proc Natl Acad Sci U S A

HetR is an essential regulator of heterocyst development in cyanobacteria. Many mutations in HetR render Anabaena incapable of nitrogen fixation. The protein binds to a DNA palindrome upstream of hetP and other genes. We have determined the crystal structures of HetR complexed with palindromic DNA targets, 21, 23, and 29 bp at 2.50-, 3.00-, and 3.25-A resolution, respectively. The highest-resolution structure shows fine details of specific protein-DNA interactions. The lower-resolution structures with longer DNA duplexes have similar interaction patterns and show how the flap domains interact with DNA in a sequence nonspecific fashion. Fifteen of 15 protein-DNA contacts predicted on the basis of the structure were confirmed by single amino acid mutations that abolished binding in vitro and complementation in vivo. A striking feature of the structure is the association of glutamate 71 from each subunit of the HetR dimer with three successive cytosines in each arm of the palindromic target, a feature that is conserved among all known heterocyst-forming cyanobacteria sequenced to date.

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Origin of intrinsic irregular firing in cortical interneurons.

Publication Date: 2013 May 7 PMID: 23610409
Authors: Stiefel, K. M. - Englitz, B. - Sejnowski, T. J.
Journal: Proc Natl Acad Sci U S A

Cortical spike trains are highly irregular both during ongoing, spontaneous activity and when driven at high firing rates. There is uncertainty about the source of this irregularity, ranging from intrinsic noise sources in neurons to collective effects in large-scale cortical networks. Cortical interneurons display highly irregular spike times (coefficient of variation of the interspike intervals >1) in response to dc-current injection in vitro. This is in marked contrast to cortical pyramidal cells, which spike highly irregularly in vivo, but regularly in vitro. We show with in vitro recordings and computational models that this is due to the fast activation kinetics of interneuronal K(+) currents. This explanation holds over a wide parameter range and with Gaussian white, power-law, and Ornstein-Uhlenbeck noise. The intrinsically irregular spiking of interneurons could contribute to the irregularity of the cortical network.

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A 4-gigabase physical map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the wheat D-genome progenitor.

Publication Date: 2013 May 7 PMID: 23610408
Authors: Luo, M. C. - Gu, Y. Q. - You, F. M. - Deal, K. R. - Ma, Y. - Hu, Y. - Huo, N. - Wang, Y. - Wang, J. - Chen, S. - Jorgensen, C. M. - Zhang, Y. - McGuire, P. E. - Pasternak, S. - Stein, J. C. - Ware, D. - Kramer, M. - McCombie, W. R. - Kianian, S. F. - Martis, M. M. - Mayer, K. F. - Sehgal, S. K. - Li, W. - Gill, B. S. - Bevan, M. W. - Simkova, H. - Dolezel, J. - Weining, S. - Lazo, G. R. - Anderson, O. D. - Dvorak, J.
Journal: Proc Natl Acad Sci U S A

The current limitations in genome sequencing technology require the construction of physical maps for high-quality draft sequences of large plant genomes, such as that of Aegilops tauschii, the wheat D-genome progenitor. To construct a physical map of the Ae. tauschii genome, we fingerprinted 461,706 bacterial artificial chromosome clones, assembled contigs, designed a 10K Ae. tauschii Infinium SNP array, constructed a 7,185-marker genetic map, and anchored on the map contigs totaling 4.03 Gb. Using whole genome shotgun reads, we extended the SNP marker sequences and found 17,093 genes and gene fragments. We showed that collinearity of the Ae. tauschii genes with Brachypodium distachyon, rice, and sorghum decreased with phylogenetic distance and that structural genome evolution rates have been high across all investigated lineages in subfamily Pooideae, including that of Brachypodieae. We obtained additional information about the evolution of the seven Triticeae chromosomes from 12 ancestral chromosomes and uncovered a pattern of centromere inactivation accompanying nested chromosome insertions in grasses. We showed that the density of noncollinear genes along the Ae. tauschii chromosomes positively correlates with recombination rates, suggested a cause, and showed that new genes, exemplified by disease resistance genes, are preferentially located in high-recombination chromosome regions.

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Drosophila ORB protein in two mushroom body output neurons is necessary for long-term memory formation.

Publication Date: 2013 May 7 PMID: 23610406
Authors: Pai, T. P. - Chen, C. C. - Lin, H. H. - Chin, A. L. - Lai, J. S. - Lee, P. T. - Tully, T. - Chiang, A. S.
Journal: Proc Natl Acad Sci U S A

Memory is initially labile and gradually consolidated over time through new protein synthesis into a long-lasting stable form. Studies of odor-shock associative learning in Drosophila have established the mushroom body (MB) as a key brain structure involved in olfactory long-term memory (LTM) formation. Exactly how early neural activity encoded in thousands of MB neurons is consolidated into protein-synthesis-dependent LTM remains unclear. Here, several independent lines of evidence indicate that changes in two MB vertical lobe V3 (MB-V3) extrinsic neurons are required and contribute to an extended neural network involved in olfactory LTM: (i) inhibiting protein synthesis in MB-V3 neurons impairs LTM; (ii) MB-V3 neurons show enhanced neural activity after spaced but not massed training; (iii) MB-V3 dendrites, synapsing with hundreds of MB alpha/beta neurons, exhibit dramatic structural plasticity after removal of olfactory inputs; (iv) neurotransmission from MB-V3 neurons is necessary for LTM retrieval; and (v) RNAi-mediated down-regulation of oo18 RNA-binding protein (involved in local regulation of protein translation) in MB-V3 neurons impairs LTM. Our results suggest a model of long-term memory formation that includes a systems-level consolidation process, wherein an early, labile olfactory memory represented by neural activity in a sparse subset of MB neurons is converted into a stable LTM through protein synthesis in dendrites of MB-V3 neurons synapsed onto MB alpha lobes.

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TFEB-mediated autophagy rescues midbrain dopamine neurons from alpha-synuclein toxicity.

Publication Date: 2013 May 7 PMID: 23610405
Authors: Decressac, M. - Mattsson, B. - Weikop, P. - Lundblad, M. - Jakobsson, J. - Bjorklund, A.
Journal: Proc Natl Acad Sci U S A

The aggregation of alpha-synuclein plays a major role in Parkinson disease (PD) pathogenesis. Recent evidence suggests that defects in the autophagy-mediated clearance of alpha-synuclein contribute to the progressive loss of nigral dopamine neurons. Using an in vivo model of alpha-synuclein toxicity, we show that the PD-like neurodegenerative changes induced by excess cellular levels of alpha-synuclein in nigral dopamine neurons are closely linked to a progressive decline in markers of lysosome function, accompanied by cytoplasmic retention of transcription factor EB (TFEB), a major transcriptional regulator of the autophagy-lysosome pathway. The changes in lysosomal function, observed in the rat model as well as in human PD midbrain, were reversed by overexpression of TFEB, which afforded robust neuroprotection via the clearance of alpha-synuclein oligomers, and were aggravated by microRNA-128-mediated repression of TFEB in both A9 and A10 dopamine neurons. Delayed activation of TFEB function through inhibition of mammalian target of rapamycin blocked alpha-synuclein induced neurodegeneration and further disease progression. The results provide a mechanistic link between alpha-synuclein toxicity and impaired TFEB function, and highlight TFEB as a key player in the induction of alpha-synuclein-induced toxicity and PD pathogenesis, thus identifying TFEB as a promising target for therapies aimed at neuroprotection and disease modification in PD.

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Neutral forces acting on intragenomic variability shape the Escherichia coli regulatory network topology.

Publication Date: 2013 May 7 PMID: 23610404
Authors: Ruths, T. - Nakhleh, L.
Journal: Proc Natl Acad Sci U S A

Cis-regulatory networks (CRNs) play a central role in cellular decision making. Like every other biological system, CRNs undergo evolution, which shapes their properties by a combination of adaptive and nonadaptive evolutionary forces. Teasing apart these forces is an important step toward functional analyses of the different components of CRNs, designing regulatory perturbation experiments, and constructing synthetic networks. Although tests of neutrality and selection based on molecular sequence data exist, no such tests are currently available based on CRNs. In this work, we present a unique genotype model of CRNs that is grounded in a genomic context and demonstrate its use in identifying portions of the CRN with properties explainable by neutral evolutionary forces at the system, subsystem, and operon levels. We leverage our model against experimentally derived data from Escherichia coli. The results of this analysis show statistically significant and substantial neutral trends in properties previously identified as adaptive in origin-degree distribution, clustering coefficient, and motifs-within the E. coli CRN. Our model captures the tightly coupled genome-interactome of an organism and enables analyses of how evolutionary events acting at the genome level, such as mutation, and at the population level, such as genetic drift, give rise to neutral patterns that we can quantify in CRNs.

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All-optical control of a solid-state spin using coherent dark states.

Publication Date: 2013 May 7 PMID: 23610403
Authors: Yale, C. G. - Buckley, B. B. - Christle, D. J. - Burkard, G. - Heremans, F. J. - Bassett, L. C. - Awschalom, D. D.
Journal: Proc Natl Acad Sci U S A

The study of individual quantum systems in solids, for use as quantum bits (qubits) and probes of decoherence, requires protocols for their initialization, unitary manipulation, and readout. In many solid-state quantum systems, these operations rely on disparate techniques that can vary widely depending on the particular qubit structure. One such qubit, the nitrogen-vacancy (NV) center spin in diamond, can be initialized and read out through its special spin-selective intersystem crossing, while microwave electron spin resonance techniques provide unitary spin rotations. Instead, we demonstrate an alternative, fully optical approach to these control protocols in an NV center that does not rely on its intersystem crossing. By tuning an NV center to an excited-state spin anticrossing at cryogenic temperatures, we use coherent population trapping and stimulated Raman techniques to realize initialization, readout, and unitary manipulation of a single spin. Each of these techniques can be performed directly along any arbitrarily chosen quantum basis, removing the need for extra control steps to map the spin to and from a preferred basis. Combining these protocols, we perform measurements of the NV center's spin coherence, a demonstration of this full optical control. Consisting solely of optical pulses, these techniques enable control within a smaller footprint and within photonic networks. Likewise, this unified approach obviates the need for both electron spin resonance manipulation and spin addressability through the intersystem crossing. This method could therefore be applied to a wide range of potential solid-state qubits, including those which currently lack a means to be addressed.

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Limits in decision making arise from limits in memory retrieval.

Publication Date: 2013 May 7 PMID: 23610402
Authors: Giguere, G. - Love, B. C.
Journal: Proc Natl Acad Sci U S A

Some decisions, such as predicting the winner of a baseball game, are challenging in part because outcomes are probabilistic. When making such decisions, one view is that humans stochastically and selectively retrieve a small set of relevant memories that provides evidence for competing options. We show that optimal performance at test is impossible when retrieving information in this fashion, no matter how extensive training is, because limited retrieval introduces noise into the decision process that cannot be overcome. One implication is that people should be more accurate in predicting future events when trained on idealized rather than on the actual distributions of items. In other words, we predict the best way to convey information to people is to present it in a distorted, idealized form. Idealization of training distributions is predicted to reduce the harmful noise induced by immutable bottlenecks in people's memory retrieval processes. In contrast, machine learning systems that selectively weight (i.e., retrieve) all training examples at test should not benefit from idealization. These conjectures are strongly supported by several studies and supporting analyses. Unlike machine systems, people's test performance on a target distribution is higher when they are trained on an idealized version of the distribution rather than on the actual target distribution. Optimal machine classifiers modified to selectively and stochastically sample from memory match the pattern of human performance. These results suggest firm limits on human rationality and have broad implications for how to train humans tasked with important classification decisions, such as radiologists, baggage screeners, intelligence analysts, and gamblers.

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Diversification of the eutherian placenta is associated with changes in the pace of life.

Publication Date: 2013 May 7 PMID: 23610401
Authors: Garratt, M. - Gaillard, J. M. - Brooks, R. C. - Lemaitre, J. F.
Journal: Proc Natl Acad Sci U S A

Few mammalian organs vary as dramatically among species as the placenta. This variation is remarkable considering that the placenta's primary function-transfer of nutrients and waste between mother and offspring-does not differ among species. Evolutionary changes in placental morphology remain poorly understood, with suggestions that parent-offspring conflict or evolutionary changes in life history might drive placental evolution. Here we demonstrate that life history differences among eutherian mammals are associated with major transitions in maternofetal interdigitation and placental invasiveness. We show that the repeated evolution of villous interdigitation is associated with reduced offspring production early in life and an increased lifespan. Further changes in placental morphology that reestablish a larger surface area are also associated with a change back to greater offspring production. After controlling for these differences in interdigitation, we also show that the least invasive placental type is associated with a fast pace of life. We predict that selection for a faster pace of life intensifies parent-offspring conflict, and that the repeated evolution of less-invasive placental structures might have allowed mothers to wrest back control of gestation from the fetus and alter their relative allocation to offspring production across life.

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Rhomboid domain-containing protein 3 is a negative regulator of TLR3-triggered natural killer cell activation.

Publication Date: 2013 May 7 PMID: 23610400
Authors: Liu, J. - Liu, S. - Xia, M. - Xu, S. - Wang, C. - Bao, Y. - Jiang, M. - Wu, Y. - Xu, T. - Cao, X.
Journal: Proc Natl Acad Sci U S A

Rhomboid domain-containing protein 3 (Rhbdd3), which belongs to a family of proteins with rhomboid domain, is widely expressed in immune cells; however, the roles of the Rhbdd members, including Rhbdd3, in immunity remain unknown. Natural killer (NK) cells are critical for host immune defense and also can mediate inflammatory diseases such as hepatitis. Although much is known about how NK cells are activated, the detailed mechanisms for negative regulation of NK cell activation remain to be fully understood. Using Rhbdd3-deficient mice, we reveal that Rhbdd3, selectively up-regulated in NK cells upon Toll-like receptor 3 (TLR3) stimulation, negatively regulates TLR3-mediated NK cell activation in a feedback manner. Rhbdd3 inhibits TLR3-triggered IFN-gamma and granzyme B expression of NK cells in cell-cell contact dependence of accessory cells such as dendritic cells and Kupffer cells. Rhbdd3 interacts with DNAX activation protein of 12 kDa and promotes its degradation, inhibiting MAPK activation in TLR3-triggered NK cells. Furthermore, Rhbdd3 plays a critical role in attenuating TLR3-triggered acute inflammation by controlling NK cell activation and accumulation in liver and disrupting NK cell-Kupffer cell interaction. Therefore, Rhbdd3 is a feedback inhibitor of TLR3-triggered NK cell activation. Our study outlines a mechanism for the negative regulation of NK cell activation and also provides clues for the function of the rhomboid proteins in immunity.

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Aurora kinase inhibitors reveal mechanisms of HURP in nucleation of centrosomal and kinetochore microtubules.

Publication Date: 2013 May 7 PMID: 23610398
Authors: Wu, J. M. - Chen, C. T. - Coumar, M. S. - Lin, W. H. - Chen, Z. J. - Hsu, J. T. - Peng, Y. H. - Shiao, H. Y. - Lin, W. H. - Chu, C. Y. - Wu, J. S. - Lin, C. T. - Chen, C. P. - Hsueh, C. C. - Chang, K. Y. - Kao, L. P. - Huang, C. Y. - Chao, Y. S. - Wu, S. Y. - Hsieh, H. P. - Chi, Y. H.
Journal: Proc Natl Acad Sci U S A

The overexpression of Aurora kinases in multiple tumors makes these kinases appealing targets for the development of anticancer therapies. This study identified two small molecules with a furanopyrimidine core, IBPR001 and IBPR002, that target Aurora kinases and induce a DFG conformation change at the ATP site of Aurora A. Our results demonstrate the high potency of the IBPR compounds in reducing tumorigenesis in a colorectal cancer xenograft model in athymic nude mice. Human hepatoma up-regulated protein (HURP) is a substrate of Aurora kinase A, which plays a crucial role in the stabilization of kinetochore fibers. This study used the IBPR compounds as well as MLN8237, a proven Aurora A inhibitor, as chemical probes to investigate the molecular role of HURP in mitotic spindle formation. These compounds effectively eliminated HURP phosphorylation, thereby revealing the coexistence and continuous cycling of HURP between unphosphorylated and phosphorylated forms that are associated, respectively, with microtubules emanating from centrosomes and kinetochores. Furthermore, these compounds demonstrate a spatial hierarchical preference for HURP in the attachment of microtubules extending from the mother to the daughter centrosome. The finding of inequality in the centrosomal microtubules revealed by these small molecules provides a versatile tool for the discovery of new cell-division molecules for the development of antitumor drugs.

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Mutual antagonism between hypoxia-inducible factors 1alpha and 2alpha regulates oxygen sensing and cardio-respiratory homeostasis.

Publication Date: 2013 May 7 PMID: 23610397
Authors: Yuan, G. - Peng, Y. J. - Reddy, V. D. - Makarenko, V. V. - Nanduri, J. - Khan, S. A. - Garcia, J. A. - Kumar, G. K. - Semenza, G. L. - Prabhakar, N. R.
Journal: Proc Natl Acad Sci U S A

Breathing and blood pressure are under constant homeostatic regulation to maintain optimal oxygen delivery to the tissues. Chemosensory reflexes initiated by the carotid body and catecholamine secretion from the adrenal medulla are the principal mechanisms for maintaining respiratory and cardiovascular homeostasis; however, the underlying molecular mechanisms are not known. Here, we report that balanced activity of hypoxia-inducible factor-1 (HIF-1) and HIF-2 is critical for oxygen sensing by the carotid body and adrenal medulla, and for their control of cardio-respiratory function. In Hif2alpha(+/-) mice, partial HIF-2alpha deficiency increased levels of HIF-1alpha and NADPH oxidase 2, leading to an oxidized intracellular redox state, exaggerated hypoxic sensitivity, and cardio-respiratory abnormalities, which were reversed by treatment with a HIF-1alpha inhibitor or a superoxide anion scavenger. Conversely, in Hif1alpha(+/-) mice, partial HIF-1alpha deficiency increased levels of HIF-2alpha and superoxide dismutase 2, leading to a reduced intracellular redox state, blunted oxygen sensing, and impaired carotid body and ventilatory responses to chronic hypoxia, which were corrected by treatment with a HIF-2alpha inhibitor. None of the abnormalities observed in Hif1alpha(+/-) mice or Hif2alpha(+/-) mice were observed in Hif1alpha(+/-);Hif2alpha(+/-) mice. These observations demonstrate that redox balance, which is determined by mutual antagonism between HIF-alpha isoforms, establishes the set point for hypoxic sensing by the carotid body and adrenal medulla, and is required for maintenance of cardio-respiratory homeostasis.

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Precise timing of ATPase activation drives targeting of tail-anchored proteins.

Publication Date: 2013 May 7 PMID: 23610396
Authors: Rome, M. E. - Rao, M. - Clemons, W. M. - Shan, S. O.
Journal: Proc Natl Acad Sci U S A

The localization of tail-anchored (TA) proteins, whose transmembrane domain resides at the extreme C terminus, presents major challenges to cellular protein targeting machineries. In eukaryotic cells, the highly conserved ATPase, guided entry of tail-anchored protein 3 (Get3), coordinates the delivery of TA proteins to the endoplasmic reticulum. How Get3 uses its ATPase cycle to drive this fundamental process remains unclear. Here, we establish a quantitative framework for the Get3 ATPase cycle and show that ATP specifically induces multiple conformational changes in Get3 that culminate in its ATPase activation through tetramerization. Further, upstream and downstream components actively regulate the Get3 ATPase cycle to ensure the precise timing of ATP hydrolysis in the pathway: the Get4/5 TA loading complex locks Get3 in the ATP-bound state and primes it for TA protein capture, whereas the TA substrate induces tetramerization of Get3 and activates its ATPase reaction 100-fold. Our results establish a precise model for how Get3 harnesses the energy from ATP to drive the membrane localization of TA proteins and illustrate how dimerization-activated nucleotide hydrolases regulate diverse cellular processes.

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Nuclear receptor corepressor (NCOR1) regulates in vivo actions of a mutated thyroid hormone receptor alpha.

Publication Date: 2013 May 7 PMID: 23610395
Authors: Fozzatti, L. - Kim, D. W. - Park, J. W. - Willingham, M. C. - Hollenberg, A. N. - Cheng, S. Y.
Journal: Proc Natl Acad Sci U S A

Genetic evidence from patients with mutations of the thyroid hormone receptor alpha gene (THRA) indicates that the dominant negative activity of mutants underlies the pathological manifestations. However, the molecular mechanisms by which TRalpha1 mutants exert dominant negative activity in vivo are not clear. We tested the hypothesis that the severe hypothyroidism in patients with THRA mutations is due to an inability of TRalpha1 mutants to properly release the nuclear corepressors (NCORs), thereby inhibiting thyroid hormone-mediated transcription activity. We crossed Thra1(PV) mice, expressing a dominant negative TRalpha1 mutant (TRalpha1PV), with mice expressing a mutant Ncor1 allele (Ncor1(DeltaID) mice) that cannot recruit the TR or PV mutant. TRalpha1PV shares the same C-terminal mutated sequences as those of patients with frameshift mutations of the THRA gene. Remarkably, NCOR1DeltaID ameliorated abnormalities in the thyroid-pituitary axis of Thra1(PV/+) mice. The severe retarded growth, infertility, and delayed bone development were partially reverted in Thra1(PV/+) mice expressing NCOR1DeltaID. The impaired adipogenesis was partially corrected by de-repression of peroxisome-proliferator activated receptor gamma and CCAAT/enhancer-binding protein alpha gene, due to the inability of TRalpha1PV to recruit NCOR1DeltaID to form a repressor complex. Thus, the aberrant recruitment of NCOR1 by TRalpha1 mutants could lead to clinical hypothyroidism in humans. Therefore, therapies aimed at the TRalpha1-NCOR1 interaction or its downstream actions could be tested as potential targets in treating TRalpha1 mutant-mediated hypothyroidism in patients.

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Targeted delivery of lipid antigen to macrophages via the CD169/sialoadhesin endocytic pathway induces robust invariant natural killer T cell activation.

Publication Date: 2013 May 7 PMID: 23610394
Authors: Kawasaki, N. - Vela, J. L. - Nycholat, C. M. - Rademacher, C. - Khurana, A. - van Rooijen, N. - Crocker, P. R. - Kronenberg, M. - Paulson, J. C.
Journal: Proc Natl Acad Sci U S A

Invariant natural killer T (iNKT) cells induce a protective immune response triggered by foreign glycolipid antigens bound to CD1d on antigen-presenting cells (APCs). A limitation of using glycolipid antigens to stimulate immune responses in human patients has been the inability to target them to the most effective APCs. Recent studies have implicated phagocytic CD169(+) macrophages as major APCs in lymph nodes for priming iNKT cells in mice immunized with glycolipid antigen in particulate form. CD169 is known as sialoadhesin (Sn), a macrophage-specific adhesion and endocytic receptor of the siglec family that recognizes sialic acid containing glycans as ligands. We have recently developed liposomes decorated with glycan ligands for CD169/Sn suitable for targeted delivery to macrophages via CD169/Sn-mediated endocytosis. Here we show that targeted delivery of a lipid antigen to CD169(+) macrophages in vivo results in robust iNKT cell activation in liver and spleen using nanogram amounts of antigen. Activation of iNKT cells is abrogated in Cd169(-/-) mice and is macrophage-dependent, demonstrating that targeting CD169(+) macrophages is sufficient for systemic activation of iNKT cells. When pulsed with targeted liposomes, human monocyte-derived dendritic cells expressing CD169/Sn activated human iNKT cells, demonstrating the conservation of the CD169/Sn endocytic pathway capable of presenting lipid antigens to iNKT cells.

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Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.

Publication Date: 2013 May 7 PMID: 23610393
Authors: Michelucci, A. - Cordes, T. - Ghelfi, J. - Pailot, A. - Reiling, N. - Goldmann, O. - Binz, T. - Wegner, A. - Tallam, A. - Rausell, A. - Buttini, M. - Linster, C. L. - Medina, E. - Balling, R. - Hiller, K.
Journal: Proc Natl Acad Sci U S A

Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production.

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Identification and characterization of a previously undescribed family of sequence-specific DNA-binding domains.

Publication Date: 2013 May 7 PMID: 23610392
Authors: Lohse, M. B. - Hernday, A. D. - Fordyce, P. M. - Noiman, L. - Sorrells, T. R. - Hanson-Smith, V. - Nobile, C. J. - Derisi, J. L. - Johnson, A. D.
Journal: Proc Natl Acad Sci U S A

Sequence-specific DNA-binding proteins are among the most important classes of gene regulatory proteins, controlling changes in transcription that underlie many aspects of biology. In this work, we identify a transcriptional regulator from the human fungal pathogen Candida albicans that binds DNA specifically but has no detectable homology with any previously described DNA- or RNA-binding protein. This protein, named White-Opaque Regulator 3 (Wor3), regulates white-opaque switching, the ability of C. albicans to switch between two heritable cell types. We demonstrate that ectopic overexpression of WOR3 results in mass conversion of white cells to opaque cells and that deletion of WOR3 affects the stability of opaque cells at physiological temperatures. Genome-wide chromatin immunoprecipitation of Wor3 and gene expression profiling of a wor3 deletion mutant strain indicate that Wor3 is highly integrated into the previously described circuit regulating white-opaque switching and that it controls a subset of the opaque transcriptional program. We show by biochemical, genetic, and microfluidic experiments that Wor3 binds directly to DNA in a sequence-specific manner, and we identify the set of cis-regulatory sequences recognized by Wor3. Bioinformatic analyses indicate that the Wor3 family arose more recently in evolutionary time than most previously described DNA-binding domains; it is restricted to a small number of fungi that include the major fungal pathogens of humans. These observations show that new families of sequence-specific DNA-binding proteins may be restricted to small clades and suggest that current annotations-which rely on deep conservation-underestimate the fraction of genes coding for transcriptional regulators.

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Analogues of simple and complex cells in rhesus monkey auditory cortex.

Publication Date: 2013 May 7 PMID: 23610391
Authors: Tian, B. - Kusmierek, P. - Rauschecker, J. P.
Journal: Proc Natl Acad Sci U S A

Receptive fields (RFs) of neurons in primary visual cortex have traditionally been subdivided into two major classes: "simple" and "complex" cells. Simple cells were originally defined by the existence of segregated subregions within their RF that respond to either the on- or offset of a light bar and by spatial summation within each of these regions, whereas complex cells had ON and OFF regions that were coextensive in space [Hubel DH, et al. (1962) J Physiol 160:106-154]. Although other definitions based on the linearity of response modulation have been proposed later [Movshon JA, et al. (1978) J Physiol 283:53-77; Skottun BC, et al. (1991) Vision Res 31(7-8):1079-1086], the segregation of ON and OFF subregions has remained an important criterion for the distinction between simple and complex cells. Here we report that response profiles of neurons in primary auditory cortex of monkeys show a similar distinction: one group of cells has segregated ON and OFF subregions in frequency space; and another group shows ON and OFF responses within largely overlapping response profiles. This observation is intriguing for two reasons: (i) spectrotemporal dissociation in the auditory domain provides a basic neural mechanism for the segregation of sounds, a fundamental prerequisite for auditory figure-ground discrimination; and (ii) the existence of similar types of RF organization in visual and auditory cortex would support the existence of a common canonical processing algorithm within cortical columns.

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Generic theory for channel sinuosity.

Publication Date: 2013 May 21 PMID: 23610390
Authors: Lazarus, E. D. - Constantine, J. A.
Journal: Proc Natl Acad Sci U S A

Sinuous patterns traced by fluid flows are a ubiquitous feature of physical landscapes on Earth, Mars, the volcanic floodplains of the Moon and Venus, and other planetary bodies. Typically discussed as a consequence of migration processes in meandering rivers, sinuosity is also expressed in channel types that show little or no indication of meandering. Sinuosity is sometimes described as "inherited" from a preexisting morphology, which still does not explain where the inherited sinuosity came from. For a phenomenon so universal as sinuosity, existing models of channelized flows do not explain the occurrence of sinuosity in the full variety of settings in which it manifests, or how sinuosity may originate. Here we present a generic theory for sinuous flow patterns in landscapes. Using observations from nature and a numerical model of flow routing, we propose that flow resistance (representing landscape roughness attributable to topography or vegetation density) relative to surface slope exerts a fundamental control on channel sinuosity that is effectively independent of internal flow dynamics. Resistance-dominated surfaces produce channels with higher sinuosity than those of slope-dominated surfaces because increased resistance impedes downslope flow. Not limited to rivers, the hypothesis we explore pertains to sinuosity as a geomorphic pattern. The explanation we propose is inclusive enough to account for a wide variety of sinuous channel types in nature, and can serve as an analytical tool for determining the sinuosity a landscape might support.

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Centrality in primate-parasite networks reveals the potential for the transmission of emerging infectious diseases to humans.

Publication Date: 2013 May 7 PMID: 23610389
Authors: Gomez, J. M. - Nunn, C. L. - Verdu, M.
Journal: Proc Natl Acad Sci U S A

Most emerging infectious diseases (EIDs) in humans have arisen from animals. Identifying high-risk hosts is therefore vital for the control and surveillance of these diseases. Viewing hosts as connected through the parasites they share, we use network tools to investigate predictors of parasitism and sources of future EIDs. We generated host-parasite networks that link hosts when they share a parasite, using nonhuman primates as a model system because-owing to their phylogenetic proximity and ecological overlap with humans-they are an important source of EIDs to humans. We then tested whether centrality in the network of host species-a measurement of the importance of a given node (i.e., host species) in the network-is associated with that host serving as a potential EID source. We found that centrality covaries with key predictors of parasitism, such as population density and geographic range size. Importantly, we also found that primate species having higher values of centrality in the primate-parasite network harbored more parasites identified as EIDs in humans and had parasite communities more similar to those found in humans. These relationships were robust to the use of different centrality metrics and to multiple ways of controlling for variation in how well each species has been studied (i.e., sampling effort). Centrality may therefore estimate the role of a host as a source of EIDs to humans in other multispecific host-parasite networks.

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Origin of seasonal predictability for summer climate over the Northwestern Pacific.

Publication Date: 2013 May 7 PMID: 23610388
Authors: Kosaka, Y. - Xie, S. P. - Lau, N. C. - Vecchi, G. A.
Journal: Proc Natl Acad Sci U S A

Summer climate in the Northwestern Pacific (NWP) displays large year-to-year variability, affecting densely populated Southeast and East Asia by impacting precipitation, temperature, and tropical cyclones. The Pacific-Japan (PJ) teleconnection pattern provides a crucial link of high predictability from the tropics to East Asia. Using coupled climate model experiments, we show that the PJ pattern is the atmospheric manifestation of an air-sea coupled mode spanning the Indo-NWP warm pool. The PJ pattern forces the Indian Ocean (IO) via a westward propagating atmospheric Rossby wave. In response, IO sea surface temperature feeds back and reinforces the PJ pattern via a tropospheric Kelvin wave. Ocean coupling increases both the amplitude and temporal persistence of the PJ pattern. Cross-correlation of ocean-atmospheric anomalies confirms the coupled nature of this PJIO mode. The ocean-atmosphere feedback explains why the last echoes of El Nino-Southern Oscillation are found in the IO-NWP in the form of the PJIO mode. We demonstrate that the PJIO mode is indeed highly predictable; a characteristic that can enable benefits to society.

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Early (300-100 B.C.) temple precinct in the Valley of Oaxaca, Mexico.

Publication Date: 2013 May 7 PMID: 23610387
Authors: Redmond, E. M. - Spencer, C. S.
Journal: Proc Natl Acad Sci U S A

Archaeological investigations during the past two decades in Mexico's Valley of Oaxaca have documented the appearance of key public buildings, such as the royal palace and multiroom temple, associated with the rise of an archaic state at ca. 300-100 B.C. A fuller picture is now emerging from the site of El Palenque, where recent excavations have defined a temple precinct on the east side of the site's plaza. This precinct exhibits characteristics similar to those of the temple precincts of later Mesoamerican states described by Colonial period sources. The excavation data document a walled enclosure containing three multiroom temples, two special residences identified as priests' residences, and an array of ritual features and activity areas. The temple precinct's components are interpreted as comprising a hierarchy of temples staffed by a specialized priesthood. A series of radiocarbon dates indicate that the precinct's differentiated components were all in use during the 300-100 B.C. period of archaic state emergence. The El Palenque temple precinct is the earliest temple precinct excavated thus far in the Valley of Oaxaca.

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Coupling social attention to the self forms a network for personal significance.

Publication Date: 2013 May 7 PMID: 23610386
Authors: Sui, J. - Rotshtein, P. - Humphreys, G. W.
Journal: Proc Natl Acad Sci U S A

Prior social psychological studies show that newly assigned personal significance can modulate high-level cognitive processes, e.g., memory and social evaluation, with self- and other-related information processed in dissociated prefrontal structure: ventral vs. dorsal, respectively. Here, we demonstrate the impact of personal significance on perception and show the neural network that supports this effect. We used an associative learning procedure in which we "tag" a neutral shape with a self-relevant label. Participants were instructed to associate three neutral shapes with labels for themselves, their best friend, or an unfamiliar other. Functional magnetic resonance imaging data were acquired while participants judged whether the shape-label pairs were maintained or swapped. Behaviorally, participants rapidly tagged a neutral stimulus with self-relevance, showing a robust advantage for self-tagged stimuli. Self-tagging responses were associated with enhanced activity in brain regions linked to self-representation [the ventral medial prefrontal cortex (vmPFC)] and to sensory-driven regions associated with social attention [the left posterior superior temporal sulcus (LpSTS)]. In contrast, associations formed with other people recruited a dorsal frontoparietal control network, with the two networks being inversely correlated. Responses in the vmPFC and LpSTS predicted behavioral self-bias effects. Effective connectivity analyses showed that the vmPFC and the LpSTS were functionally coupled, with the strength of coupling associated with behavioral self-biases. The data show that assignment of personal social significance affects perceptual matching by coupling internal self-representations to brain regions modulating attentional responses to external stimuli.

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Evolutionary remodeling of global regulatory networks during long-term bacterial adaptation to human hosts.

Publication Date: 2013 May 7 PMID: 23610385
Authors: Damkiaer, S. - Yang, L. - Molin, S. - Jelsbak, L.
Journal: Proc Natl Acad Sci U S A

The genetic basis of bacterial adaptation to a natural environment has been investigated in a highly successful Pseudomonas aeruginosa lineage (DK2) that evolved within the airways of patients with cystic fibrosis (CF) for more than 35 y. During evolution in the CF airways, the DK2 lineage underwent substantial phenotypic changes, which correlated with temporal fixation of specific mutations in the genes mucA (frame-shift), algT (substitution), rpoN (substitution), lasR (deletion), and rpoD (in-frame deletion), all encoding regulators of large gene networks. To clarify the consequences of these genetic changes, we moved the specific mutations, alone and in combination, to the genome of the reference strain PAO1. The phenotypes of the engineered PAO1 derivatives showed striking similarities with phenotypes observed among the DK2 isolates. The phenotypes observed in the DK2 isolates and PAO1 mutants were the results of individual, additive and epistatic effects of the regulatory mutations. The mutations fixed in the sigma factor encoding genes algT, rpoN, and rpoD caused minor changes in sigma factor activity, resulting in remodeling of the regulatory networks to facilitate generation of unexpected phenotypes. Our results suggest that adaptation to a highly selective environment, such as the CF airways, is a highly dynamic and complex process, which involves continuous optimization of existing regulatory networks to match the fluctuations in the environment.

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Histone chaperone FACT action during transcription through chromatin by RNA polymerase II.

Publication Date: 2013 May 7 PMID: 23610384
Authors: Hsieh, F. K. - Kulaeva, O. I. - Patel, S. S. - Dyer, P. N. - Luger, K. - Reinberg, D. - Studitsky, V. M.
Journal: Proc Natl Acad Sci U S A

FACT (facilitates chromatin transcription) is a histone chaperone that promotes chromatin recovery during transcription, with additional roles in cell differentiation. Although several models of the action of FACT during transcription have been proposed, they remain to be experimentally evaluated. Here we show that human FACT (hFACT) facilitates transcription through chromatin and promotes nucleosome recovery in vitro. FACT action depends on the presence of histone H2A/H2B dimers in the nucleosome. Kinetic analysis suggests that hFACT decreases the lifetime of nonproductive RNA polymerase II (Pol II)-nucleosome complexes and facilitates the formation of productive complexes containing nucleosomal DNA partially uncoiled from the octamer. Taken together, our data suggest that hFACT interacts with DNA-binding surfaces of H2A/H2B dimers, facilitating uncoiling of DNA from the histone octamer. Thus, hFACT-H2A/H2B interactions play a key role in overcoming the nucleosomal barrier by Pol II and promoting nucleosome survival during transcription.

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Position effect on FGF13 associated with X-linked congenital generalized hypertrichosis.

Publication Date: 2013 May 7 PMID: 23603273
Authors: Destefano, G. M. - Fantauzzo, K. A. - Petukhova, L. - Kurban, M. - Tadin-Strapps, M. - Levy, B. - Warburton, D. - Cirulli, E. T. - Han, Y. - Sun, X. - Shen, Y. - Shirazi, M. - Jobanputra, V. - Cepeda-Valdes, R. - Cesar Salas-Alanis, J. - Christiano, A. M.
Journal: Proc Natl Acad Sci U S A

X-linked congenital generalized hypertrichosis (Online Mendelian Inheritance in Man 307150) is an extremely rare condition of hair overgrowth on different body sites. We previously reported linkage in a large Mexican family with X-linked congenital generalized hypertrichosis cosegregating with deafness and with dental and palate anomalies to Xq24-27. Using SNP oligonucleotide microarray analysis and whole-genome sequencing, we identified a 389-kb interchromosomal insertion at an extragenic palindrome site at Xq27.1 that completely cosegregates with the disease. Among the genes surrounding the insertion, we found that Fibroblast Growth Factor 13 (FGF13) mRNA levels were significantly reduced in affected individuals, and immunofluorescence staining revealed a striking decrease in FGF13 localization throughout the outer root sheath of affected hair follicles. Taken together, our findings suggest a role for FGF13 in hair follicle growth and in the hair cycle.

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Vaccinia virus F1L protein promotes virulence by inhibiting inflammasome activation.

Publication Date: 2013 May 7 PMID: 23603272
Authors: Gerlic, M. - Faustin, B. - Postigo, A. - Yu, E. C. - Proell, M. - Gombosuren, N. - Krajewska, M. - Flynn, R. - Croft, M. - Way, M. - Satterthwait, A. - Liddington, R. C. - Salek-Ardakani, S. - Matsuzawa, S. - Reed, J. C.
Journal: Proc Natl Acad Sci U S A

Host innate immune responses to DNA viruses involve members of the nucleotide-binding domain, leucine-rich repeat and pyrin domain containing protein (NLRP) family, which form "inflammasomes" that activate caspase-1, resulting in proteolytic activation of cytokines interleukin (IL)-1beta and IL-18. We hypothesized that DNA viruses would target inflammasomes to overcome host defense. A Vaccinia virus (VACV) B-cell CLL/lymphoma 2 (Bcl-2) homolog, F1L, was demonstrated to bind and inhibit the NLR family member NLRP1 in vitro. Moreover, infection of macrophages in culture with virus lacking F1L (DeltaF1L) caused increased caspase-1 activation and IL-1beta secretion compared with wild-type virus. Virulence of DeltaF1L virus was attenuated in vivo, causing altered febrile responses, increased proteolytic processing of caspase-1, and more rapid inflammation in lungs of infected mice without affecting cell death or virus replication. Furthermore, we found that a hexapeptide from F1L is necessary and sufficient for inhibiting the NLRP1 inflammasome in vitro, thus identifying a peptidyl motif required for binding and inhibiting NLRP1. The functional importance of this NLRP1-binding motif was further confirmed by studies of recombinant DeltaF1L viruses reconstituted either with the wild-type F1L or a F1L mutant that fails to bind NLRP1. Cellular infection with wild-type F1L reconstituted virus-suppressed IL-1beta production, whereas mutant F1L did not. In contrast, both wild-type and mutant versions of F1L equally suppressed apoptosis. In vivo, the NLR nonbinding F1L mutant virus exhibited an attenuated phenotype similar to DeltaF1L virus, thus confirming the importance of F1L interactions with NLRP1 for viral pathogenicity in mice. Altogether, these findings reveal a unique viral mechanism for evading host innate immune responses.

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Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry.

Publication Date: 2013 May 7 PMID: 23603271
Authors: Hu, W. - Walters, B. T. - Kan, Z. Y. - Mayne, L. - Rosen, L. E. - Marqusee, S. - Englander, S. W.
Journal: Proc Natl Acad Sci U S A

The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on native-like interfoldon interactions orders the pathway.

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Faulty protocols yield contaminated samples, unconfirmed results.

Publication Date: 2013 Apr 30 PMID: 23599285
Authors: Boslough, M.
Journal: Proc Natl Acad Sci U S A

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Mechanism of tyrosine D oxidation in Photosystem II.

Publication Date: 2013 May 7 PMID: 23599284
Authors: Saito, K. - Rutherford, A. W. - Ishikita, H.
Journal: Proc Natl Acad Sci U S A

Using quantum mechanics/molecular mechanics calculations and the 1.9-A crystal structure of Photosystem II [Umena Y, Kawakami K, Shen J-R, Kamiya N (2011) Nature 473(7345):55-60], we investigated the H-bonding environment of the redox-active tyrosine D (TyrD) and obtained insights that help explain its slow redox kinetics and the stability of TyrD(*). The water molecule distal to TyrD, located approximately 4 A away from the phenolic O of TyrD, corresponds to the presence of the tyrosyl radical state. The water molecule proximal to TyrD, in H-bonding distance to the phenolic O of TyrD, corresponds to the presence of the unoxidized tyrosine. The H(+) released on oxidation of TyrD is transferred to the proximal water, which shifts to the distal position, triggering a concerted proton transfer pathway involving D2-Arg180 and a series of waters, through which the proton reaches the aqueous phase at D2-His61. The water movement linked to the ejection of the proton from the hydrophobic environment near TyrD makes oxidation slow and quasiirreversible, explaining the great stability of the TyrD(*). A symmetry-related proton pathway associated with tyrosine Z is pointed out, and this is associated with one of the Cl(-) sites. This may represent a proton pathway functional in the water oxidation cycle.

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Protection against malaria after immunization by chloroquine prophylaxis and sporozoites is mediated by preerythrocytic immunity.

Publication Date: 2013 May 7 PMID: 23599283
Authors: Bijker, E. M. - Bastiaens, G. J. - Teirlinck, A. C. - van Gemert, G. J. - Graumans, W. - van de Vegte-Bolmer, M. - Siebelink-Stoter, R. - Arens, T. - Teelen, K. - Nahrendorf, W. - Remarque, E. J. - Roeffen, W. - Jansens, A. - Zimmerman, D. - Vos, M. - van Schaijk, B. C. - Wiersma, J. - van der Ven, A. J. - de Mast, Q. - van Lieshout, L. - Verweij, J. J. - Hermsen, C. C. - Scholzen, A. - Sauerwein, R. W.
Journal: Proc Natl Acad Sci U S A

Volunteers immunized under chloroquine chemoprophylaxis with Plasmodium falciparum sporozoites (CPS) develop complete, long-lasting protection against homologous sporozoite challenge. Chloroquine affects neither sporozoites nor liver-stages, but kills only asexual forms in erythrocytes once released from the liver into the circulation. Consequently, CPS immunization exposes the host to antigens from both preerythrocytic and blood stages, and induced immunity might target either of these stages. We therefore explored the life cycle stage specificity of CPS-induced protection. Twenty-five malaria-naive volunteers were enrolled in a clinical trial, 15 of whom received CPS immunization. Five immunized subjects and five controls received a sporozoite challenge by mosquito bites, whereas nine immunized and five control subjects received an i.v. challenge with P. falciparum-infected erythrocytes. The latter approach completely bypasses preerythrocytic stages, enabling a direct comparison of protection against either life cycle stage. CPS-immunized subjects (13 of 14) developed anticircumsporozoite antibodies, whereas only one volunteer generated minimal titers against typical blood-stage antigens. IgG from CPS-immunized volunteers did not inhibit asexual blood-stage growth in vitro. All CPS-immunized subjects (5 of 5) were protected against sporozoite challenge. In contrast, nine of nine CPS-immunized subjects developed parasitemia after blood-stage challenge, with identical prepatent periods and blood-stage multiplication rates compared with controls. Intravenously challenged CPS-immunized subjects showed earlier fever and increased plasma concentrations of inflammatory markers D-dimer, IFN-gamma, and monokine induced by IFN-gamma than i.v. challenged controls. The complete lack of protection against blood-stage challenge indicates that CPS-induced protection is mediated by immunity against preerythrocytic stages. However, evidence is presented for immune recognition of P. falciparum-infected erythrocytes, suggesting memory responses unable to generate functional immunity.

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Leptin resistance is a secondary consequence of the obesity in ciliopathy mutant mice.

Publication Date: 2013 May 7 PMID: 23599282
Authors: Berbari, N. F. - Pasek, R. C. - Malarkey, E. B. - Yazdi, S. M. - McNair, A. D. - Lewis, W. R. - Nagy, T. R. - Kesterson, R. A. - Yoder, B. K.
Journal: Proc Natl Acad Sci U S A

Although primary cilia are well established as important sensory and signaling structures, their function in most tissues remains unknown. Obesity is a feature associated with some syndromes of cilia dysfunction, such as Bardet-Biedl syndrome (BBS) and Alstrom syndrome, as well as in several cilia mutant mouse models. Recent data indicate that obesity in BBS mutant mice is due to defects in leptin receptor trafficking and leptin resistance. Furthermore, induction of cilia loss in leptin-responsive proopiomelanocortin neurons results in obesity, implicating cilia on hypothalamic neurons in regulating feeding behavior. Here, we directly test the importance of the cilium as a mediator of the leptin response. In contrast to the current dogma, a longitudinal study of conditional Ift88 cilia mutant mice under different states of adiposity indicates that leptin resistance is present only when mutants are obese. Our studies show that caloric restriction leads to an altered anticipatory feeding behavior that temporarily abrogates the anorectic actions of leptin despite normalized circulating leptin levels. Interestingly, preobese Bbs4 mutant mice responded to the anorectic effects of leptin and did not display other phenotypes associated with defective leptin signaling. Furthermore, thermoregulation and activity measurements in cilia mutant mice are inconsistent with phenotypes previously observed in leptin deficient ob/ob mice. Collectively, these data indicate that cilia are not directly involved in leptin responses and that a defect in the leptin signaling axis is not the initiating event leading to hyperphagia and obesity associated with cilia dysfunction.

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HPV virions hitchhike a ride on retromer complexes.

Publication Date: 2013 Apr 30 PMID: 23599281
Authors: Sapp, M. J.
Journal: Proc Natl Acad Sci U S A

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Molecular view of an electron transfer process essential for iron-sulfur protein biogenesis.

Publication Date: 2013 Apr 30 PMID: 23596212
Authors: Banci, L. - Bertini, I. - Calderone, V. - Ciofi-Baffoni, S. - Giachetti, A. - Jaiswal, D. - Mikolajczyk, M. - Piccioli, M. - Winkelmann, J.
Journal: Proc Natl Acad Sci U S A

Biogenesis of iron-sulfur cluster proteins is a highly regulated process that requires complex protein machineries. In the cytosolic iron-sulfur protein assembly machinery, two human key proteins-NADPH-dependent diflavin oxidoreductase 1 (Ndor1) and anamorsin-form a stable complex in vivo that was proposed to provide electrons for assembling cytosolic iron-sulfur cluster proteins. The Ndor1-anamorsin interaction was also suggested to be implicated in the regulation of cell survival/death mechanisms. In the present work we unravel the molecular basis of recognition between Ndor1 and anamorsin and of the electron transfer process. This is based on the structural characterization of the two partner proteins, the investigation of the electron transfer process, and the identification of those protein regions involved in complex formation and those involved in electron transfer. We found that an unstructured region of anamorsin is essential for the formation of a specific and stable protein complex with Ndor1, whereas the C-terminal region of anamorsin, containing the [2Fe-2S] redox center, transiently interacts through complementary charged residues with the FMN-binding site region of Ndor1 to perform electron transfer. Our results propose a molecular model of the electron transfer process that is crucial for understanding the functional role of this interaction in human cells.

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Genome-wide diel growth state transitions in the diatom Thalassiosira pseudonana.

Publication Date: 2013 Apr 30 PMID: 23596211
Authors: Ashworth, J. - Coesel, S. - Lee, A. - Armbrust, E. V. - Orellana, M. V. - Baliga, N. S.
Journal: Proc Natl Acad Sci U S A

Marine diatoms are important primary producers that thrive in diverse and dynamic environments. They do so, in theory, by sensing changing conditions and adapting their physiology accordingly. Using the model species Thalassiosira pseudonana, we conducted a detailed physiological and transcriptomic survey to measure the recurrent transcriptional changes that characterize typical diatom growth in batch culture. Roughly 40% of the transcriptome varied significantly and recurrently, reflecting large, reproducible cell-state transitions between four principal states: (i) "dawn," following 12 h of darkness; (ii) "dusk," following 12 h of light; (iii) exponential growth and nutrient repletion; and (iv) stationary phase and nutrient depletion. Increases in expression of thousands of genes at the end of the reoccurring dark periods (dawn), including those involved in photosynthesis (e.g., ribulose-1,5-bisphosphate carboxylase oxygenase genes rbcS and rbcL), imply large-scale anticipatory circadian mechanisms at the level of gene regulation. Repeated shifts in the transcript levels of hundreds of genes encoding sensory, signaling, and regulatory functions accompanied the four cell-state transitions, providing a preliminary map of the highly coordinated gene regulatory program under varying conditions. Several putative light sensing and signaling proteins were associated with recurrent diel transitions, suggesting that these genes may be involved in light-sensitive and circadian regulation of cell state. These results begin to explain, in comprehensive detail, how the diatom gene regulatory program operates under varying environmental conditions. Detailed knowledge of this dynamic molecular process will be invaluable for new hypothesis generation and the interpretation of genetic, environmental, and metatranscriptomic data from field studies.

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TRPM8 activation attenuates inflammatory responses in mouse models of colitis.

Publication Date: 2013 Apr 30 PMID: 23596210
Authors: Ramachandran, R. - Hyun, E. - Zhao, L. - Lapointe, T. K. - Chapman, K. - Hirota, C. L. - Ghosh, S. - McKemy, D. D. - Vergnolle, N. - Beck, P. L. - Altier, C. - Hollenberg, M. D.
Journal: Proc Natl Acad Sci U S A

Transient Receptor Potential Melastatin-8 (TRPM8), a recently identified member of the transient receptor potential (TRP) family of ion channels, is activated by mild cooling and by chemical compounds such as the supercooling agent, icilin. Since cooling, possibly involving TRPM8 stimulation, diminishes injury-induced peripheral inflammation, we hypothesized that TRPM8 activation may also attenuate systemic inflammation. We thus studied the involvement of TRPM8 in regulating colonic inflammation using two mouse models of chemically induced colitis. TRPM8 expression, localized immunohistochemically in transgenic TRPM8(GFP) mouse colon, was up-regulated in both human- and murine-inflamed colon samples, as measured by real-time PCR. Wild-type mice (but not TRPM8-nulls) treated systemically with the TRPM8 agonist, icilin showed an attenuation of chemically induced colitis, as reflected by a decrease in macroscopic and microscopic damage scores, bowel thickness, and myeloperoxidase activity compared with untreated animals. Furthermore, icilin treatment reduced the 2,4,6-trinitrobenzenesulfonic acid-induced increase in levels of inflammatory cytokines and chemokines in the colon. In comparison with wild-type mice, Dextran Sodium Sulfate (DSS)-treated TRPM8 knockout mice showed elevated colonic levels of the inflammatory neuropeptide calcitonin-gene-related peptide, although inflammatory indices were equivalent for both groups. Further, TRPM8 activation by icilin blocked capsaicin-triggered calcitonin-gene-related peptide release from colon tissue ex vivo and blocked capsaicin-triggered calcium signaling in Transient Receptor Potential Vaniloid-1 (TRPV1) and TRPM8 transfected HEK cells. Our data document an anti-inflammatory role for TRPM8 activation, in part due to an inhibiton of neuropeptide release, pointing to a novel therapeutic target for colitis and other inflammatory diseases.

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Correction for Shankarappa et al., Prolonged nerve blockade delays the onset of neuropathic pain.

Publication Date: 2013 May 7 PMID: 23596209
Authors:
Journal: Proc Natl Acad Sci U S A

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Correction for Barber et al., Association of RIG-I with innate immunity of ducks to influenza.

Publication Date: 2013 May 7 PMID: 23596208
Authors:
Journal: Proc Natl Acad Sci U S A

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X-ray snapshots of possible intermediates in the time course of synthesis and degradation of protein-bound Fe4S4 clusters.

Publication Date: 2013 Apr 30 PMID: 23596207
Authors: Nicolet, Y. - Rohac, R. - Martin, L. - Fontecilla-Camps, J. C.
Journal: Proc Natl Acad Sci U S A

Fe4S4 clusters are very common versatile prosthetic groups in proteins. Their redox property of being sensitive to O2-induced oxidative damage is, for instance, used by the cell to sense oxygen levels and switch between aerobic and anaerobic metabolisms, as exemplified by the fumarate, nitrate reduction regulator (FNR). Using the hydrogenase maturase HydE from Thermotoga maritima as a template, we obtained several unusual forms of FeS clusters, some of which are associated with important structural changes. These structures represent intermediate states relevant to both FeS cluster assembly and degradation. We observe one Fe2S2 cluster bound by two cysteine persulfide residues. This observation lends structural support to a very recent Raman study, which reported that Fe4S4-to-Fe2S2 cluster conversion upon oxygen exposure in FNR resulted in concomitant production of cysteine persulfide as cluster ligands. Similar persulfide ligands have been observed in vitro for several other Fe4S4 cluster-containing proteins. We have also monitored FeS cluster conversion directly in our protein crystals. Our structures indicate that the Fe4S4-to-Fe2S2 change requires large structural modifications, which are most likely responsible for the dimer-monomer transition in FNR.

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Tertiary model of a plant cellulose synthase.

Publication Date: 2013 Apr 30 PMID: 23592721
Authors: Sethaphong, L. - Haigler, C. H. - Kubicki, J. D. - Zimmer, J. - Bonetta, D. - Debolt, S. - Yingling, Y. G.
Journal: Proc Natl Acad Sci U S A

A 3D atomistic model of a plant cellulose synthase (CESA) has remained elusive despite over forty years of experimental effort. Here, we report a computationally predicted 3D structure of 506 amino acids of cotton CESA within the cytosolic region. Comparison of the predicted plant CESA structure with the solved structure of a bacterial cellulose-synthesizing protein validates the overall fold of the modeled glycosyltransferase (GT) domain. The coaligned plant and bacterial GT domains share a six-stranded beta-sheet, five alpha-helices, and conserved motifs similar to those required for catalysis in other GT-2 glycosyltransferases. Extending beyond the cross-kingdom similarities related to cellulose polymerization, the predicted structure of cotton CESA reveals that plant-specific modules (plant-conserved region and class-specific region) fold into distinct subdomains on the periphery of the catalytic region. Computational results support the importance of the plant-conserved region and/or class-specific region in CESA oligomerization to form the multimeric cellulose-synthesis complexes that are characteristic of plants. Relatively high sequence conservation between plant CESAs allowed mapping of known mutations and two previously undescribed mutations that perturb cellulose synthesis in Arabidopsis thaliana to their analogous positions in the modeled structure. Most of these mutation sites are near the predicted catalytic region, and the confluence of other mutation sites supports the existence of previously undefined functional nodes within the catalytic core of CESA. Overall, the predicted tertiary structure provides a platform for the biochemical engineering of plant CESAs.

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ATP-gated ion channels mediate adaptation to elevated sound levels.

Publication Date: 2013 Apr 30 PMID: 23592720
Authors: Housley, G. D. - Morton-Jones, R. - Vlajkovic, S. M. - Telang, R. S. - Paramananthasivam, V. - Tadros, S. F. - Wong, A. C. - Froud, K. E. - Cederholm, J. M. - Sivakumaran, Y. - Snguanwongchai, P. - Khakh, B. S. - Cockayne, D. A. - Thorne, P. R. - Ryan, A. F.
Journal: Proc Natl Acad Sci U S A

The sense of hearing is remarkable for its auditory dynamic range, which spans more than 10(12) in acoustic intensity. The mechanisms that enable the cochlea to transduce high sound levels without damage are of key interest, particularly with regard to the broad impact of industrial, military, and recreational auditory overstimulation on hearing disability. We show that ATP-gated ion channels assembled from P2X2 receptor subunits in the cochlea are necessary for the development of temporary threshold shift (TTS), evident in auditory brainstem response recordings as sound levels rise. In mice null for the P2RX2 gene (encoding the P2X2 receptor subunit), sustained 85-dB noise failed to elicit the TTS that wild-type (WT) mice developed. ATP released from the tissues of the cochlear partition with elevation of sound levels likely activates the broadly distributed P2X2 receptors on epithelial cells lining the endolymphatic compartment. This purinergic signaling is supported by significantly greater noise-induced suppression of distortion product otoacoustic emissions derived from outer hair cell transduction and decreased suprathreshold auditory brainstem response input/output gain in WT mice compared with P2RX2-null mice. At higher sound levels (>/=95 dB), additional processes dominated TTS, and P2RX2-null mice were more vulnerable than WT mice to permanent hearing loss due to hair cell synapse disruption. P2RX2-null mice lacked ATP-gated conductance across the cochlear partition, including loss of ATP-gated inward current in hair cells. These data indicate that a significant component of TTS represents P2X2 receptor-dependent purinergic hearing adaptation that underpins the upper physiological range of hearing.

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Rac-specific guanine nucleotide exchange factor DOCK1 is a critical regulator of HER2-mediated breast cancer metastasis.

Publication Date: 2013 Apr 30 PMID: 23592719
Authors: Laurin, M. - Huber, J. - Pelletier, A. - Houalla, T. - Park, M. - Fukui, Y. - Haibe-Kains, B. - Muller, W. J. - Cote, J. F.
Journal: Proc Natl Acad Sci U S A

Progression of solid tumors to the metastatic stage is accountable for the majority of cancer-related deaths. Further understanding of the molecular mechanisms governing metastasis is essential for the development of antimetastatic regimens. Here, we aimed to identify Rac activators that could promote metastasis downstream of human epithelial growth factor receptor 2 (HER2). We investigated if Dedicator of Cytokinesis 1 (DOCK1), based on its evolutionarily conserved role in receptor tyrosine kinases (RTKs)-mediated Rac activation and cell invasion, could be a regulator of metastasis. We report that high expression of DOCK1 in HER2(+) and basal breast cancer subtypes inversely correlates with human patients' survival. Mechanistically, DOCK1 interacts with HER2 and promotes HER2-induced Rac activation and cell migration. To gain further insight, we developed a HER2 breast cancer mouse model with mammary-gland-specific inactivation of DOCK1. In this in vivo model, a significant decrease in tumor growth and metastasis in lungs was found in animals where DOCK1 is inactivated. Furthermore, we found that DOCK1 is required for maximal activation of two HER2 effectors, c-JUN and STAT3. Using an unbiased gene profiling approach, we identified a mammary tumor DOCK1-associated gene signature enriched for genes implicated in response to IFN type I. This analysis revealed a unique set of genes, including Receptor Transporter Protein 4 (RTP4) and STAT1, for which the expression levels can be used to independently predict breast cancer outcome in HER2(+) patients. Our work demonstrates DOCK1-Rac signaling as an HER2 effector pathway essential for HER2-mediated breast cancer progression to metastasis and offers a therapeutic opportunity to limit the spread of metastatic breast cancers.

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Structural basis for angiopoietin-1-mediated signaling initiation.

Publication Date: 2013 Apr 30 PMID: 23592718
Authors: Yu, X. - Seegar, T. C. - Dalton, A. C. - Tzvetkova-Robev, D. - Goldgur, Y. - Rajashankar, K. R. - Nikolov, D. B. - Barton, W. A.
Journal: Proc Natl Acad Sci U S A

Angiogenesis is a complex cellular process involving multiple regulatory growth factors and growth factor receptors. Among them, the ligands for the endothelial-specific tunica intima endothelial receptor tyrosine kinase 2 (Tie2) receptor kinase, angiopoietin-1 (Ang1) and Ang2, play essential roles in balancing vessel stability and regression during both developmental and tumor-induced angiogenesis. Despite possessing a high degree of sequence identity, Ang1 and Ang2 have distinct functional roles and cell-signaling characteristics. Here, we present the crystal structures of Ang1 both unbound and in complex with the Tie2 ectodomain. Comparison of the Ang1-containing structures with their Ang2-containing counterparts provide insight into the mechanism of receptor activation and reveal molecular surfaces important for interactions with Tie2 coreceptors and associated signaling proteins. Using structure-based mutagenesis, we identify a loop within the angiopoietin P domain, adjacent to the receptor-binding interface, which confers the specific agonist/antagonist properties of the molecule. We demonstrate using cell-based assays that an Ang2 chimera containing the Ang1 loop sequence behaves functionally similarly to Ang1 as a constitutive Tie2 agonist, able to efficiently dissociate the inhibitory Tie1/Tie2 complex and elicit Tie2 clustering and downstream signaling.

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E-selectin ligand 1 regulates bone remodeling by limiting bioactive TGF-beta in the bone microenvironment.

Publication Date: 2013 Apr 30 PMID: 23589896
Authors: Yang, T. - Grafe, I. - Bae, Y. - Chen, S. - Chen, Y. - Bertin, T. K. - Jiang, M. M. - Ambrose, C. G. - Lee, B.
Journal: Proc Natl Acad Sci U S A

TGF-beta is abundantly produced in the skeletal system and plays a crucial role in skeletal homeostasis. E-selectin ligand-1 (ESL-1), a Golgi apparatus-localized protein, acts as a negative regulator of TGF-beta bioavailability by attenuating maturation of pro-TGF-beta during cartilage homeostasis. However, whether regulation of intracellular TGF-beta maturation by ESL-1 is also crucial during bone homeostasis has not been well defined. Here, we show that Esl-1(-/-) mice exhibit a severe osteopenia with elevated bone resorption and decreased bone mineralization. In primary culture, Esl-1(-/-) osteoclast progenitors show no difference in osteoclastogenesis. However, Esl-1(-/-) osteoblasts show delayed differentiation and mineralization and stimulate osteoclastogenesis more potently in the osteoblast-osteoclast coculture, suggesting that ESL-1 primarily acts in osteoblasts to regulate bone homeostasis. In addition, Esl-1(-/-) calvaria exhibit an elevated mature TGF-beta/pro-TGF-beta ratio, with increased expression of TGF-beta downstream targets (plasminogen activator inhibitor-1, parathyroid hormone-related peptide, connective tissue growth factor, and matrix metallopeptidase 13, etc.) and a key regulator of osteoclastogenesis (receptor activator of nuclear factor kappaB ligand). Moreover, in vivo treatment with 1D11, a pan-TGF-beta antibody, significantly improved the low bone mass of Esl-1(-/-) mice, suggesting that elevated TGF-beta signaling is the major cause of osteopenia in Esl-1(-/-) mice. In summary, our study identifies ESL-1 as an important regulator of bone remodeling and demonstrates that the modulation of TGF-beta maturation is pivotal in the maintenance of a homeostatic bone microenvironment and for proper osteoblast-osteoclast coupling.

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Role of p63 and the Notch pathway in cochlea development and sensorineural deafness.

Publication Date: 2013 Apr 30 PMID: 23589895
Authors: Terrinoni, A. - Serra, V. - Bruno, E. - Strasser, A. - Valente, E. - Flores, E. R. - van Bokhoven, H. - Lu, X. - Knight, R. A. - Melino, G.
Journal: Proc Natl Acad Sci U S A

The ectodermal dysplasias are a group of inherited autosomal dominant syndromes associated with heterozygous mutations in the Tumor Protein p63 (TRP63) gene. Here we show that, in addition to their epidermal pathology, a proportion of these patients have distinct levels of deafness. Accordingly, p63 null mouse embryos show marked cochlea abnormalities, and the transactivating isoform of p63 (TAp63) protein is normally found in the organ of Corti. TAp63 transactivates hairy and enhancer of split 5 (Hes5) and atonal homolog 1 (Atoh1), components of the Notch pathway, known to be involved in cochlear neuroepithelial development. Strikingly, p63 null mice show morphological defects of the organ of Corti, with supernumerary hair cells, as also reported for Hes5 null mice. This phenotype is related to loss of a differentiation property of TAp63 and not to loss of its proapoptotic function, because cochleas in mice lacking the critical Bcl-2 homology domain (BH-3) inducers of p53- and p63-mediated apoptosis-Puma, Noxa, or both-are normal. Collectively, these data demonstrate that TAp63, acting via the Notch pathway, is crucial for the development of the organ of Corti, providing a molecular explanation for the sensorineural deafness in ectodermal dysplasia patients with TRP63 mutations.

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Murine natural killer cell licensing and regulation by T regulatory cells in viral responses.

Publication Date: 2013 Apr 30 PMID: 23589894
Authors: Sungur, C. M. - Tang-Feldman, Y. J. - Ames, E. - Alvarez, M. - Chen, M. - Longo, D. L. - Pomeroy, C. - Murphy, W. J.
Journal: Proc Natl Acad Sci U S A

Natural killer (NK) cells show differential functionality based on their capability of binding to self-MHC consistent with licensing. Here we show in vivo confirmation of the physiologic effects of licensing with differential effects of NK subsets on anti-murine cytomegalovirus (anti-MCMV) responses after syngeneic hematopoietic stem cell transplantation (HSCT) or regulatory T-cell (Treg) depletion. After HSCT, depletion of licensed NK cells led to far greater viral loads in target organs early after infection compared with nondepleted and unlicensed depleted mice. There was a preferential expansion of licensed, C-type lectin-like activating receptor Ly49H+ NK cells with increased IFNgamma production after infection in nondepleted mice post-HSCT and after Treg depletion. Adoptive transfer of licensed NK subsets into immunodeficient hosts provided significantly greater MCMV resistance compared with transfer of total NK populations or unlicensed subsets. In non-HSCT mice, only concurrent depletion of Tregs or TGF-beta neutralization resulted in detection of NK licensing effects. This suggests that licensed NK cells are the initial and rapidly responding population of NK cells to MCMV infection, but are highly regulated by Tregs and TGF-beta.

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Deficiency of beta-arrestin1 ameliorates collagen-induced arthritis with impaired TH17 cell differentiation.

Publication Date: 2013 Apr 30 PMID: 23589893
Authors: Li, J. - Wei, B. - Guo, A. - Liu, C. - Huang, S. - Du, F. - Fan, W. - Bao, C. - Pei, G.
Journal: Proc Natl Acad Sci U S A

Rheumatoid arthritis (RA) is an inflammatory disease in which interleukin 17 (IL-17)-producing T helper 17 (TH17) cells have been critically involved. We show that in patients with RA, the expression of a multifunctional regulator beta-arrestin1 was significantly up-regulated in peripheral and synovial CD4(+) T cells, which correlated well with active phases of RA. In collagen-induced arthritis, deficiency of beta-arrestin1 ameliorated disease with decreased TH17 cell differentiation, proinflammatory cytokine production, synovitis, and cartilage and bone destruction. Further mechanistic study reveals that beta-arrestin1 promoted signal transducer and activator of transcription 3 (STAT3) activation required for TH17 cell differentiation through scaffolding the interaction of Janus kinase 1 and STAT3. These findings indicate a critical role for beta-arrestin1 in the pathogenesis of collagen-induced arthritis and TH17 cell differentiation and suggest beta-arrestin1 as a potential diagnostic biomarker and therapeutic target for RA.

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Quantum critical point and spin fluctuations in lower-mantle ferropericlase.

Publication Date: 2013 Apr 30 PMID: 23589892
Authors: Lyubutin, I. S. - Struzhkin, V. V. - Mironovich, A. A. - Gavriliuk, A. G. - Naumov, P. G. - Lin, J. F. - Ovchinnikov, S. G. - Sinogeikin, S. - Chow, P. - Xiao, Y. - Hemley, R. J.
Journal: Proc Natl Acad Sci U S A

Ferropericlase [(Mg,Fe)O] is one of the most abundant minerals of the earth's lower mantle. The high-spin (HS) to low-spin (LS) transition in the Fe(2+) ions may dramatically alter the physical and chemical properties of (Mg,Fe)O in the deep mantle. To understand the effects of compression on the ground electronic state of iron, electronic and magnetic states of Fe(2+) in (Mg0.75Fe0.25)O have been investigated using transmission and synchrotron Mossbauer spectroscopy at high pressures and low temperatures (down to 5 K). Our results show that the ground electronic state of Fe(2+) at the critical pressure Pc of the spin transition close to T = 0 is governed by a quantum critical point (T = 0, P = Pc) at which the energy required for the fluctuation between HS and LS states is zero. Analysis of the data gives Pc = 55 GPa. Thermal excitation within the HS or LS states (T > 0 K) is expected to strongly influence the magnetic as well as physical properties of ferropericlase. Multielectron theoretical calculations show that the existence of the quantum critical point at temperatures approaching zero affects not only physical properties of ferropericlase at low temperatures but also its properties at P-T of the earth's lower mantle.

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Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms.

Publication Date: 2013 Apr 30 PMID: 23589891
Authors: Sarangapani, K. K. - Akiyoshi, B. - Duggan, N. M. - Biggins, S. - Asbury, C. L.
Journal: Proc Natl Acad Sci U S A

During mitosis, multiprotein complexes called kinetochores orchestrate chromosome segregation by forming load-bearing attachments to dynamic microtubule tips, and by participating in phosphoregulatory error correction. The conserved kinase Aurora B phosphorylates the major microtubule-binding kinetochore subcomplexes, Ndc80 and (in yeast) Dam1, to promote release of erroneous attachments, giving another chance for proper attachments to form. It is unknown whether Aurora B phosphorylation promotes release directly, by increasing the rate of kinetochore detachment, or indirectly, by destabilizing the microtubule tip. Moreover, the relative importance of phosphorylation of Ndc80 vs. Dam1 in the context of whole kinetochores is unclear. To address these uncertainties, we isolated native yeast kinetochore particles carrying phosphomimetic mutations on Ndc80 and Dam1, and applied advanced laser-trapping techniques to measure the strength and stability of their attachments to individual dynamic microtubule tips. Rupture forces were reduced by phosphomimetic mutations on both subcomplexes, in an additive manner, indicating that both subcomplexes make independent contributions to attachment strength. Phosphomimetics on either subcomplex reduced attachment lifetimes under constant force, primarily by accelerating detachment during microtubule growth. Phosphomimetics on Dam1 also increased the likelihood of switches from microtubule growth into shortening, further promoting release in an indirect manner. Taken together, our results suggest that, in vivo, Aurora B releases kinetochores via at least two mechanisms: by weakening the kinetochore-microtubule interface and also by destabilizing the kinetochore-attached microtubule tip.

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Physical and genetic-interaction density reveals functional organization and informs significance cutoffs in genome-wide screens.

Publication Date: 2013 Apr 30 PMID: 23589890
Authors: Dittmar, J. C. - Pierce, S. - Rothstein, R. - Reid, R. J.
Journal: Proc Natl Acad Sci U S A

Genome-wide experiments often measure quantitative differences between treated and untreated cells to identify affected strains. For these studies, statistical models are typically used to determine significance cutoffs. We developed a method termed "CLIK" (Cutoff Linked to Interaction Knowledge) that overlays biological knowledge from the interactome on screen results to derive a cutoff. The method takes advantage of the fact that groups of functionally related interacting genes often respond similarly to experimental conditions and, thus, cluster in a ranked list of screen results. We applied CLIK analysis to five screens of the yeast gene disruption library and found that it defined a significance cutoff that differed from traditional statistics. Importantly, verification experiments revealed that the CLIK cutoff correlated with the position in the rank order where the rate of true positives drops off significantly. In addition, the gene sets defined by CLIK analysis often provide further biological perspectives. For example, applying CLIK analysis retrospectively to a screen for cisplatin sensitivity allowed us to identify the importance of the Hrq1 helicase in DNA crosslink repair. Furthermore, we demonstrate the utility of CLIK to determine optimal treatment conditions by analyzing genome-wide screens at multiple rapamycin concentrations. We show that CLIK is an extremely useful tool for evaluating screen quality, determining screen cutoffs, and comparing results between screens. Furthermore, because CLIK uses previously annotated interaction data to determine biologically informed cutoffs, it provides additional insights into screen results, which supplement traditional statistical approaches.

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Early quality assessment lessens pheromone specificity in a moth.

Publication Date: 2013 Apr 30 PMID: 23589889
Authors: Karpati, Z. - Tasin, M. - Carde, R. T. - Dekker, T.
Journal: Proc Natl Acad Sci U S A

Pheromone orientation in moths is an exemplar of olfactory acuity. To avoid heterospecific mating, males respond to female-produced blends with high specificity and temporal resolution. A finely tuned sensory to projection neuron network secures specificity, and this network is thought to assess pheromone quality continually during orientation. We tested whether male moths do indeed evaluate each pheromone encounter and surprisingly found that male European corn borer moths instead generalize across successive encounters. Although initially highly ratio specific, once "locked on" to the pheromone plume the acceptable ratio can vary widely, and even unattractive blends can become attractive. We further found that this "mental shortcut" may be a consequence of the fact that sensory neurons exposed to frequent encounters do not reliably encode blend ratios. Neurons tuned to either of the two pheromone components adapt differentially in plumes containing the preferred blend ratio (97:3) and cause the olfactory sensory signal to "evolve," even in narrowly tuned pheromonal circuits. However, apparently the brain interprets these shifting signals as invariant "gestalts." Generalization in pheromone perception may mitigate stabilizing selection and allow introgression between sympatric strains, such as in the European corn borer, that otherwise appear isolated by pheromonal differences. Generalization may also be important in responses to general odorants, as circuits underlying these display vast sensitivity differences, complex interactions, and temporal intricacies.

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SK4 Ca2+ activated K+ channel is a critical player in cardiac pacemaker derived from human embryonic stem cells.

Publication Date: 2013 Apr 30 PMID: 23589888
Authors: Weisbrod, D. - Peretz, A. - Ziskind, A. - Menaker, N. - Oz, S. - Barad, L. - Eliyahu, S. - Itskovitz-Eldor, J. - Dascal, N. - Khananshvili, D. - Binah, O. - Attali, B.
Journal: Proc Natl Acad Sci U S A

Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. Two main mechanisms have been proposed: (i) the "voltage-clock," where the hyperpolarization-activated funny current If causes diastolic depolarization that triggers action potential cycling; and (ii) the "Ca(2+) clock," where cyclical release of Ca(2+) from Ca(2+) stores depolarizes the membrane during diastole via activation of the Na(+)-Ca(2+) exchanger. Nonetheless, these mechanisms remain controversial. Here, we used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study their autonomous beating mechanisms. Combined current- and voltage-clamp recordings from the same cell showed the so-called "voltage and Ca(2+) clock" pacemaker mechanisms to operate in a mutually exclusive fashion in different cell populations, but also to coexist in other cells. Blocking the "voltage or Ca(2+) clock" produced a similar depolarization of the maximal diastolic potential (MDP) that culminated by cessation of action potentials, suggesting that they converge to a common pacemaker component. Using patch-clamp recording, real-time PCR, Western blotting, and immunocytochemistry, we identified a previously unrecognized Ca(2+)-activated intermediate K(+) conductance (IKCa, KCa3.1, or SK4) in young and old stage-derived hESC-CMs. IKCa inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IKCa appears to play a crucial role in human embryonic cardiac automaticity.

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Ocean acidification alters the otoliths of a pantropical fish species with implications for sensory function.

Publication Date: 2013 Apr 30 PMID: 23589887
Authors: Bignami, S. - Enochs, I. C. - Manzello, D. P. - Sponaugle, S. - Cowen, R. K.
Journal: Proc Natl Acad Sci U S A

Ocean acidification affects a wide diversity of marine organisms and is of particular concern for vulnerable larval stages critical to population replenishment and connectivity. Whereas it is well known that ocean acidification will negatively affect a range of calcareous taxa, the study of fishes is more limited in both depth of understanding and diversity of study species. We used new 3D microcomputed tomography to conduct in situ analysis of the impact of ocean acidification on otolith (ear stone) size and density of larval cobia (Rachycentron canadum), a large, economically important, pantropical fish species that shares many life history traits with a diversity of high-value, tropical pelagic fishes. We show that 2,100 muatm partial pressure of carbon dioxide (pCO2) significantly increased not only otolith size (up to 49% greater volume and 58% greater relative mass) but also otolith density (6% higher). Estimated relative mass in 800 muatm pCO2 treatments was 14% greater, and there was a similar but nonsignificant trend for otolith size. Using a modeling approach, we demonstrate that these changes could affect auditory sensitivity including a approximately 50% increase in hearing range at 2,100 muatm pCO2, which may alter the perception of auditory information by larval cobia in a high-CO2 ocean. Our results indicate that ocean acidification has a graded effect on cobia otoliths, with the potential to substantially influence the dispersal, survival, and recruitment of a pelagic fish species. These results have important implications for population maintenance/replenishment, connectivity, and conservation efforts for other valuable fish stocks that are already being deleteriously impacted by overfishing.

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Crystal structure of a prokaryotic (6-4) photolyase with an Fe-S cluster and a 6,7-dimethyl-8-ribityllumazine antenna chromophore.

Publication Date: 2013 Apr 30 PMID: 23589886
Authors: Zhang, F. - Scheerer, P. - Oberpichler, I. - Lamparter, T. - Krauss, N.
Journal: Proc Natl Acad Sci U S A

The (6-4) photolyases use blue light to reverse UV-induced (6-4) photoproducts in DNA. This (6-4) photorepair was thought to be restricted to eukaryotes. Here we report a prokaryotic (6-4) photolyase, PhrB from Agrobacterium tumefaciens, and propose that (6-4) photolyases are broadly distributed in prokaryotes. The crystal structure of photolyase related protein B (PhrB) at 1.45 A resolution suggests a DNA binding mode different from that of the eukaryotic counterparts. A His-His-X-X-Arg motif is located within the proposed DNA lesion contact site of PhrB. This motif is structurally conserved in eukaryotic (6-4) photolyases for which the second His is essential for the (6-4) photolyase function. The PhrB structure contains 6,7-dimethyl-8-ribityllumazine as an antenna chromophore and a [4Fe-4S] cluster bound to the catalytic domain. A significant part of the Fe-S fold strikingly resembles that of the large subunit of eukaryotic and archaeal primases, suggesting that the PhrB-like photolyases branched at the base of the evolution of the cryptochrome/photolyase family. Our study presents a unique prokaryotic (6-4) photolyase and proposes that the prokaryotic (6-4) photolyases are the ancestors of the cryptochrome/photolyase family.

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Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development.

Publication Date: 2013 Apr 30 PMID: 23589885
Authors: Kucharzewska, P. - Christianson, H. C. - Welch, J. E. - Svensson, K. J. - Fredlund, E. - Ringner, M. - Morgelin, M. - Bourseau-Guilmain, E. - Bengzon, J. - Belting, M.
Journal: Proc Natl Acad Sci U S A

Hypoxia, or low oxygen tension, is a major regulator of tumor development and aggressiveness. However, how cancer cells adapt to hypoxia and communicate with their surrounding microenvironment during tumor development remain important questions. Here, we show that secreted vesicles with exosome characteristics mediate hypoxia-dependent intercellular signaling of the highly malignant brain tumor glioblastoma multiforme (GBM). In vitro hypoxia experiments with glioma cells and studies with patient materials reveal the enrichment in exosomes of hypoxia-regulated mRNAs and proteins (e.g., matrix metalloproteinases, IL-8, PDGFs, caveolin 1, and lysyl oxidase), several of which were associated with poor glioma patient prognosis. We show that exosomes derived from GBM cells grown at hypoxic compared with normoxic conditions are potent inducers of angiogenesis ex vivo and in vitro through phenotypic modulation of endothelial cells. Interestingly, endothelial cells were programmed by GBM cell-derived hypoxic exosomes to secrete several potent growth factors and cytokines and to stimulate pericyte PI3K/AKT signaling activation and migration. Moreover, exosomes derived from hypoxic compared with normoxic conditions showed increased autocrine, promigratory activation of GBM cells. These findings were correlated with significantly enhanced induction by hypoxic compared with normoxic exosomes of tumor vascularization, pericyte vessel coverage, GBM cell proliferation, as well as decreased tumor hypoxia in a mouse xenograft model. We conclude that the proteome and mRNA profiles of exosome vesicles closely reflect the oxygenation status of donor glioma cells and patient tumors, and that the exosomal pathway constitutes a potentially targetable driver of hypoxia-dependent intercellular signaling during tumor development.

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Cost-effectiveness of a community-based intervention for reducing the transmission of Schistosoma haematobium and HIV in Africa.

Publication Date: 2013 May 7 PMID: 23589884
Authors: Ndeffo Mbah, M. L. - Kjetland, E. F. - Atkins, K. E. - Poolman, E. M. - Orenstein, E. W. - Meyers, L. A. - Townsend, J. P. - Galvani, A. P.
Journal: Proc Natl Acad Sci U S A

Epidemiological studies from sub-Saharan Africa show that genital infection with Schistosoma hematobium may increase the risk for HIV infection in young women. Therefore, preventing schistosomiasis has the potential to reduce HIV transmission in sub-Saharan Africa. We developed a transmission model of female genital schistosomiasis and HIV infections that we fit to epidemiological data of HIV and female genital schistosomiasis prevalence and coinfection in rural Zimbabwe. We used the model to evaluate the cost-effectiveness of a multifaceted community-based intervention for preventing schistosomiasis and, consequently, HIV infections in rural Zimbabwe, from the perspective of a health payer. The community-based intervention combined provision of clean water, sanitation, and health education (WSH) with administration of praziquantel to school-aged children. Considering variation in efficacy between 10% and 70% of WSH for reducing S. hematobium transmission, our model predicted that community-based intervention is likely to be cost-effective in Zimbabwe at an aggregated WSH cost corresponding to US $725-$1,000 per individual over a 20-y intervention period. These costs compare favorably with empirical measures of WSH provision in developing countries, indicating that integrated community-based intervention for reducing the transmission of S. hematobium is an economically attractive strategy for reducing schistosomiasis and HIV transmission in sub-Saharan Africa that would have a powerful impact on averting infections and saving lives.

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Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents.

Publication Date: 2013 Apr 30 PMID: 23589883
Authors: Dezi, M. - Di Cicco, A. - Bassereau, P. - Levy, D.
Journal: Proc Natl Acad Sci U S A

Giant unilamellar vesicles (GUVs) are convenient biomimetic systems of the same size as cells that are increasingly used to quantitatively address biophysical and biochemical processes related to cell functions. However, current approaches to incorporate transmembrane proteins in the membrane of GUVs are limited by the amphiphilic nature or proteins. Here, we report a method to incorporate transmembrane proteins in GUVs, based on concepts developed for detergent-mediated reconstitution in large unilamellar vesicles. Reconstitution is performed either by direct incorporation from proteins purified in detergent micelles or by fusion of purified native vesicles or proteoliposomes in preformed GUVs. Lipid compositions of the membrane and the ionic, protein, or DNA compositions in the internal and external volumes of GUVs can be controlled. Using confocal microscopy and functional assays, we show that proteins are unidirectionally incorporated in the GUVs and keep their functionality. We have successfully tested our method with three types of transmembrane proteins. GUVs containing bacteriorhodopsin, a photoactivable proton pump, can generate large transmembrane pH and potential gradients that are light-switchable and stable for hours. GUVs with FhuA, a bacterial porin, were used to follow the DNA injection by T5 phage upon binding to its transmembrane receptor. GUVs incorporating BmrC/BmrD, a bacterial heterodimeric ATP-binding cassette efflux transporter, were used to demonstrate the protein-dependent translocation of drugs and their interactions with encapsulated DNA. Our method should thus apply to a wide variety of membrane or peripheral proteins for producing more complex biomimetic GUVs.

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Adaptation to steady light by intrinsically photosensitive retinal ganglion cells.

Publication Date: 2013 Apr 30 PMID: 23589882
Authors: Do, M. T. - Yau, K. W.
Journal: Proc Natl Acad Sci U S A

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are recently discovered photoreceptors in the mammalian eye. These photoreceptors mediate primarily nonimage visual functions, such as pupillary light reflex and circadian photoentrainment, which are generally expected to respond to the absolute light intensity. The classical rod and cone photoreceptors, on the other hand, mediate image vision by signaling contrast, accomplished by adaptation to light. Experiments by others have indicated that the ipRGCs do, in fact, light-adapt. We found the same but, in addition, have now quantified this light adaptation for the M1 ipRGC subtype. Interestingly, in incremental-flash-on-background experiments, the ipRGC's receptor current showed a flash sensitivity that adapted in background light according to the Weber-Fechner relation, well known to describe the adaptation behavior of rods and cones. Part of this light adaptation by ipRGCs appeared to be triggered by a Ca(2+) influx, in that the flash response elicited in the absence of extracellular Ca(2+) showed a normal rising phase but a slower decay phase, resulting in longer time to peak and higher sensitivity. There is, additionally, a prominent Ca(2+)-independent component of light adaptation not typically seen in rods and cones or in invertebrate rhabdomeric photoreceptors.

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Camouflage mismatch in seasonal coat color due to decreased snow duration.

Publication Date: 2013 Apr 30 PMID: 23589881
Authors: Mills, L. S. - Zimova, M. - Oyler, J. - Running, S. - Abatzoglou, J. T. - Lukacs, P. M.
Journal: Proc Natl Acad Sci U S A

Most examples of seasonal mismatches in phenology span multiple trophic levels, with timing of animal reproduction, hibernation, or migration becoming detached from peak food supply. The consequences of such mismatches are difficult to link to specific future climate change scenarios because the responses across trophic levels have complex underlying climate drivers often confounded by other stressors. In contrast, seasonal coat color polyphenism creating camouflage against snow is a direct and potentially severe type of seasonal mismatch if crypsis becomes compromised by the animal being white when snow is absent. It is unknown whether plasticity in the initiation or rate of coat color change will be able to reduce mismatch between the seasonal coat color and an increasingly snow-free background. We find that natural populations of snowshoe hares exposed to 3 y of widely varying snowpack have plasticity in the rate of the spring white-to-brown molt, but not in either the initiation dates of color change or the rate of the fall brown-to-white molt. Using an ensemble of locally downscaled climate projections, we also show that annual average duration of snowpack is forecast to decrease by 29-35 d by midcentury and 40-69 d by the end of the century. Without evolution in coat color phenology, the reduced snow duration will increase the number of days that white hares will be mismatched on a snowless background by four- to eightfold by the end of the century. This novel and visually compelling climate change-induced stressor likely applies to >9 widely distributed mammals with seasonal coat color.

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Interference with ERKThr188 phosphorylation impairs pathological but not physiological cardiac hypertrophy.

Publication Date: 2013 Apr 30 PMID: 23589880
Authors: Ruppert, C. - Deiss, K. - Herrmann, S. - Vidal, M. - Oezkur, M. - Gorski, A. - Weidemann, F. - Lohse, M. J. - Lorenz, K.
Journal: Proc Natl Acad Sci U S A

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are central mediators of cardiac hypertrophy and are discussed as potential therapeutic targets. However, direct inhibition of ERK1/2 leads to exacerbated cardiomyocyte death and impaired heart function. We have previously identified ERK(Thr188) autophosphorylation as a regulatory phosphorylation of ERK1/2 that is a key factor in cardiac hypertrophy. Here, we investigated whether interference with ERK(Thr188) phosphorylation permits the impairment of ERK1/2-mediated cardiac hypertrophy without increasing cardiomyocyte death. The impact of ERK(Thr188) phosphorylation on cardiomyocyte hypertrophy and cell survival was analyzed in isolated cells and in mice using the mutant ERK2(T188A), which is dominant-negative for ERK(Thr188) signaling. ERK2(T188A) efficiently attenuated cardiomyocyte hypertrophic responses to phenylephrine and to chronic pressure overload, but it affected neither antiapoptotic ERK1/2 signaling nor overall physiological cardiac function. In contrast to its inhibition of pathological hypertrophy, ERK2(T188A) did not interfere with physiological cardiac growth occurring with age or upon voluntary exercise. A preferential role of ERK(Thr188) phosphorylation in pathological types of hypertrophy was also seen in patients with aortic valve stenosis: ERK(Thr188) phosphorylation was increased 8.5 +/- 1.3-fold in high-gradient, rapidly progressing cases (>/=40 mmHg gradient), whereas in low-gradient, slowly progressing cases, the increase was not significant. Because interference with ERK(Thr188) phosphorylation (i) inhibits pathological hypertrophy and (ii) does not impair antiapoptotic ERK1/2 signaling and because ERK(Thr188) phosphorylation shows strong prevalence for aortic stenosis patients with rapidly progressing course, we conclude that interference with ERK(Thr188) phosphorylation offers the possibility to selectively address pathological types of cardiac hypertrophy.

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Structural evidence for a bifurcated mode of action in the antibody-mediated neutralization of hepatitis C virus.

Publication Date: 2013 Apr 30 PMID: 23589879
Authors: Deng, L. - Zhong, L. - Struble, E. - Duan, H. - Ma, L. - Harman, C. - Yan, H. - Virata-Theimer, M. L. - Zhao, Z. - Feinstone, S. - Alter, H. - Zhang, P.
Journal: Proc Natl Acad Sci U S A

Hepatitis C virus (HCV) envelope glycoprotein E2 has been considered as a major target for vaccine design. Epitope II, mapped between residues 427-446 within the E2 protein, elicits antibodies that are either neutralizing or nonneutralizing. The fundamental mechanism of antibody-mediated neutralization at epitope II remains to be defined at the atomic level. Here we report the crystal structure of the epitope II peptide in complex with a monoclonal antibody (mAb#8) capable of neutralizing HCV. The complex structure revealed that this neutralizing antibody engages epitope II via interactions with both the C-terminal alpha-helix and the N-terminal loop using a bifurcated mode of action. Our structural insights into the key determinants for the antibody-mediated neutralization may contribute to the immune prophylaxis of HCV infection and the development of an effective HCV vaccine.

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A thin polymer membrane, nano-suit, enhancing survival across the continuum between air and high vacuum.

Publication Date: 2013 May 7 PMID: 23589878
Authors: Takaku, Y. - Suzuki, H. - Ohta, I. - Ishii, D. - Muranaka, Y. - Shimomura, M. - Hariyama, T.
Journal: Proc Natl Acad Sci U S A

Most multicellular organisms can only survive under atmospheric pressure. The reduced pressure of a high vacuum usually leads to rapid dehydration and death. Here we show that a simple surface modification can render multicellular organisms strongly tolerant to high vacuum. Animals that collapsed under high vacuum continued to move following exposure of their natural extracellular surface layer (or that of an artificial coat-like polysorbitan monolaurate) to an electron beam or plasma ionization (i.e., conditions known to enhance polymer formation). Transmission electron microscopic observations revealed the existence of a thin polymerized extra layer on the surface of the animal. The layer acts as a flexible "nano-suit" barrier to the passage of gases and liquids and thus protects the organism. Furthermore, the biocompatible molecule, the component of the nano-suit, was fabricated into a "biomimetic" free-standing membrane. This concept will allow biology-related fields especially to use these membranes for several applications.

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Hammondia hammondi, an avirulent relative of Toxoplasma gondii, has functional orthologs of known T. gondii virulence genes.

Publication Date: 2013 Apr 30 PMID: 23589877
Authors: Walzer, K. A. - Adomako-Ankomah, Y. - Dam, R. A. - Herrmann, D. C. - Schares, G. - Dubey, J. P. - Boyle, J. P.
Journal: Proc Natl Acad Sci U S A

Toxoplasma gondii is a ubiquitous protozoan parasite capable of infecting all warm-blooded animals, including humans. Its closest extant relative, Hammondia hammondi, has never been found to infect humans and, in contrast to T. gondii, is highly attenuated in mice. To better understand the genetic bases for these phenotypic differences, we sequenced the genome of a H. hammondi isolate (HhCatGer041) and found the genomic synteny between H. hammondi and T. gondii to be >95%. We used this genome to determine the H. hammondi primary sequence of two major T. gondii mouse virulence genes, TgROP5 and TgROP18. When we expressed these genes in T. gondii, we found that H. hammondi orthologs of TgROP5 and TgROP18 were functional. Similar to T. gondii, the HhROP5 locus is expanded, and two distinct HhROP5 paralogs increased the virulence of a T. gondii TgROP5 knockout strain. We also identified a 107 base pair promoter region, absent only in type III TgROP18, which is necessary for TgROP18 expression. This result indicates that the ROP18 promoter was active in the most recent common ancestor of these two species and that it was subsequently inactivated in progenitors of the type III lineage. Overall, these data suggest that the virulence differences between these species are not solely due to the functionality of these key virulence factors. This study provides evidence that other mechanisms, such as differences in gene expression or the lack of currently uncharacterized virulence factors, may underlie the phenotypic differences between these species.

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Allosteric mechanisms can be distinguished using structural mass spectrometry.

Publication Date: 2013 Apr 30 PMID: 23589876
Authors: Dyachenko, A. - Gruber, R. - Shimon, L. - Horovitz, A. - Sharon, M.
Journal: Proc Natl Acad Sci U S A

The activity of many proteins, including metabolic enzymes, molecular machines, and ion channels, is often regulated by conformational changes that are induced or stabilized by ligand binding. In cases of multimeric proteins, such allosteric regulation has often been described by the concerted Monod-Wyman-Changeux and sequential Koshland-Nemethy-Filmer classic models of cooperativity. Despite the important functional implications of the mechanism of cooperativity, it has been impossible in many cases to distinguish between these various allosteric models using ensemble measurements of ligand binding in bulk protein solutions. Here, we demonstrate that structural MS offers a way to break this impasse by providing the full distribution of ligand-bound states of a protein complex. Given this distribution, it is possible to determine all the binding constants of a ligand to a highly multimeric cooperative system, and thereby infer its allosteric mechanism. Our approach to the dissection of allosteric mechanisms relies on advances in MS-which provide the required resolution of ligand-bound states-and in data analysis. We validated our approach using the well-characterized Escherichia coli chaperone GroEL, a double-heptameric ring containing 14 ATP binding sites, which has become a paradigm for molecular machines. The values of the 14 binding constants of ATP to GroEL were determined, and the ATP-loading pathway of the chaperone was characterized. The methodology and analyses presented here are directly applicable to numerous other cooperative systems and are therefore expected to promote further research on allosteric systems.

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High-performance hollow sulfur nanostructured battery cathode through a scalable, room temperature, one-step, bottom-up approach.

Publication Date: 2013 Apr 30 PMID: 23589875
Authors: Li, W. - Zheng, G. - Yang, Y. - Seh, Z. W. - Liu, N. - Cui, Y.
Journal: Proc Natl Acad Sci U S A

Sulfur is an exciting cathode material with high specific capacity of 1,673 mAh/g, more than five times the theoretical limits of its transition metal oxides counterpart. However, successful applications of sulfur cathode have been impeded by rapid capacity fading caused by multiple mechanisms, including large volume expansion during lithiation, dissolution of intermediate polysulfides, and low ionic/electronic conductivity. Tackling the sulfur cathode problems requires a multifaceted approach, which can simultaneously address the challenges mentioned above. Herein, we present a scalable, room temperature, one-step, bottom-up approach to fabricate monodisperse polymer (polyvinylpyrrolidone)-encapsulated hollow sulfur nanospheres for sulfur cathode, allowing unprecedented control over electrode design from nanoscale to macroscale. We demonstrate high specific discharge capacities at different current rates (1,179, 1,018, and 990 mAh/g at C/10, C/5, and C/2, respectively) and excellent capacity retention of 77.6% (at C/5) and 73.4% (at C/2) after 300 and 500 cycles, respectively. Over a long-term cycling of 1,000 cycles at C/2, a capacity decay as low as 0.046% per cycle and an average coulombic efficiency of 98.5% was achieved. In addition, a simple modification on the sulfur nanosphere surface with a layer of conducting polymer, poly(3,4-ethylenedioxythiophene), allows the sulfur cathode to achieve excellent high-rate capability, showing a high reversible capacity of 849 and 610 mAh/g at 2C and 4C, respectively.

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Cell-trappable fluorescent probes for endogenous hydrogen sulfide signaling and imaging H2O2-dependent H2S production.

Publication Date: 2013 Apr 30 PMID: 23589874
Authors: Lin, V. S. - Lippert, A. R. - Chang, C. J.
Journal: Proc Natl Acad Sci U S A

Hydrogen sulfide (H2S) is a reactive small molecule generated in the body that can be beneficial or toxic owing to its potent redox activity. In living systems, disentangling the pathways responsible for H2S production and their physiological and pathological consequences remains a challenge in part due to a lack of methods for monitoring changes in endogenous H2S fluxes. The development of fluorescent probes with appropriate selectivity and sensitivity for monitoring production of H2S at biologically relevant signaling levels offers opportunities to explore its roles in a variety of systems. Here we report the design, synthesis, and application of a family of azide-based fluorescent H2S indicators, Sulfidefluor-4, Sulfidefluor-5 acetoxymethyl ester, and Sulfidefluor-7 acetoxymethyl ester, which offer the unique capability to image H2S generated at physiological signaling levels. These probes are optimized for cellular imaging and feature enhanced sensitivity and cellular retention compared with our previously reported molecules. In particular, Sulfidefluor-7 acetoxymethyl ester allows for direct, real-time visualization of endogenous H2S produced in live human umbilical vein endothelial cells upon stimulation with vascular endothelial growth factor (VEGF). Moreover, we show that H2S production is dependent on NADPH oxidase-derived hydrogen peroxide (H2O2), which attenuates VEGF receptor 2 phosphorylation and establishes a link for H2S/H2O2 crosstalk.

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Europe's other debt crisis caused by the long legacy of future extinctions.

Publication Date: 2013 Apr 30 PMID: 23589873
Authors: Dullinger, S. - Essl, F. - Rabitsch, W. - Erb, K. H. - Gingrich, S. - Haberl, H. - Hulber, K. - Jarosik, V. - Krausmann, F. - Kuhn, I. - Pergl, J. - Pysek, P. - Hulme, P. E.
Journal: Proc Natl Acad Sci U S A

Rapid economic development in the past century has translated into severe pressures on species survival as a result of increasing land-use change, environmental pollution, and the spread of invasive alien species. However, though the impact of these pressures on biodiversity is substantial, it could be seriously underestimated if population declines of plants and animals lag behind contemporary environmental degradation. Here, we test for such a delay in impact by relating numbers of threatened species appearing on national red lists to historical and contemporary levels of socioeconomic pressures. Across 22 European countries, the proportions of vascular plants, bryophytes, mammals, reptiles, dragonflies, and grasshoppers facing medium-to-high extinction risks are more closely matched to indicators of socioeconomic pressures (i.e., human population density, per capita gross domestic product, and a measure of land use intensity) from the early or mid-, rather than the late, 20th century. We conclude that, irrespective of recent conservation actions, large-scale risks to biodiversity lag considerably behind contemporary levels of socioeconomic pressures. The negative impact of human activities on current biodiversity will not become fully realized until several decades into the future. Mitigating extinction risks might be an even greater challenge if temporal delays mean many threatened species might already be destined toward extinction.

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The cardiac Na+-Ca2+ exchanger has two cytoplasmic ion permeation pathways.

Publication Date: 2013 Apr 30 PMID: 23589872
Authors: John, S. A. - Liao, J. - Jiang, Y. - Ottolia, M.
Journal: Proc Natl Acad Sci U S A

The Na(+)-Ca(2+) exchanger (NCX) is a ubiquitously expressed plasma membrane protein. It plays a fundamental role in Ca(2+) homeostasis by moving Ca(2+) out of the cell using the electrochemical gradient of Na(+) as the driving force. Recent structural studies of a homologous archaebacterial exchanger, NCX_Mj, revealed its outward configuration with two potential ion permeation pathways exposed to the extracellular environment. Based on the symmetry of NCX_Mj structure, an atomic model of an inward-facing conformation was generated showing similar pathways but directed to the cytoplasm. The presence of these water-filled cavities has yet to be confirmed experimentally, and it is unknown if the mammalian exchanger adopts the same structure. In this study, we mutated multiple residues within transmembrane segments 2 and 7 of NCX1.1 (cardiac isoform) to cysteines, allowing us to investigate their sensitivity to membrane-impermeable sulfhydryl reagents as exchanger current block. By trapping NCX1.1 in the inward-facing configuration, we have mapped two differently sized cytoplasmic aqueous cavities, the access of which is modified during exchange. This data reveals movements of the protein associated with ion transport. Electrophysiological characterization shows that the conserved residues within transmembrane segments 2 and 7, coordinating Na(+) and Ca(2+) ions in NCX_Mj, play a fundamental role in NCX1.1. Our results suggest a similar architecture between the mammalian and archaebacterial exchangers.

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Phosphoinositides and membrane curvature switch the mode of actin polymerization via selective recruitment of toca-1 and Snx9.

Publication Date: 2013 Apr 30 PMID: 23589871
Authors: Gallop, J. L. - Walrant, A. - Cantley, L. C. - Kirschner, M. W.
Journal: Proc Natl Acad Sci U S A

The membrane-cytosol interface is the major locus of control of actin polymerization. At this interface, phosphoinositides act as second messengers to recruit membrane-binding proteins. We show that curved membranes, but not flat ones, can use phosphatidylinositol 3-phosphate [PI(3)P] along with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to stimulate actin polymerization. In this case, actin polymerization requires the small GTPase cell cycle division 42 (Cdc42), the nucleation-promoting factor neural Wiskott-Aldrich syndrome protein (N-WASP) and the actin nucleator the actin-related protein (Arp) 2/3 complex. In liposomes containing PI(4,5)P2 as the sole phosphoinositide, actin polymerization requires transducer of Cdc42 activation-1 (toca-1). In the presence of phosphatidylinositol 3-phosphate, polymerization is both more efficient and independent of toca-1. Under these conditions, sorting nexin 9 (Snx9) can be implicated as a specific adaptor that replaces toca-1 to mobilize neural Wiskott-Aldrich syndrome protein and the Arp2/3 complex. This switch in phosphoinositide and adaptor specificity for actin polymerization from membranes has implications for how different types of actin structures are generated at precise times and locations in the cell.

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A strategy to capture and characterize the synaptic transcriptome.

Publication Date: 2013 Apr 30 PMID: 23589870
Authors: Puthanveettil, S. V. - Antonov, I. - Kalachikov, S. - Rajasethupathy, P. - Choi, Y. B. - Kohn, A. B. - Citarella, M. - Yu, F. - Karl, K. A. - Kinet, M. - Morozova, I. - Russo, J. J. - Ju, J. - Moroz, L. L. - Kandel, E. R.
Journal: Proc Natl Acad Sci U S A

Here we describe a strategy designed to identify RNAs that are actively transported to synapses during learning. Our approach is based on the characterization of RNA transport complexes carried by molecular motor kinesin. Using this strategy in Aplysia, we have identified 5,657 unique sequences consisting of both coding and noncoding RNAs from the CNS. Several of these RNAs have key roles in the maintenance of synaptic function and growth. One of these RNAs, myosin heavy chain, is critical in presynaptic sensory neurons for the establishment of long-term facilitation, but not for its persistence.

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Protein-DNA complexes are the primary sources of replication fork pausing in Escherichia coli.

Publication Date: 2013 Apr 30 PMID: 23589869
Authors: Gupta, M. K. - Guy, C. P. - Yeeles, J. T. - Atkinson, J. - Bell, H. - Lloyd, R. G. - Marians, K. J. - McGlynn, P.
Journal: Proc Natl Acad Sci U S A

Replication fork pausing drives genome instability, because any loss of paused replisome activity creates a requirement for reloading of the replication machinery, a potentially mutagenic process. Despite this importance, the relative contributions to fork pausing of different replicative barriers remain unknown. We show here that Deinococcus radiodurans RecD2 helicase inactivates Escherichia coli replisomes that are paused but still functional in vitro, preventing continued fork movement upon barrier removal or bypass, but does not inactivate elongating forks. Using RecD2 to probe replisome pausing in vivo, we demonstrate that most pausing events do not lead to replisome inactivation, that transcription complexes are the primary sources of this pausing, and that an accessory replicative helicase is critical for minimizing the frequency and/or duration of replisome pauses. These findings reveal the hidden potential for replisome inactivation, and hence genome instability, inside cells. They also demonstrate that efficient chromosome duplication requires mechanisms that aid resumption of replication by paused replisomes, especially those halted by protein-DNA barriers such as transcription complexes.

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NIF-type iron-sulfur cluster assembly system is duplicated and distributed in the mitochondria and cytosol of Mastigamoeba balamuthi.

Publication Date: 2013 Apr 30 PMID: 23589868
Authors: Nyvltova, E. - Sutak, R. - Harant, K. - Sedinova, M. - Hrdy, I. - Paces, J. - Vlcek, C. - Tachezy, J.
Journal: Proc Natl Acad Sci U S A

In most eukaryotes, the mitochondrion is the main organelle for the formation of iron-sulfur (FeS) clusters. This function is mediated through the iron-sulfur cluster assembly machinery, which was inherited from the alpha-proteobacterial ancestor of mitochondria. In Archamoebae, including pathogenic Entamoeba histolytica and free-living Mastigamoeba balamuthi, the complex iron-sulfur cluster machinery has been replaced by an epsilon-proteobacterial nitrogen fixation (NIF) system consisting of two components: NifS (cysteine desulfurase) and NifU (scaffold protein). However, the cellular localization of the NIF system and the involvement of mitochondria in archamoebal FeS assembly are controversial. Here, we show that the genes for both NIF components are duplicated within the M. balamuthi genome. One paralog of each protein contains an amino-terminal extension that targets proteins to mitochondria (NifS-M and NifU-M), and the second paralog lacks a targeting signal, thereby reflecting the cytosolic form of the NIF machinery (NifS-C and NifU-C). The dual localization of the NIF system corresponds to the presence of FeS proteins in both cellular compartments, including detectable hydrogenase activity in Mastigamoeba cytosol and mitochondria. In contrast, E. histolytica possesses only single genes encoding NifS and NifU, respectively, and there is no evidence for the presence of the NIF machinery in its reduced mitochondria. Thus, M. balamuthi is unique among eukaryotes in that its FeS cluster formation is mediated through two most likely independent NIF machineries present in two cellular compartments.

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Atypical lipid composition in the purified relict plastid (apicoplast) of malaria parasites.

Publication Date: 2013 Apr 30 PMID: 23589867
Authors: Botte, C. Y. - Yamaryo-Botte, Y. - Rupasinghe, T. W. - Mullin, K. A. - Macrae, J. I. - Spurck, T. P. - Kalanon, M. - Shears, M. J. - Coppel, R. L. - Crellin, P. K. - Marechal, E. - McConville, M. J. - McFadden, G. I.
Journal: Proc Natl Acad Sci U S A

The human malaria parasite Plasmodium falciparum harbors a relict, nonphotosynthetic plastid of algal origin termed the apicoplast. Although considerable progress has been made in defining the metabolic functions of the apicoplast, information on the composition and biogenesis of the four delimiting membranes of this organelle is limited. Here, we report an efficient method for preparing highly purified apicoplasts from red blood cell parasite stages and the comprehensive lipidomic analysis of this organelle. Apicoplasts were prepared from transgenic parasites expressing an epitope-tagged triosephosphate transporter and immunopurified on magnetic beads. Gas and liquid chromatography MS analyses of isolated apicoplast lipids indicated significant differences compared with total parasite lipids. In particular, apicoplasts were highly enriched in phosphatidylinositol, consistent with a suggested role for phosphoinositides in targeting membrane vesicles to apicoplasts. Apicoplast phosphatidylinositol and other phospholipids were also enriched in saturated fatty acids, which could reflect limited acyl exchange with other membrane phospholipids and/or a requirement for specific physical properties. Lipids atypical for plastids (sphingomyelins, ceramides, and cholesterol) were detected in apicoplasts. The presence of cholesterol in apicoplast membranes was supported by filipin staining of isolated apicoplasts. Galactoglycerolipids, dominant in plant and algal plastids, were not detected in P. falciparum apicoplasts, suggesting that these glycolipids are a hallmark of photosynthetic plastids and were lost when these organisms assumed a parasitic lifestyle. Apicoplasts thus contain an atypical melange of lipids scavenged from the human host alongside lipids remodeled by the parasite cytoplasm, and stable isotope labeling shows some apicoplast lipids are generated de novo by the organelle itself.

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Lineage tracing with Axin2 reveals distinct developmental and adult populations of Wnt/beta-catenin-responsive neural stem cells.

Publication Date: 2013 Apr 30 PMID: 23589866
Authors: Bowman, A. N. - van Amerongen, R. - Palmer, T. D. - Nusse, R.
Journal: Proc Natl Acad Sci U S A

Since the discovery of neural stem cells in the mammalian brain, there has been significant interest in understanding their contribution to tissue homeostasis at both the cellular and molecular level. Wnt/beta-catenin signaling is crucial for development of the central nervous system and has been implicated in stem cell maintenance in multiple tissues. Based on this, we hypothesized that the Wnt pathway likely controls neural stem cell maintenance and differentiation along the entire developmental continuum. To test this, we performed lineage tracing experiments using the recently developed tamoxifen-inducible Cre at Axin2 mouse strain to follow the developmental fate of Wnt/beta-catenin-responsive cells in both the embryonic and postnatal mouse brain. From as early as embryonic day 8.5 onwards, Axin2(+) cells can give rise to spatially and functionally restricted populations of adult neural stem cells in the subventricular zone. Similarly, progeny from Axin2(+) cells labeled from E12.5 contribute to both the subventricular zone and the dentate gyrus of the hippocampus. Labeling in the postnatal brain, in turn, demonstrates the persistence of long-lived, Wnt/beta-catenin-responsive stem cells in both of these sites. These results demonstrate the continued importance of Wnt/beta-catenin signaling for neural stem and progenitor cell formation and function throughout developmental time.

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Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries.

Publication Date: 2013 Apr 30 PMID: 23589865
Authors: Yi, L. - Gebhard, M. C. - Li, Q. - Taft, J. M. - Georgiou, G. - Iverson, B. L.
Journal: Proc Natl Acad Sci U S A

Myriad new applications of proteases would be enabled by an ability to fine-tune substrate specificity and activity. Herein we present a general strategy for engineering protease selectivity and activity by capitalizing on sequestration of the protease to be engineered within the yeast endoplasmic reticulum (ER). A substrate fusion protein composed of yeast adhesion receptor subunit Aga2, selection and counterselection substrate sequences, multiple intervening epitope tag sequences, and a C-terminal ER retention sequence is coexpressed with a protease library. Cleavage of the substrate fusion protein by the protease eliminates the ER retention sequence, facilitating transport to the yeast surface. Yeast cells that display Aga2 fusions in which only the selection substrate is cleaved are isolated by multicolor FACS with fluorescently labeled antiepitope tag antibodies. Using this system, the Tobacco Etch Virus protease (TEV-P), which strongly prefers Gln at P1 of its canonical ENLYFQ downward arrowS substrate, was engineered to recognize selectively Glu or His at P1. Kinetic analysis indicated an overall 5,000-fold and 1,100-fold change in selectivity, respectively, for the Glu- and His-specific TEV variants, both of which retained high catalytic turnover. Human granzyme K and the hepatitis C virus protease were also shown to be amenable to this unique approach. Further, by adjusting the signaling strategy to identify phosphorylated as opposed to cleaved sequences, this unique system was shown to be compatible with the human Abelson tyrosine kinase.

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Coordinated elevation of mitochondrial oxidative phosphorylation and autophagy help drive hepatocyte polarization.

Publication Date: 2013 Apr 30 PMID: 23589864
Authors: Fu, D. - Mitra, K. - Sengupta, P. - Jarnik, M. - Lippincott-Schwartz, J. - Arias, I. M.
Journal: Proc Natl Acad Sci U S A

Cell polarization requires increased cellular energy and metabolic output, but how these energetic demands are met by polarizing cells is unclear. To address these issues, we investigated the roles of mitochondrial bioenergetics and autophagy during cell polarization of hepatocytes cultured in a collagen sandwich system. We found that as the hepatocytes begin to polarize, they use oxidative phosphorylation to raise their ATP levels, and this energy production is required for polarization. After the cells are polarized, the hepatocytes shift to become more dependent on glycolysis to produce ATP. Along with this central reliance on oxidative phosphorylation as the main source of ATP production in polarizing cultures, several other metabolic processes are reprogrammed during the time course of polarization. As the cells polarize, mitochondria elongate and mitochondrial membrane potential increases. In addition, lipid droplet abundance decreases over time. These findings suggest that polarizing cells are reliant on fatty acid oxidation, which is supported by pharmacologic inhibition of beta-oxidation by etomoxir. Finally, autophagy is up-regulated during cell polarization, with inhibition of autophagy retarding cell polarization. Taken together, our results describe a metabolic shift involving a number of coordinated metabolic pathways that ultimately serve to increase energy production during cell polarization.

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Dosage compensation and inverse effects in triple X metafemales of Drosophila.

Publication Date: 2013 Apr 30 PMID: 23589863
Authors: Sun, L. - Johnson, A. F. - Donohue, R. C. - Li, J. - Cheng, J. - Birchler, J. A.
Journal: Proc Natl Acad Sci U S A

Dosage compensation, the equalized X chromosome gene expression between males and females in Drosophila, has also been found in triple X metafemales. Inverse dosage effects, produced by genomic imbalance, are believed to account for this modulated expression, but they have not been studied on a global level. Here, we show a global expression comparison of metafemales (XXX; AA) with normal females (XX; AA) with high-throughput RNA-sequencing. We found that the majority of the X-linked genes in metafemales exhibit dosage compensation with an expression level similar to that of normal diploid females. In parallel, most of the autosomal genes were expressed at about two-thirds the level of normal females, the ratio of inverse dosage effects produced by the extra X chromosome. Both compensation and inverse effects were further confirmed by combination of X-linked and autosomally located miniwhite reporter genes in metafemales and relative quantitative PCR of selected genes. These data provide evidence for an inverse dosage component to X chromosome compensation.

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Rational design of thermostable vaccines by engineered peptide-induced virus self-biomineralization under physiological conditions.

Publication Date: 2013 May 7 PMID: 23589862
Authors: Wang, G. - Cao, R. Y. - Chen, R. - Mo, L. - Han, J. F. - Wang, X. - Xu, X. - Jiang, T. - Deng, Y. Q. - Lyu, K. - Zhu, S. Y. - Qin, E. D. - Tang, R. - Qin, C. F.
Journal: Proc Natl Acad Sci U S A

The development of vaccines against infectious diseases represents one of the most important contributions to medical science. However, vaccine-preventable diseases still cause millions of deaths each year due to the thermal instability and poor efficacy of vaccines. Using the human enterovirus type 71 vaccine strain as a model, we suggest a combined, rational design approach to improve the thermostability and immunogenicity of live vaccines by self-biomineralization. The biomimetic nucleating peptides are rationally integrated onto the capsid of enterovirus type 71 by reverse genetics so that calcium phosphate mineralization can be biologically induced onto vaccine surfaces under physiological conditions, generating a mineral exterior. This engineered self-biomineralized virus was characterized in detail for its unique structural, virological, and chemical properties. Analogous to many exteriors, the mineral coating confers some new properties on enclosed vaccines. The self-biomineralized vaccine can be stored at 26 degrees C for more than 9 d and at 37 degrees C for approximately 1 wk. Both in vitro and in vivo experiments demonstrate that this engineered vaccine can be used efficiently after heat treatment or ambient temperature storage, which reduces the dependence on a cold chain. Such a combination of genetic technology and biomineralization provides an economic solution for current vaccination programs, especially in developing countries that lack expensive refrigeration infrastructures.

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Non-Fermi liquid regimes with and without quantum criticality in Ce1-xYbxCoIn5.

Publication Date: 2013 Apr 30 PMID: 23589861
Authors: Hu, T. - Singh, Y. P. - Shu, L. - Janoschek, M. - Dzero, M. - Maple, M. B. - Almasan, C. C.
Journal: Proc Natl Acad Sci U S A

One of the greatest challenges to Landau's Fermi liquid theory-the standard theory of metals-is presented by complex materials with strong electronic correlations. In these materials, non-Fermi liquid transport and thermodynamic properties are often explained by the presence of a continuous quantum phase transition that happens at a quantum critical point (QCP). A QCP can be revealed by applying pressure, magnetic field, or changing the chemical composition. In the heavy-fermion compound CeCoIn5, the QCP is assumed to play a decisive role in defining the microscopic structure of both normal and superconducting states. However, the question of whether a QCP must be present in the material's phase diagram to induce non-Fermi liquid behavior and trigger superconductivity remains open. Here, we show that the full suppression of the field-induced QCP in CeCoIn5 by doping with Yb has surprisingly little impact on both unconventional superconductivity and non-Fermi liquid behavior. This implies that the non-Fermi liquid metallic behavior could be a new state of matter in its own right rather than a consequence of the underlying quantum phase transition.

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Agricultural intensification escalates future conservation costs.

Publication Date: 2013 May 7 PMID: 23589860
Authors: Phelps, J. - Carrasco, L. R. - Webb, E. L. - Koh, L. P. - Pascual, U.
Journal: Proc Natl Acad Sci U S A

The supposition that agricultural intensification results in land sparing for conservation has become central to policy formulations across the tropics. However, underlying assumptions remain uncertain and have been little explored in the context of conservation incentive schemes such as policies for Reducing Emissions from Deforestation and forest Degradation, conservation, sustainable management, and enhancement of carbon stocks (REDD+). Incipient REDD+ forest carbon policies in a number of countries propose agricultural intensification measures to replace extensive "slash-and-burn" farming systems. These may result in conservation in some contexts, but will also increase future agricultural land rents as productivity increases, creating new incentives for agricultural expansion and deforestation. While robust governance can help to ensure land sparing, we propose that conservation incentives will also have to increase over time, tracking future agricultural land rents, which might lead to runaway conservation costs. We present a conceptual framework that depicts these relationships, supported by an illustrative model of the intensification of key crops in the Democratic Republic of Congo, a leading REDD+ country. A von Thunen land rent model is combined with geographic information systems mapping to demonstrate how agricultural intensification could influence future conservation costs. Once postintensification agricultural land rents are considered, the cost of reducing forest sector emissions could significantly exceed current and projected carbon credit prices. Our analysis highlights the importance of considering escalating conservation costs from agricultural intensification when designing conservation initiatives.

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Domain cooperativity in the beta1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle.

Publication Date: 2013 Apr 30 PMID: 23589859
Authors: Dayal, A. - Bhat, V. - Franzini-Armstrong, C. - Grabner, M.
Journal: Proc Natl Acad Sci U S A

The dihydropyridine receptor (DHPR) beta1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRalpha1S charge movement-the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target alpha1S into triadic junctions and tetradic arrays, the neuronal isoform beta3 was unable to restore considerable charge movement (measure of alpha1S voltage sensing) upon expression in beta1-null zebrafish relaxed myotubes, unlike the other three vertebrate beta-isoforms (beta1a, beta2a, and beta4). Thus, we used beta3 for chimerization with beta1a to investigate whether any of the five distinct molecular regions of beta1a is dominantly involved in inducing the voltage-sensing function of alpha1S. Surprisingly, systematic domain swapping between beta1a and beta3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of beta1a in charge movement restoration. More interestingly, beta1a SH3 domain and C terminus, when simultaneously engineered into beta3 sequence background, were able to fully restore charge movement together with proper intracellular Ca(2+) release, suggesting cooperativity of these two domains in induction of the alpha1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of beta1a (also of beta2a and beta4) fully obstructed alpha1S charge movement. Consequently, we postulate a model according to which beta subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the alpha1S conformation apt for voltage sensing in skeletal muscle.

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Relationship of DNA degradation by Saccharomyces cerevisiae Exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection.

Publication Date: 2013 Apr 30 PMID: 23589858
Authors: Cannavo, E. - Cejka, P. - Kowalczykowski, S. C.
Journal: Proc Natl Acad Sci U S A

Significance The repair of double-strand DNA breaks by homologous recombination is initiated by the nucleolytic resection of the 5'-terminated strand at the DNA break. Genetic work in Saccharomyces cerevisiae identified three DNA end resection pathways: Mre11-Rad50-Xrs2, Dna2-Sgs1-Top3-Rmi1, and Exo1. Here we investigated the relationship between the three nucleolytic complexes in vitro. With a focus on Exo1, we show that it is stimulated by the single-strand DNA-binding protein, RPA, and also by the Mre11-Rad50-Xrs2 complex. Furthermore, our analysis provides biochemical evidence for the view that Exo1 and Dna2-Sgs1-Top3-Rmi1 function downstream of Mre11-Rad50-Xrs2 as independent and mutually exclusive DNA end-processing pathways.

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SoxB1-driven transcriptional network underlies neural-specific interpretation of morphogen signals.

Publication Date: 2013 Apr 30 PMID: 23589857
Authors: Oosterveen, T. - Kurdija, S. - Enstero, M. - Uhde, C. W. - Bergsland, M. - Sandberg, M. - Sandberg, R. - Muhr, J. - Ericson, J.
Journal: Proc Natl Acad Sci U S A

The reiterative deployment of a small cadre of morphogen signals underlies patterning and growth of most tissues during embyogenesis, but how such inductive events result in tissue-specific responses remains poorly understood. By characterizing cis-regulatory modules (CRMs) associated with genes regulated by Sonic hedgehog (Shh), retinoids, or bone morphogenetic proteins in the CNS, we provide evidence that the neural-specific interpretation of morphogen signaling reflects a direct integration of these pathways with SoxB1 proteins at the CRM level. Moreover, expression of SoxB1 proteins in the limb bud confers on mesodermal cells the potential to activate neural-specific target genes upon Shh, retinoid, or bone morphogenetic protein signaling, and the collocation of binding sites for SoxB1 and morphogen-mediatory transcription factors in CRMs faithfully predicts neural-specific gene activity. Thus, an unexpectedly simple transcriptional paradigm appears to conceptually explain the neural-specific interpretation of pleiotropic signaling during vertebrate development. Importantly, genes induced in a SoxB1-dependent manner appear to constitute repressive gene regulatory networks that are directly interlinked at the CRM level to constrain the regional expression of patterning genes. Accordingly, not only does the topology of SoxB1-driven gene regulatory networks provide a tissue-specific mode of gene activation, but it also determines the spatial expression pattern of target genes within the developing neural tube.

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High NaCl- and urea-induced posttranslational modifications that increase glycerophosphocholine by inhibiting GDPD5 phosphodiesterase.

Publication Date: 2013 Apr 30 PMID: 23589856
Authors: Topanurak, S. - Ferraris, J. D. - Li, J. - Izumi, Y. - Williams, C. K. - Gucek, M. - Wang, G. - Zhou, X. - Burg, M. B.
Journal: Proc Natl Acad Sci U S A

Glycerophosphocholine (GPC) is high in cells of the renal inner medulla where high interstitial NaCl and urea power concentration of the urine. GPC protects inner medullary cells against the perturbing effects of high NaCl and urea by stabilizing intracellular macromolecules. Degradation of GPC is catalyzed by the glycerophosphocholine phosphodiesterase activity of glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). We previously found that inhibitory posttranslational modification (PTM) of GDPD5 contributes to high NaCl- and urea-induced increase of GPC. The purpose of the present studies was to identify the PTM(s). We find at least three such PTMs in HEK293 cells: (i) Formation of a disulfide bond between C25 and C571. High NaCl and high urea increase reactive oxygen species (ROS). The ROS increase disulfide bonding between GDPD5-C25 and -C571, which inhibits GDPD5 activity, as supported by the findings that the antioxidant N-acetylcysteine prevents high NaCl- and urea-induced inhibition of GDPD5; GDPD5-C25S/C571S mutation or over expression of peroxiredoxin increases GDPD5 activity; H2O2 inhibits activity of wild type GDPD5, but not of GDPD5-C25S/C571S; and peroxiredoxin is relatively low in the renal inner medulla where GPC is high. (ii) Dephosphorylation of GDPD5-T587. GDPD5 threonine 587 is constitutively phosphorylated. High NaCl and high urea dephosphorylate GDPD5-T587. Mutation of GDPD5-T587 to alanine, which cannot be phosphorylated, decreases GPC-PDE activity of GDPD5. (iii) Alteration at an unknown site mediated by CDK1. Inhibition of CDK1 protein kinase reduces GDE-PDE activity of GDPD5 without altering phosphorylation at T587, and CDK1/5 inhibitor reduces activity of GDPD5- C25S/C571S-T587A.

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Critical role for miR-181a/b-1 in agonist selection of invariant natural killer T cells.

Publication Date: 2013 Apr 30 PMID: 23589855
Authors: Zietara, N. - Lyszkiewicz, M. - Witzlau, K. - Naumann, R. - Hurwitz, R. - Langemeier, J. - Bohne, J. - Sandrock, I. - Ballmaier, M. - Weiss, S. - Prinz, I. - Krueger, A.
Journal: Proc Natl Acad Sci U S A

T-cell receptor (TCR) signal strength determines selection and lineage fate at the CD4(+)CD8(+) double-positive stage of intrathymic T-cell development. Members of the miR-181 family constitute the most abundantly expressed microRNA at this stage of T-cell development. Here we show that deletion of miR-181a/b-1 reduced the responsiveness of double-positive thymocytes to TCR signals and virtually abrogated early invariant natural killer T (iNKT) cell development, resulting in a dramatic reduction in iNKT cell numbers in thymus as well as in the periphery. Increased concentrations of agonist ligand rescued iNKT cell development in miR-181a/b-1(-/-) mice. Our results define a critical role of miR-181a/b-1 in early iNKT cell development and show that miR-181a/b-1 sets a TCR signaling threshold for agonist selection.

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Structural basis for encapsidation of genomic RNA by La Crosse Orthobunyavirus nucleoprotein.

Publication Date: 2013 Apr 30 PMID: 23589854
Authors: Reguera, J. - Malet, H. - Weber, F. - Cusack, S.
Journal: Proc Natl Acad Sci U S A

The nucleoprotein (NP) of segmented negative-strand RNA viruses such as Orthomyxo-, Arena-, and Bunyaviruses coats the genomic viral RNA and together with the polymerase forms ribonucleoprotein particles (RNPs), which are both the template for replication and transcription and are packaged into new virions. Here we describe the crystal structure of La Crosse Orthobunyavirus NP both RNA free and a tetrameric form with single-stranded RNA bound. La Crosse Orthobunyavirus NP is a largely helical protein with a fold distinct from other bunyavirus genera NPs. It binds 11 RNA nucleotides in the positively charged groove between its two lobes, and hinged N- and C-terminal arms mediate oligomerization, allowing variable protein-protein interface geometry. Oligomerization and RNA binding are mediated by residues conserved in the Orthobunyavirus genus. In the twofold symmetric tetramer, 44 nucleotides bind in a closed ring with sharp bends at the NP-NP interfaces. The RNA is largely inaccessible within a continuous internal groove. Electron microscopy of RNPs released from virions shows them capable of forming a hierarchy of more or less compact irregular helical structures. We discuss how the planar, tetrameric NP-RNA structure might relate to a polar filament that upon supercoiling could be packaged into virions. This work gives insight into the RNA encapsidation and protection function of bunyavirus NP, but also highlights the need for dynamic rearrangements of the RNP to give the polymerase access to the template RNA.

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Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain.

Publication Date: 2013 Apr 30 PMID: 23589853
Authors: Muretta, J. M. - Petersen, K. J. - Thomas, D. D.
Journal: Proc Natl Acad Sci U S A

We have used transient kinetics, nanosecond time-resolved fluorescence resonance energy transfer (FRET), and kinetics simulations to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with the force-transducing rotation of the myosin light-chain domain. Previous research in the absence of actin showed that ATP binding to myosin induces a dynamic equilibrium between a bent prepower stroke state of the relay helix and a straight postpower stroke state, which dominates in the absence of ATP or when ADP is bound. We now ask whether actin binding reverses this transition and if so, how this reversal is coordinated with actin-activated phosphate release. We labeled a Cys-lite Dictyostelium myosin II motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We then performed transient time-resolved FRET following stopped-flow mixing of actin with labeled myosin, preincubated with ATP. We determined the kinetics of actin-activated phosphate release, using fluorescent phosphate-binding protein. The results show that actin binding to the myosin.ADP.P complex straightens the relay helix before phosphate dissociation. This actin-activated relay helix straightening is reversible, but phosphate irreversibly dissociates from the postpower stroke state, preventing reversal of the power stroke. Thus, relay helix straightening gates phosphate dissociation, whereas phosphate dissociation provides the thermodynamic driving force underlying force production.

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Selective degradation of mRNAs by the HSV host shutoff RNase is regulated by the UL47 tegument protein.

Publication Date: 2013 Apr 30 PMID: 23589852
Authors: Shu, M. - Taddeo, B. - Zhang, W. - Roizman, B.
Journal: Proc Natl Acad Sci U S A

Herpes simplex virus 1 (HSV-1) encodes an endoribonuclease that is responsible for the shutoff of host protein synthesis [virion host shutoff (VHS)-RNase]. The VHS-RNase released into cells during infection targets differentially four classes of mRNAs. Thus, (a) VHS-RNase degrades stable cellular mRNAs and alpha (immediate early) viral mRNAs; (b) it stabilizes host stress response mRNAs after deadenylation and subsequent cleavage near the adenylate-uridylate (AU)-rich elements; (c) it does not effectively degrade viral beta or gamma mRNAs; and (d) it selectively spares from degradation a small number of cellular mRNAs. Current evidence suggests that several viral and at least one host protein (tristetraprolin) regulate its activity. Thus, virion protein (VP) 16 and VP22 neutralize the RNase activity at late times after infection. By binding to AU-rich elements via its interaction with tristetraprolin, the RNase deadenylates and cleaves the mRNAs in proximity to the AU-rich elements. In this report we show that another virion protein, UL47, brought into the cell during infection, attenuates the VHS-RNase activity with respect to stable host and viral alpha mRNAs and effectively blocks the degradation of beta and gamma mRNAs, but it has no effect on the processing of AU-rich mRNAs. The properties of UL47 suggest that it, along with the alpha protein infected cell protein 27, attenuates degradation of mRNAs by the VHS-RNase through interaction with the enzyme in polyribosomes. Mutants lacking both VHS-RNase and UL47 overexpress alpha genes and delay the expression of beta and gamma genes, suggesting that overexpression of alpha genes inhibits the downstream expression of early and late genes.

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Acetyl-CoA induces transcription of the key G1 cyclin CLN3 to promote entry into the cell division cycle in Saccharomyces cerevisiae.

Publication Date: 2013 Apr 30 PMID: 23589851
Authors: Shi, L. - Tu, B. P.
Journal: Proc Natl Acad Sci U S A

In budding yeast cells, nutrient repletion induces rapid exit from quiescence and entry into a round of growth and division. The G1 cyclin CLN3 is one of the earliest genes activated in response to nutrient repletion. Subsequent to its activation, hundreds of cell-cycle genes can then be expressed, including the cyclins CLN1/2 and CLB5/6. Although much is known regarding how CLN3 functions to activate downstream targets, the mechanism through which nutrients activate CLN3 transcription in the first place remains poorly understood. Here we show that a central metabolite of glucose catabolism, acetyl-CoA, induces CLN3 transcription by promoting the acetylation of histones present in its regulatory region. Increased rates of acetyl-CoA synthesis enable the Gcn5p-containing Spt-Ada-Gcn5-acetyltransferase transcriptional coactivator complex to catalyze histone acetylation at the CLN3 locus alongside ribosomal and other growth genes to promote entry into the cell division cycle.

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LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus.

Publication Date: 2013 Apr 30 PMID: 23589850
Authors: Finkelshtein, D. - Werman, A. - Novick, D. - Barak, S. - Rubinstein, M.
Journal: Proc Natl Acad Sci U S A

Vesicular stomatitis virus (VSV) exhibits a remarkably robust and pantropic infectivity, mediated by its coat protein, VSV-G. Using this property, recombinant forms of VSV and VSV-G-pseudotyped viral vectors are being developed for gene therapy, vaccination, and viral oncolysis and are extensively used for gene transduction in vivo and in vitro. The broad tropism of VSV suggests that it enters cells through a highly ubiquitous receptor, whose identity has so far remained elusive. Here we show that the LDL receptor (LDLR) serves as the major entry port of VSV and of VSV-G-pseudotyped lentiviral vectors in human and mouse cells, whereas other LDLR family members serve as alternative receptors. The widespread expression of LDLR family members accounts for the pantropism of VSV and for the broad applicability of VSV-G-pseudotyped viral vectors for gene transduction.

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Prognostic microRNA/mRNA signature from the integrated analysis of patients with invasive breast cancer.

Publication Date: 2013 Apr 30 PMID: 23589849
Authors: Volinia, S. - Croce, C. M.
Journal: Proc Natl Acad Sci U S A

The optimal management of breast cancer (BC) presents challenges due to the heterogeneous molecular classification of the disease. We performed survival analysis on a cohort of 466 patients with primary invasive ductal carcinoma (IDC), the most frequent type of BC, by integrating mRNA, microRNA (miRNA), and DNA methylation next-generation sequencing data from The Cancer Genome Atlas (TCGA). Expression data from eight other BC cohorts were used for validation. The prognostic value of the resulting miRNA/mRNA signature was compared with that of other prognostic BC signatures. Thirty mRNAs and seven miRNAs were associated with overall survival across different clinical and molecular subclasses of a 466-patient IDC cohort from TCGA. The prognostic RNAs included PIK3CA, one of the two most frequently mutated genes in IDC, and miRNAs such as hsa-miR-328, hsa-miR-484, and hsa-miR-874. The area under the curve of the receiver-operator characteristic for the IDC risk predictor in the TCGA cohort was 0.74 at 60 mo of overall survival (P < 0.001). Most relevant for clinical application, the integrated signature had the highest prognostic value in early stage I and II tumors (receiver-operator characteristic area under the curve = 0.77, P value < 0.001). The genes in the RNA risk predictor had an independent prognostic value compared with the clinical covariates, as shown by multivariate analysis. The integrated RNA signature was successfully validated on eight BC cohorts, comprising a total of 2,399 patients, and it had superior performance for risk stratification with respect to other RNA predictors, including the mRNAs used in MammaPrint and Oncotype DX assays.

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Structural effect of size on interracial friendship.

Publication Date: 2013 Apr 30 PMID: 23589848
Authors: Cheng, S. - Xie, Y.
Journal: Proc Natl Acad Sci U S A

Social contexts exert structural effects on individuals' social relationships, including interracial friendships. In this study, we posit that, net of group composition, total context size has a distinct effect on interracial friendship. Under the assumptions of (i) maximization of preference in choosing a friend, (ii) multidimensionality of preference, and (iii) preference for same-race friends, we conducted analyses using microsimulation that yielded three main findings. First, increased context size decreases the likelihood of forming an interracial friendship. Second, the size effect increases with the number of preference dimensions. Third, the size effect is diluted by noise, i.e., the random component affecting friendship formation. Analysis of actual friendship data among 4,745 American high school students yielded results consistent with the main conclusion that increased context size promotes racial segregation and discourages interracial friendship.

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In vitro activity of the nisin dehydratase NisB.

Publication Date: 2013 Apr 30 PMID: 23589847
Authors: Garg, N. - Salazar-Ocampo, L. M. - van der Donk, W. A.
Journal: Proc Natl Acad Sci U S A

The biosynthesis of several classes of ribosomally synthesized and posttranslationally modified peptides involves dehydration of serine and threonine residues. For class I lantibiotics, thiopeptides, and goadsporin, this dehydration is catalyzed by lanthionine biosynthetic enzyme B (LanB) or LanB-like proteins. Although LanB proteins have been studied since 1992, in vitro reconstitution of their dehydration activity has been elusive. We show here the in vitro activity of the dehydratase involved in the biosynthesis of the food preservative nisin (NisB). In vitro, NisB dehydrated its substrate peptide NisA eight times in the presence of glutamate, ATP, Mg(2+), and the ribosomal/membrane fraction of bacterial cell extract. Mutation of 23 highly conserved residues of NisB identified a number of amino acids that are essential for dehydration activity. In addition, these mutagenesis studies identified three mutants, R786A, R826A, and H961A, that result in multiple glutamylations of the NisA substrate. Glutamylation was observed during both Escherichia coli coexpression of NisA with these mutants and in vitro assays. Treatment of the glutamylated substrate with WT NisB results in dehydrated NisA, suggesting that the glutamylated peptide is an intermediate in dehydration. Collectively, these studies suggest that dehydration involves glutamylation of the side chains of Ser and Thr followed by elimination. The latter step has precedent in the virginiamycin resistance protein virginiamycin B lyase. These studies will facilitate investigation of other LanB proteins involved in the biosynthesis of lantibiotics, thiopeptides, and goadsporin.

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Increased photosystem II stability promotes H2 production in sulfur-deprived Chlamydomonas reinhardtii.

Publication Date: 2013 Apr 30 PMID: 23589846
Authors: Volgusheva, A. - Styring, S. - Mamedov, F.
Journal: Proc Natl Acad Sci U S A

Photobiological H2 production is an attractive option for renewable solar fuels. Sulfur-deprived cells of Chlamydomonas reinhardtii have been shown to produce hydrogen with the highest efficiency among photobiological systems. We have investigated the photosynthetic reactions during sulfur deprivation and H2 production in the wild-type and state transition mutant 6 (Stm6) mutant of Chlamydomonas reinhardtii. The incubation period (130 h) was dissected into different phases, and changes in the amount and functional status of photosystem II (PSII) were investigated in vivo by electron paramagnetic resonance spectroscopy and variable fluorescence measurements. In the wild type it was found that the amount of PSII is decreased to 25% of the original level; the electron transport from PSII was completely blocked during the anaerobic phase preceding H2 formation. This block was released during the H2 production phase, indicating that the hydrogenase withdraws electrons from the plastoquinone pool. This partly removes the block in PSII electron transport, thereby permitting electron flow from water oxidation to hydrogenase. In the Stm6 mutant, which has higher respiration and H2 evolution than the wild type, PSII was analogously but much less affected. The addition of the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea revealed that approximately 80% of the H2 production was inhibited in both strains. We conclude that (i) at least in the earlier stages, most of the electrons delivered to the hydrogenase originate from water oxidation by PSII, (ii) a faster onset of anaerobiosis preserves PSII from irreversible photoinhibition, and (iii) mutants with enhanced respiratory activity should be considered for better photobiological H2 production.

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Targeted mutagenesis of mitochondrial carbonic anhydrases VA and VB implicates both enzymes in ammonia detoxification and glucose metabolism.

Publication Date: 2013 Apr 30 PMID: 23589845
Authors: Shah, G. N. - Rubbelke, T. S. - Hendin, J. - Nguyen, H. - Waheed, A. - Shoemaker, J. D. - Sly, W. S.
Journal: Proc Natl Acad Sci U S A

Prior studies with carbonic anhydrase (CA) inhibitors implicated mitochondrial CA in ureagenesis and gluconeogenesis. Subsequent studies identified two mitochondrial CAs. To distinguish the contribution of each enzyme, we studied the effects of targeted disruption of the murine CA genes, called Car5A and Car5B. The Car5A mutation had several deleterious consequences. Car5A null mice were smaller than wild-type littermates and bred poorly. However, on sodium-potassium citrate-supplemented water, they produced offspring in expected numbers. Their blood ammonia concentrations were markedly elevated, but their fasting blood sugars were normal. By contrast, Car5B null mice showed normal growth and normal blood ammonia levels. They too had normal fasting blood sugars. Car5A/B double-knockout (DKO) mice showed additional abnormalities. Impaired growth was more severe than for Car5A null mice. Hyperammonemia was even greater as well. Although fertile, DKO animals were produced in less-than-predicted numbers even when supplemented with sodium-potassium citrate in their drinking water. Survival after weaning was also reduced, especially for males. In addition, fasting blood glucose levels for DKO mice were significantly lower than for controls (153 +/- 33 vs. 230 +/- 24 mg/dL). The enhanced hyperammonemia and lower fasting blood sugar, which are both seen in the DKO mice, indicate that both Car5A and Car5B contribute to both ammonia detoxification (ureagenesis) and regulation of fasting blood sugar (gluconeogenesis). Car5A, which is expressed mainly in liver, clearly has the predominant role in ammonia detoxification. The contribution of Car5B to ureagenesis and gluconeogenesis was evident only on a Car5A null background.

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Widespread distribution and unexpected variation among science faculty with education specialties (SFES) across the United States.

Publication Date: 2013 Apr 30 PMID: 23589844
Authors: Bush, S. D. - Pelaez, N. J. - Rudd, J. A. 2nd - Stevens, M. T. - Tanner, K. D. - Williams, K. S.
Journal: Proc Natl Acad Sci U S A

College and university science departments are increasingly taking an active role in improving science education. Perhaps as a result, a new type of specialized science faculty position within science departments is emerging-referred to here as science faculty with education specialties (SFES)-where individual scientists focus their professional efforts on strengthening undergraduate science education, improving kindergarten-through-12th grade science education, and conducting discipline-based education research. Numerous assertions, assumptions, and questions about SFES exist, yet no national studies have been published. Here, we present findings from a large-scale study of US SFES, who are widespread and increasing in numbers. Contrary to many assumptions, SFES were indeed found across the nation, across science disciplines, and, most notably, across primarily undergraduate, master of science-granting, and PhD-granting institutions. Data also reveal unexpected variations among SFES by institution type. Among respondents, SFES at master of science-granting institutions were almost twice as likely to have formal training in science education compared with other SFES. In addition, SFES at PhD-granting institutions were much more likely to have obtained science education funding. Surprisingly, formal training in science education provided no advantage in obtaining science education funding. Our findings show that the SFES phenomenon is likely more complex and diverse than anticipated, with differences being more evident across institution types than across science disciplines. These findings raise questions about the origins of differences among SFES and are useful to science departments interested in hiring SFES, scientific trainees preparing for SFES careers, and agencies awarding science education funding.

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Structure of the 26S proteasome with ATP-gammaS bound provides insights into the mechanism of nucleotide-dependent substrate translocation.

Publication Date: 2013 Apr 30 PMID: 23589842
Authors: Sledz, P. - Unverdorben, P. - Beck, F. - Pfeifer, G. - Schweitzer, A. - Forster, F. - Baumeister, W.
Journal: Proc Natl Acad Sci U S A

The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-gammaS induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6.

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T box RNA decodes both the information content and geometry of tRNA to affect gene expression.

Publication Date: 2013 Apr 30 PMID: 23589841
Authors: Grigg, J. C. - Chen, Y. - Grundy, F. J. - Henkin, T. M. - Pollack, L. - Ke, A.
Journal: Proc Natl Acad Sci U S A

The T box leader sequence is an RNA element that controls gene expression by binding directly to a specific tRNA and sensing its aminoacylation state. This interaction controls expression of amino acid-related genes in a negative feedback loop. The T box RNA structure is highly conserved, but its tRNA binding mechanism is only partially understood. Known sequence elements are the specifier sequence, which recognizes the tRNA anticodon, and the antiterminator bulge, which base pairs with the tRNA acceptor end. Here, we reveal the crucial function of the highly conserved stem I distal region in tRNA recognition and report its 2.65-A crystal structure. The apex of this region contains an intricately woven loop-loop interaction between two conserved motifs, the Adenine-guanine (AG) bulge and the distal loop. This loop-loop structure presents a base triple on its surface that is optimally positioned for base-stacking interactions. Mutagenesis, cross-linking, and small-angle X-ray scattering data demonstrate that the apical base triple serves as a binding platform to dock the tRNA D- and T-loops. Strikingly, the binding platform strongly resembles the D- and T-loop binding elements from RNase P and the ribosome exit site, suggesting that this loop-loop structure may represent a widespread tRNA recognition platform. We propose a two-checkpoint molecular ruler model for tRNA decoding in which the information content of tRNA is first examined through specifier sequence-anticodon interaction, and the length of the tRNA anticodon arm is then measured by the distal loop-loop platform. When both conditions are met, tRNA is secured, and its aminoacylation state is sensed.

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Enzymatic transformation of nonfood biomass to starch.

Publication Date: 2013 Apr 30 PMID: 23589840
Authors: You, C. - Chen, H. - Myung, S. - Sathitsuksanoh, N. - Ma, H. - Zhang, X. Z. - Li, J. - Zhang, Y. H.
Journal: Proc Natl Acad Sci U S A

The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world's future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture's environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma.

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Myc-induced AMPK-phospho p53 pathway activates Bak to sensitize mitochondrial apoptosis.

Publication Date: 2013 May 14 PMID: 23589839
Authors: Nieminen, A. I. - Eskelinen, V. M. - Haikala, H. M. - Tervonen, T. A. - Yan, Y. - Partanen, J. I. - Klefstrom, J.
Journal: Proc Natl Acad Sci U S A

Oncogenic transcription factor Myc deregulates the cell cycle and simultaneously reprograms cellular metabolism to meet the biosynthetic and bioenergetic needs of proliferation. Myc also sensitizes cells to mitochondria-dependent apoptosis. Although metabolic reprogramming has been circumstantially connected to vulnerability to apoptosis, the connecting molecular pathways have remained poorly defined. Here, we show that Myc-induced altered glutamine metabolism involves ATP depletion and activation of the energy sensor AMP-activated protein kinase (AMPK), which induces stabilizing phosphorylation of p53 at Ser15. Under influence of Myc, AMPK-stabilized tumor suppressor protein p53 accumulates in the mitochondria and interacts with the protein complex comprised of B-cell lymphoma 2 (Bcl-2) antagonist/killer (BAK) and Bcl2-like 1 (Bcl-xL). Mitochondrial p53 induces conformational activation of proapoptotic Bak without disrupting the Bak-Bcl-xL interaction. Further liberation of Bak specifically from the p53-activated Bak-Bcl-xL complex leads to spontaneous oligomerization of Bak and apoptosis. Thus, Myc-induced metabolic changes are coupled via AMPK and phospho-p53 to the mitochondrial apoptosis effector Bak, demonstrating a cell-intrinsic mechanism to counteract uncontrolled proliferation.

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Epigenetics: Core misconcept.

Publication Date: 2013 Apr 30 PMID: 23584020
Authors: Ptashne, M.
Journal: Proc Natl Acad Sci U S A

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GroEL and CCT are catalytic unfoldases mediating out-of-cage polypeptide refolding without ATP.

Publication Date: 2013 Apr 30 PMID: 23584019
Authors: Priya, S. - Sharma, S. K. - Sood, V. - Mattoo, R. U. - Finka, A. - Azem, A. - De Los Rios, P. - Goloubinoff, P.
Journal: Proc Natl Acad Sci U S A

Chaperonins are cage-like complexes in which nonnative polypeptides prone to aggregation are thought to reach their native state optimally. However, they also may use ATP to unfold stably bound misfolded polypeptides and mediate the out-of-cage native refolding of large proteins. Here, we show that even without ATP and GroES, both GroEL and the eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) can unfold stable misfolded polypeptide conformers and readily release them from the access ways to the cage. Reconciling earlier disparate experimental observations to ours, we present a comprehensive model whereby following unfolding on the upper cavity, in-cage confinement is not needed for the released intermediates to slowly reach their native state in solution. As over-sticky intermediates occasionally stall the catalytic unfoldase sites, GroES mobile loops and ATP are necessary to dissociate the inhibitory species and regenerate the unfolding activity. Thus, chaperonin rings are not obligate confining antiaggregation cages. They are polypeptide unfoldases that can iteratively convert stable off-pathway conformers into functional proteins.

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