PNAS

Targeting the microbiota-gut-brain axis to modulate behavior: Which bacterial strain will translate best to humans?

Publication Date: 2012 Jan 13 PMID: 22247294
Authors: McLean, P. G. - Bergonzelli, G. E. - Collins, S. M. - Bercik, P.
Journal: Proc Natl Acad Sci U S A

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Gut bacteria and brain function: The challenges of a growing field.

Publication Date: 2012 Jan 13 PMID: 22247293
Authors: Burnet, P. W.
Journal: Proc Natl Acad Sci U S A

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Alternative reading frame selection mediated by a tRNA-like domain of an internal ribosome entry site.

Publication Date: 2012 Jan 13 PMID: 22247292
Authors: Ren, Q. - Wang, Q. S. - Firth, A. E. - Chan, M. M. - Gouw, J. W. - Guarna, M. M. - Foster, L. J. - Atkins, J. F. - Jan, E.
Journal: Proc Natl Acad Sci U S A

The dicistrovirus intergenic region internal ribosome entry site (IRES) utilizes a unique mechanism, involving P-site tRNA mimicry, to directly assemble 80S ribosomes and initiate translation at a specific non-AUG codon in the ribosomal A site. A subgroup of dicistrovirus genomes contains an additional stem-loop 5'-adjacent to the IRES and a short open reading frame (ORFx) that overlaps the viral structural polyprotein ORF (ORF2) in the +1 reading frame. Using mass spectrometry and extensive mutagenesis, we show that, besides directing ORF2 translation, the Israeli acute paralysis dicistrovirus IRES also directs ORFx translation. The latter is mediated by a UG base pair adjacent to the P-site tRNA-mimicking domain. An ORFx peptide was detected in virus-infected honey bees by multiple reaction monitoring mass spectrometry. Finally, the 5' stem-loop increases IRES activity and may couple translation of the two major ORFs of the virus. This study reveals a novel viral strategy in which a tRNA-like IRES directs precise, initiator Met-tRNA-independent translation of two overlapping ORFs.

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Correction for Kirshenbaum et al., Mania-like behavior induced by genetic dysfunction of the neuron-specific Na+,K+-ATPase alpha3 sodium pump.

Publication Date: 2012 Jan 13 PMID: 22247291
Authors:
Journal: Proc Natl Acad Sci U S A

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Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin.

Publication Date: 2012 Jan 12 PMID: 22247290
Authors: Westfall, P. J. - Pitera, D. J. - Lenihan, J. R. - Eng, D. - Woolard, F. X. - Regentin, R. - Horning, T. - Tsuruta, H. - Melis, D. J. - Owens, A. - Fickes, S. - Diola, D. - Benjamin, K. R. - Keasling, J. D. - Leavell, M. D. - McPhee, D. J. - Renninger, N. S. - Newman, J. D. - Paddon, C. J.
Journal: Proc Natl Acad Sci U S A

Malaria, caused by Plasmodium sp, results in almost one million deaths and over 200 million new infections annually. The World Health Organization has recommended that artemisinin-based combination therapies be used for treatment of malaria. Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua. However, the supply and price of artemisinin fluctuate greatly, and an alternative production method would be valuable to increase availability. We describe progress toward the goal of developing a supply of semisynthetic artemisinin based on production of the artemisinin precursor amorpha-4,11-diene by fermentation from engineered Saccharomyces cerevisiae, and its chemical conversion to dihydroartemisinic acid, which can be subsequently converted to artemisinin. Previous efforts to produce artemisinin precursors used S. cerevisiae S288C overexpressing selected genes of the mevalonate pathway [Ro et al. (2006) Nature 440:940-943]. We have now overexpressed every enzyme of the mevalonate pathway to ERG20 in S. cerevisiae CEN.PK2, and compared production to CEN.PK2 engineered identically to the previously engineered S288C strain. Overexpressing every enzyme of the mevalonate pathway doubled artemisinic acid production, however, amorpha-4,11-diene production was 10-fold higher than artemisinic acid. We therefore focused on amorpha-4,11-diene production. Development of fermentation processes for the reengineered CEN.PK2 amorpha-4,11-diene strain led to production of > 40 g/L product. A chemical process was developed to convert amorpha-4,11-diene to dihydroartemisinic acid, which could subsequently be converted to artemisinin. The strains and procedures described represent a complete process for production of semisynthetic artemisinin.

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Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand Tfh cells and promote germinal center induction.

Publication Date: 2012 Jan 12 PMID: 22247289
Authors: Moon, J. J. - Suh, H. - Li, A. V. - Ockenhouse, C. F. - Yadava, A. - Irvine, D. J.
Journal: Proc Natl Acad Sci U S A

For subunit vaccines, adjuvants play a key role in shaping immunological memory. Nanoparticle (NP) delivery systems for antigens and/or molecular danger signals are promising adjuvants capable of promoting both cellular and humoral immune responses, but in most cases the mechanisms of action of these materials are poorly understood. Here, we studied the immune response elicited by NPs composed of multilamellar "stapled" lipid vesicles carrying a recombinant Plasmodium vivax circumsporozoite antigen, VMP001, both entrapped in the aqueous core and anchored to the lipid bilayer surfaces. Immunization with these particles and monophosphoryl lipid A (MPLA), a US Food and Drug Administration-approved immunostimulatory agonist for Toll-like receptor-4, promoted high-titer, high-avidity antibody responses against VMP001, lasting more than 1 y in mice at 10-fold lower doses than conventional adjuvants. Compared to soluble VMP001 mixed with MPLA, VMP001-NPs promoted broader humoral responses, targeting multiple epitopes of the protein and a more balanced Th1/Th2 cytokine profile from antigen-specific T cells. To begin to understand the underlying mechanisms, we examined components of the B-cell response and found that NPs promoted robust germinal center (GC) formation at low doses of antigen where no GC induction occurred with soluble protein immunization, and that GCs nucleated near depots of NPs accumulating in the draining lymph nodes over time. In parallel, NP vaccination enhanced the expansion of antigen-specific follicular helper T cells (T(fh)), compared to vaccinations with soluble VMP001 or alum. Thus, NP vaccines may be a promising strategy to enhance the durability, breadth, and potency of humoral immunity by enhancing key elements of the B-cell response.

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Isoprenoid biosynthesis is required for miRNA function and affects membrane association of ARGONAUTE 1 in Arabidopsis.

Publication Date: 2012 Jan 12 PMID: 22247288
Authors: Brodersen, P. - Sakvarelidze-Achard, L. - Schaller, H. - Khafif, M. - Schott, G. - Bendahmane, A. - Voinnet, O.
Journal: Proc Natl Acad Sci U S A

Plant and metazoan microRNAs (miRNAs) guide ARGONAUTE (AGO) protein complexes to regulate expression of complementary RNAs via base pairing. In the plant Arabidopsis thaliana, the main miRNA effector is AGO1, but few other factors required for miRNA activity are known. Here, we isolate the genes defined by the previously described miRNA action deficient (mad) mutants, mad3 and mad4. Both genes encode enzymes involved in isoprenoid biosynthesis. MAD3 encodes 3-hydroxy-3-methylglutaryl CoA reductase (HMG1), which functions in the initial C(5) building block biogenesis that precedes isoprenoid metabolism. HMG1 is a key regulatory enzyme that controls the amounts of isoprenoid end products. MAD4 encodes sterol C-8 isomerase (HYDRA1) that acts downstream in dedicated sterol biosynthesis. Using yeast complementation assays and in planta application of lovastatin, a competitive inhibitor of HMG1, we show that defects in HMG1 catalytic activity are sufficient to inhibit miRNA activity. Many isoprenoid derivatives are indispensable structural and signaling components, and especially sterols are essential membrane constituents. Accordingly, we provide evidence that AGO1 is a peripheral membrane protein. Moreover, specific hypomorphic mutant alleles of AGO1 display compromised membrane association and AGO1-membrane interaction is reduced upon knockdown of HMG1/MAD3. These results suggest a possible basis for the requirement of isoprenoid biosynthesis for the activity of plant miRNAs, and unravel mechanistic features shared with their metazoan counterparts.

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Rosalyn Sussman Yalow (1921-2011).

Publication Date: 2012 Jan 11 PMID: 22238428
Authors: Kahn, C. R. - Roth, J.
Journal: Proc Natl Acad Sci U S A

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Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development.

Publication Date: 2012 Jan 11 PMID: 22238427
Authors: Smaczniak, C. - Immink, R. G. - Muino, J. M. - Blanvillain, R. - Busscher, M. - Busscher-Lange, J. - Dinh, Q. D. - Liu, S. - Westphal, A. H. - Boeren, S. - Parcy, F. - Xu, L. - Carles, C. C. - Angenent, G. C. - Kaufmann, K.
Journal: Proc Natl Acad Sci U S A

Floral organs are specified by the combinatorial action of MADS-domain transcription factors, yet the mechanisms by which MADS-domain proteins activate or repress the expression of their target genes and the nature of their cofactors are still largely unknown. Here, we show using affinity purification and mass spectrometry that five major floral homeotic MADS-domain proteins (AP1, AP3, PI, AG, and SEP3) interact in floral tissues as proposed in the "floral quartet" model. In vitro studies confirmed a flexible composition of MADS-domain protein complexes depending on relative protein concentrations and DNA sequence. In situ bimolecular fluorescent complementation assays demonstrate that MADS-domain proteins interact during meristematic stages of flower development. By applying a targeted proteomics approach we were able to establish a MADS-domain protein interactome that strongly supports a mechanistic link between MADS-domain proteins and chromatin remodeling factors. Furthermore, members of other transcription factor families were identified as interaction partners of floral MADS-domain proteins suggesting various specific combinatorial modes of action.

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Synthetic in vivo validation of gene network circuitry.

Publication Date: 2012 Jan 11 PMID: 22238426
Authors: Damle, S. S. - Davidson, E. H.
Journal: Proc Natl Acad Sci U S A

Embryonic development is controlled by networks of interacting regulatory genes. The individual linkages of gene regulatory networks (GRNs) are customarily validated by functional cis-regulatory analysis, but an additional approach to validation is to rewire GRN circuitry to test experimentally predictions derived from network structure. Here we use this synthetic method to challenge specific predictions of the sea urchin embryo endomesoderm GRN. Expression vectors generated by in vitro recombination of exogenous sequences into BACs were used to cause elements of a nonskeletogenic mesoderm GRN to be deployed in skeletogenic cells and to detect their effects. The result of reengineering the regulatory circuitry in this way was to divert the developmental program of these cells from skeletogenesis to pigment cell formation, confirming a direct prediction of the GRN. In addition, the experiment revealed previously undetected cryptic repression functions.

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Glycogen synthase kinase 3beta transfers cytoprotective signaling through connexin 43 onto mitochondrial ATP-sensitive K+ channels.

Publication Date: 2012 Jan 11 PMID: 22238425
Authors: Rottlaender, D. - Boengler, K. - Wolny, M. - Schwaiger, A. - Motloch, L. J. - Ovize, M. - Schulz, R. - Heusch, G. - Hoppe, U. C.
Journal: Proc Natl Acad Sci U S A

Despite compelling evidence supporting key roles for glycogen synthase kinase 3beta (GSK3beta), mitochondrial adenosine triphosphate-sensitive K(+) (mitoK(ATP)) channels, and mitochondrial connexin 43 (Cx43) in cytoprotection, it is not clear how these signaling modules are linked mechanistically. By patch-clamping the inner membrane of murine cardiac mitochondria, we found that inhibition of GSK3beta activated mitoK(ATP). PKC activation and protein phosphatase 2a inhibition increased the open probability of mitoK(ATP) channels through GSK3beta, and this GSK3beta signal was mediated via mitochondrial Cx43. Moreover, (i) PKC-induced phosphorylation of mitochondrial Cx43 was reduced in GSK3beta-S9A mice; (ii) Cx43 and GSK3beta proteins associated in mitochondria; and (iii) SB216763-mediated reduction of infarct size was abolished in Cx43 KO mice in vivo, consistent with the notion that GSK3beta inhibition results in mitoK(ATP) opening via mitochondrial Cx43. We therefore directly targeted mitochondrial Cx43 by the Cx43 C-terminal binding peptide RRNYRRNY for cardioprotection, circumventing further upstream pathways. RRNYRRNY activated mitoK(ATP) channels via Cx43. We directly recorded mitochondrial Cx43 channels that were activated by RRNYRRNY and blocked by the Cx43 mimetic peptide (43)GAP27. RRNYRRNY rendered isolated cardiomyocytes in vitro and the heart in vivo resistant to ischemia/reperfusion injury, indicating that mitochondrial Cx43- and/or mitoK(ATP)-mediated reduction of infarct size was not undermined by RRNYRRNY-related opening of sarcolemmal Cx43 channels. Our results demonstrate that GSK3beta transfers cytoprotective signaling through mitochondrial Cx43 onto mitoK(ATP) channels and that Cx43 functions as a channel in mitochondria, being an attractive target for drug treatment against cardiomyocyte injury.

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Classification of protein functional surfaces using structural characteristics.

Publication Date: 2012 Jan 11 PMID: 22238424
Authors: Tseng, Y. Y. - Li, W. H.
Journal: Proc Natl Acad Sci U S A

Protein structure and function are closely related, especially in functional surfaces, which are local spatial regions that perform the biological functions. Also, protein structures tend to evolve more slowly than amino acid sequences. We have therefore developed a method to classify proteins using the structures of functional surfaces; we call it protein surface classification (PSC). PSC may reflect functional relationships among proteins and may detect evolutionary relationships among highly divergent sequences. We focused on the surfaces of ligand-bound regions because they represent well-defined structures. Specifically, we used structural attributes to measure similarities between binding surfaces and constructed a PSC library of approximately 2,000 binding surface types from the bound forms. Using flavin mononucleotide-binding proteins and glycosidases as examples, we show how the evolutionary position of an uncharacterized protein can be defined and its function inferred from the characterized members of the same surface subtype. We found that proteins with the same enzyme nomenclature may be divided into subtypes and that two proteins in the same CATH (Class, Architecture, Topology, Homologous superfamily) fold may belong to two different surface types. In conclusion, our approach complements the sequence-based and fold-domain classifications and has the advantage of associating the shape of a protein with its biological function. As an expandable library, PSC provides a resource of spatial patterns for studying the evolution of protein structure and function.

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Sampling protein motion and solvent effect during ligand binding.

Publication Date: 2012 Jan 11 PMID: 22238423
Authors: Limongelli, V. - Marinelli, L. - Cosconati, S. - La Motta, C. - Sartini, S. - Mugnaini, L. - Da Settimo, F. - Novellino, E. - Parrinello, M.
Journal: Proc Natl Acad Sci U S A

An exhaustive description of the molecular recognition mechanism between a ligand and its biological target is of great value because it provides the opportunity for an exogenous control of the related process. Very often this aim can be pursued using high resolution structures of the complex in combination with inexpensive computational protocols such as docking algorithms. Unfortunately, in many other cases a number of factors, like protein flexibility or solvent effects, increase the degree of complexity of ligand/protein interaction and these standard techniques are no longer sufficient to describe the binding event. We have experienced and tested these limits in the present study in which we have developed and revealed the mechanism of binding of a new series of potent inhibitors of Adenosine Deaminase. We have first performed a large number of docking calculations, which unfortunately failed to yield reliable results due to the dynamical character of the enzyme and the complex role of the solvent. Thus, we have stepped up the computational strategy using a protocol based on metadynamics. Our approach has allowed dealing with protein motion and solvation during ligand binding and finally identifying the lowest energy binding modes of the most potent compound of the series, 4-decyl-pyrazolo[1,5-a]pyrimidin-7-one.

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Mitochondrial hexokinase II (HKII) and phosphoprotein enriched in astrocytes (PEA15) form a molecular switch governing cellular fate depending on the metabolic state.

Publication Date: 2012 Jan 10 PMID: 22233811
Authors: Mergenthaler, P. - Kahl, A. - Kamitz, A. - van Laak, V. - Stohlmann, K. - Thomsen, S. - Klawitter, H. - Przesdzing, I. - Neeb, L. - Freyer, D. - Priller, J. - Collins, T. J. - Megow, D. - Dirnagl, U. - Andrews, D. W. - Meisel, A.
Journal: Proc Natl Acad Sci U S A

The metabolic state of a cell is a key determinant in the decision to live and proliferate or to die. Consequently, balanced energy metabolism and the regulation of apoptosis are critical for the development and maintenance of differentiated organisms. Hypoxia occurs physiologically during development or exercise and pathologically in vascular disease, tumorigenesis, and inflammation, interfering with homeostatic metabolism. Here, we show that the hypoxia-inducible factor (HIF)-1-regulated glycolytic enzyme hexokinase II (HKII) acts as a molecular switch that determines cellular fate by regulating both cytoprotection and induction of apoptosis based on the metabolic state. We provide evidence for a direct molecular interactor of HKII and show that, together with phosphoprotein enriched in astrocytes (PEA15), HKII inhibits apoptosis after hypoxia. In contrast, HKII accelerates apoptosis in the absence of PEA15 and under glucose deprivation. HKII both protects cells from death during hypoxia and functions as a sensor of glucose availability during normoxia, inducing apoptosis in response to glucose depletion. Thus, HKII-mediated apoptosis may represent an evolutionarily conserved altruistic mechanism to eliminate cells during metabolic stress to the advantage of a multicellular organism.

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Deciphering the genetic architecture of variation in the immune response to Mycobacterium tuberculosis infection.

Publication Date: 2012 Jan 10 PMID: 22233810
Authors: Barreiro, L. B. - Tailleux, L. - Pai, A. A. - Gicquel, B. - Marioni, J. C. - Gilad, Y.
Journal: Proc Natl Acad Sci U S A

Tuberculosis (TB) is a major public health problem. One-third of the world's population is estimated to be infected with Mycobacterium tuberculosis (MTB), the etiological agent causing TB, and active disease kills nearly 2 million individuals worldwide every year. Several lines of evidence indicate that interindividual variation in susceptibility to TB has a heritable component, yet we still know little about the underlying genetic architecture. To address this, we performed a genome-wide mapping study of loci that are associated with functional variation in immune response to MTB. Specifically, we characterized transcript and protein expression levels and mapped expression quantitative trait loci (eQTL) in primary dendritic cells (DCs) from 65 individuals, before and after infection with MTB. We found 198 response eQTL, namely loci that were associated with variation in gene expression levels in either untreated or MTB-infected DCs, but not both. These response eQTL are associated with natural regulatory variation that likely affects (directly or indirectly) host interaction with MTB. Indeed, when we integrated our data with results from a genome-wide association study (GWAS) for pulmonary TB, we found that the response eQTL were more likely to be genetically associated with the disease. We thus identified a number of candidate loci, including the MAPK phosphatase DUSP14 in particular, that are promising susceptibility genes to pulmonary TB.

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Convergent structural alterations define SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeler as a central tumor suppressive complex in pancreatic cancer.

Publication Date: 2012 Jan 10 PMID: 22233809
Authors: Shain, A. H. - Giacomini, C. P. - Matsukuma, K. - Karikari, C. A. - Bashyam, M. D. - Hidalgo, M. - Maitra, A. - Pollack, J. R.
Journal: Proc Natl Acad Sci U S A

Defining the molecular genetic alterations underlying pancreatic cancer may provide unique therapeutic insight for this deadly disease. Toward this goal, we report here an integrative DNA microarray and sequencing-based analysis of pancreatic cancer genomes. Notable among the alterations newly identified, genomic deletions, mutations, and rearrangements recurrently targeted genes encoding components of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex, including all three putative DNA binding subunits (ARID1A, ARID1B, and PBRM1) and both enzymatic subunits (SMARCA2 and SMARCA4). Whereas alterations of each individual SWI/SNF subunit occurred at modest-frequency, as mutational "hills" in the genomic landscape, together they affected at least one-third of all pancreatic cancers, defining SWI/SNF as a major mutational "mountain." Consistent with a tumor-suppressive role, re-expression of SMARCA4 in SMARCA4-deficient pancreatic cancer cell lines reduced cell growth and promoted senescence, whereas its overexpression in a SWI/SNF-intact line had no such effect. In addition, expression profiling analyses revealed that SWI/SNF likely antagonizes Polycomb repressive complex 2, implicating this as one possible mechanism of tumor suppression. Our findings reveal SWI/SNF to be a central tumor suppressive complex in pancreatic cancer.

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Dynamic autoinoculation and the microbial ecology of a deep water hydrocarbon irruption.

Publication Date: 2012 Jan 10 PMID: 22233808
Authors: Valentine, D. L. - Mezic, I. - Macesic, S. - Crnjaric-Zic, N. - Ivic, S. - Hogan, P. J. - Fonoberov, V. A. - Loire, S.
Journal: Proc Natl Acad Sci U S A

The irruption of gas and oil into the Gulf of Mexico during the Deepwater Horizon event fed a deep sea bacterial bloom that consumed hydrocarbons in the affected waters, formed a regional oxygen anomaly, and altered the microbiology of the region. In this work, we develop a coupled physical-metabolic model to assess the impact of mixing processes on these deep ocean bacterial communities and their capacity for hydrocarbon and oxygen use. We find that observed biodegradation patterns are well-described by exponential growth of bacteria from seed populations present at low abundance and that current oscillation and mixing processes played a critical role in distributing hydrocarbons and associated bacterial blooms within the northeast Gulf of Mexico. Mixing processes also accelerated hydrocarbon degradation through an autoinoculation effect, where water masses, in which the hydrocarbon irruption had caused blooms, later returned to the spill site with hydrocarbon-degrading bacteria persisting at elevated abundance. Interestingly, although the initial irruption of hydrocarbons fed successive blooms of different bacterial types, subsequent irruptions promoted consistency in the structure of the bacterial community. These results highlight an impact of mixing and circulation processes on biodegradation activity of bacteria during the Deepwater Horizon event and suggest an important role for mixing processes in the microbial ecology of deep ocean environments.

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Chemical data quantify Deepwater Horizon hydrocarbon flow rate and environmental distribution.

Publication Date: 2012 Jan 10 PMID: 22233807
Authors: Ryerson, T. B. - Camilli, R. - Kessler, J. D. - Kujawinski, E. B. - Reddy, C. M. - Valentine, D. L. - Atlas, E. - Blake, D. R. - de Gouw, J. - Meinardi, S. - Parrish, D. D. - Peischl, J. - Seewald, J. S. - Warneke, C.
Journal: Proc Natl Acad Sci U S A

Detailed airborne, surface, and subsurface chemical measurements, primarily obtained in May and June 2010, are used to quantify initial hydrocarbon compositions along different transport pathways (i.e., in deep subsurface plumes, in the initial surface slick, and in the atmosphere) during the Deepwater Horizon oil spill. Atmospheric measurements are consistent with a limited area of surfacing oil, with implications for leaked hydrocarbon mass transport and oil drop size distributions. The chemical data further suggest relatively little variation in leaking hydrocarbon composition over time. Although readily soluble hydrocarbons made up approximately 25% of the leaking mixture by mass, subsurface chemical data show these compounds made up approximately 69% of the deep plume mass; only approximately 31% of the deep plume mass was initially transported in the form of trapped oil droplets. Mass flows along individual transport pathways are also derived from atmospheric and subsurface chemical data. Subsurface hydrocarbon composition, dissolved oxygen, and dispersant data are used to assess release of hydrocarbons from the leaking well. We use the chemical measurements to estimate that (7.8 +/- 1.9) x 10(6) kg of hydrocarbons leaked on June 10, 2010, directly accounting for roughly three-quarters of the total leaked mass on that day. The average environmental release rate of (10.1 +/- 2.0) x 10(6) kg/d derived using atmospheric and subsurface chemical data agrees within uncertainties with the official average leak rate of (10.2 +/- 1.0) x 10(6) kg/d derived using physical and optical methods.

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Kruppel-like factor 5 (KLF5) is critical for conferring uterine receptivity to implantation.

Publication Date: 2012 Jan 10 PMID: 22233806
Authors: Sun, X. - Zhang, L. - Xie, H. - Wan, H. - Magella, B. - Whitsett, J. A. - Dey, S. K.
Journal: Proc Natl Acad Sci U S A

A blastocyst will implant only when the uterus becomes receptive. Following attachment, luminal epithelial cells undergo degeneration at the site of the blastocyst. Although many genes critical for uterine receptivity are primarily regulated by ovarian hormones, Kruppel-like factor 5 (KLF5), a zinc finger-containing transcription factor, is persistently expressed in epithelial cells independently of ovarian hormones. Loss of uterine Klf5 causes female infertility due to defective implantation. Cox2 is normally expressed in the luminal epithelium and stroma at the site of blastocyst attachment, but luminal epithelial COX2 expression is absent with loss of Klf5. This is associated with the retention of the epithelium around the implantation chamber with arrested embryonic growth. These results suggest that Klf5 is indispensable for normal implantation.

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Elucidating the mechanism of selective ion adsorption to the liquid water surface.

Publication Date: 2012 Jan 10 PMID: 22233805
Authors: Otten, D. E. - Shaffer, P. R. - Geissler, P. L. - Saykally, R. J.
Journal: Proc Natl Acad Sci U S A

Adsorption of aqueous thiocyanate ions from bulk solution to the liquid/vapor interface was measured as a function of temperature by resonant UV second harmonic generation spectroscopy. The resulting adsorption enthalpy and entropy changes of this prototypical chaotrope were both determined to be negative. This surprising result is supported by molecular simulations, which clarify the microscopic origins of observed thermodynamic changes. Calculations reveal energetic influences of adsorbed ions on their surroundings to be remarkably local. Negative adsorption enthalpies thus reflect a simple repartitioning of solvent density among surface, bulk, and coordination regions. A different, and much less spatially local, mechanism underlies the concomitant loss of entropy. Simulations indicate that ions at the interface can significantly bias surface height fluctuations even several molecular diameters away, imposing restrictions consistent with the scale of measured and computed adsorption entropies. Based on these results, we expect an ion's position in the Hofmeister lyotropic series to be determined by a combination of driving forces associated with the pinning of capillary waves and with a competition between ion hydration energy and the neat liquid's surface tension.

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Ubiquitination, localization, and stability of an anti-apoptotic BCL2-like protein, BCL2L10/BCLb, are regulated by Ubiquilin1.

Publication Date: 2012 Jan 10 PMID: 22233804
Authors: Beverly, L. J. - Lockwood, W. W. - Shah, P. P. - Erdjument-Bromage, H. - Varmus, H.
Journal: Proc Natl Acad Sci U S A

We have previously shown that all six members of the anti-apoptotic BCL2 gene family can cooperate with (myelocytomatosis oncogene) MYC in a mouse model of leukemia, but three of them are significantly less potent contributors to leukemogenicity than the other three. The protein encoded by one of these less potent genes, BCL2L10/BCLb, was recently shown to vary dramatically in many primary human cancers by immunohistochemistry, and the protein levels were inversely correlated with survival in patients with several cancer types. We examined BCLb mRNA in a panel of human cancer cell lines and did not observe the extensive variation in mRNA that would be required to explain the vast differences in protein levels. We found that the levels of BCLb protein diminish quickly after inhibition of protein synthesis with cycloheximide, so we searched for interacting proteins that might affect posttranslational stability of BCLb. Using a variety of approaches, including immunoaffinity and mass spectrometry, we identified a protein, Ubiquilin1 (Ubqln), that specifically interacts with BCLb, and not with other anti-apoptotic BCL2-like proteins. Ubqln stabilizes BCLb protein, while also promoting monoubiquitination on multiple lysine residues and relocation to the cytosol. Furthermore, primary lung adencarcinomas have more Ubqln mRNA than normal adjacent lung tissue, and higher Ubqln mRNA levels are associated with shorter survival of lung cancer patients, suggesting that potentiation of the anti-apoptotic potential of BCLb through regulation of its stability by Ubqln may be an important factor in tumor progression.

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Liquid crystal self-assembly of random-sequence DNA oligomers.

Publication Date: 2012 Jan 10 PMID: 22233803
Authors: Bellini, T. - Zanchetta, G. - Fraccia, T. P. - Cerbino, R. - Tsai, E. - Smith, G. P. - Moran, M. J. - Walba, D. M. - Clark, N. A.
Journal: Proc Natl Acad Sci U S A

In biological systems and nanoscale assemblies, the self-association of DNA is typically studied and applied in the context of the evolved or directed design of base sequences that give complementary pairing, duplex formation, and specific structural motifs. Here we consider the collective behavior of DNA solutions in the distinctly different regime where DNA base sequences are chosen at random or with varying degrees of randomness. We show that in solutions of completely random sequences, corresponding to a remarkably large number of different molecules, e.g., approximately 10(12) for random 20-mers, complementary still emerges and, for a narrow range of oligomer lengths, produces a subtle hierarchical sequence of structured self-assembly and organization into liquid crystal (LC) phases. This ordering follows from the kinetic arrest of oligomer association into long-lived partially paired double helices, followed by reversible association of these pairs into linear aggregates that in turn condense into LC domains.

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Lipidomic discovery of deoxysiderophores reveals a revised mycobactin biosynthesis pathway in Mycobacterium tuberculosis.

Publication Date: 2012 Jan 9 PMID: 22232695
Authors: Madigan, C. A. - Cheng, T. Y. - Layre, E. - Young, D. C. - McConnell, M. J. - Debono, C. A. - Murry, J. P. - Wei, J. R. - Barry, C. E. 3rd - Rodriguez, G. M. - Matsunaga, I. - Rubin, E. J. - Moody, D. B.
Journal: Proc Natl Acad Sci U S A

To measure molecular changes underlying pathogen adaptation, we generated a searchable dataset of more than 12,000 mass spectrometry events, corresponding to lipids and small molecules that constitute a lipidome for Mycobacterium tuberculosis. Iron is essential for M. tuberculosis survival, and the organism imports this metal using mycobactin and carboxymycobactin siderophores. Detection of an unexpected siderophore variant and deletions of genes for iron scavenging has led to a revised mycobactin biosynthesis model. An organism-wide search of the M. tuberculosis database for hypothetical compounds predicted by this model led to the discovery of two families of previously unknown lipids, designated monodeoxymycobactins and monodeoxycarboxymycobactins. These molecules suggest a revised biosynthetic model that alters the substrates and order of action of enzymes through the mycobactin biosynthetic pathway. We tested this model genetically by solving M. tuberculosis lipidomes after deletion of the iron-dependent regulator (ideR), mycobactin synthase B (mbtB), or mycobactin synthase G (mbtG). These studies show that deoxymycobactins are actively regulated during iron starvation, and also define essential roles of MbtG in converting deoxymycobactins to mycobactin and in promoting M. tuberculosis growth. Thus, lipidomics is an efficient discovery tool that informs genetic relationships, leading to a revised general model for the biosynthesis of these virulence-conferring siderophores.

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The take-it-or-leave-it option allows small penalties to overcome social dilemmas.

Publication Date: 2012 Jan 9 PMID: 22232694
Authors: Sasaki, T. - Brannstrom, A. - Dieckmann, U. - Sigmund, K.
Journal: Proc Natl Acad Sci U S A

Self-interest frequently causes individuals engaged in joint enterprises to choose actions that are counterproductive. Free-riders can invade a society of cooperators, causing a tragedy of the commons. Such social dilemmas can be overcome by positive or negative incentives. Even though an incentive-providing institution may protect a cooperative society from invasion by free-riders, it cannot always convert a society of free-riders to cooperation. In the latter case, both norms, cooperation and defection, are stable: To avoid a collapse to full defection, cooperators must be sufficiently numerous initially. A society of free-riders is then caught in a social trap, and the institution is unable to provide an escape, except at a high, possibly prohibitive cost. Here, we analyze the interplay of (a) incentives provided by institutions and (b) the effects of voluntary participation. We show that this combination fundamentally improves the efficiency of incentives. In particular, optional participation allows institutions punishing free-riders to overcome the social dilemma at a much lower cost, and to promote a globally stable regime of cooperation. This removes the social trap and implies that whenever a society of cooperators cannot be invaded by free-riders, it will necessarily become established in the long run, through social learning, irrespective of the initial number of cooperators. We also demonstrate that punishing provides a "lighter touch" than rewarding, guaranteeing full cooperation at considerably lower cost.

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Gut inflammation can boost horizontal gene transfer between pathogenic and commensal Enterobacteriaceae.

Publication Date: 2012 Jan 9 PMID: 22232693
Authors: Stecher, B. - Denzler, R. - Maier, L. - Bernet, F. - Sanders, M. J. - Pickard, D. J. - Barthel, M. - Westendorf, A. M. - Krogfelt, K. A. - Walker, A. W. - Ackermann, M. - Dobrindt, U. - Thomson, N. R. - Hardt, W. D.
Journal: Proc Natl Acad Sci U S A

The mammalian gut harbors a dense microbial community interacting in multiple ways, including horizontal gene transfer (HGT). Pangenome analyses established particularly high levels of genetic flux between Gram-negative Enterobacteriaceae. However, the mechanisms fostering intraenterobacterial HGT are incompletely understood. Using a mouse colitis model, we found that Salmonella-inflicted enteropathy elicits parallel blooms of the pathogen and of resident commensal Escherichia coli. These blooms boosted conjugative HGT of the colicin-plasmid p2 from Salmonella enterica serovar Typhimurium to E. coli. Transconjugation efficiencies of approximately 100% in vivo were attributable to high intrinsic p2-transfer rates. Plasmid-encoded fitness benefits contributed little. Under normal conditions, HGT was blocked by the commensal microbiota inhibiting contact-dependent conjugation between Enterobacteriaceae. Our data show that pathogen-driven inflammatory responses in the gut can generate transient enterobacterial blooms in which conjugative transfer occurs at unprecedented rates. These blooms may favor reassortment of plasmid-encoded genes between pathogens and commensals fostering the spread of fitness-, virulence-, and antibiotic-resistance determinants.

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Decoupling of deforestation and soy production in the southern Amazon during the late 2000s.

Publication Date: 2012 Jan 9 PMID: 22232692
Authors: Macedo, M. N. - Defries, R. S. - Morton, D. C. - Stickler, C. M. - Galford, G. L. - Shimabukuro, Y. E.
Journal: Proc Natl Acad Sci U S A

From 2006 to 2010, deforestation in the Amazon frontier state of Mato Grosso decreased to 30% of its historical average (1996-2005) whereas agricultural production reached an all-time high. This study combines satellite data with government deforestation and production statistics to assess land-use transitions and potential market and policy drivers associated with these trends. In the forested region of the state, increased soy production from 2001 to 2005 was entirely due to cropland expansion into previously cleared pasture areas (74%) or forests (26%). From 2006 to 2010, 78% of production increases were due to expansion (22% to yield increases), with 91% on previously cleared land. Cropland expansion fell from 10 to 2% of deforestation between the two periods, with pasture expansion accounting for most remaining deforestation. Declining deforestation coincided with a collapse of commodity markets and implementation of policy measures to reduce deforestation. Soybean profitability has since increased to pre-2006 levels whereas deforestation continued to decline, suggesting that antideforestation measures may have influenced the agricultural sector. We found little evidence of direct leakage of soy expansion into cerrado in Mato Grosso during the late 2000s, although indirect land-use changes and leakage to more distant regions are possible. This study provides evidence that reduced deforestation and increased agricultural production can occur simultaneously in tropical forest frontiers, provided that land is available and policies promote the efficient use of already-cleared lands (intensification) while restricting deforestation. It remains uncertain whether government- and industry-led policies can contain deforestation if future market conditions favor another boom in agricultural expansion.

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beta3 integrin interacts directly with GluA2 AMPA receptor subunit and regulates AMPA receptor expression in hippocampal neurons.

Publication Date: 2012 Jan 9 PMID: 22232691
Authors: Pozo, K. - Cingolani, L. A. - Bassani, S. - Laurent, F. - Passafaro, M. - Goda, Y.
Journal: Proc Natl Acad Sci U S A

The integrins are transmembrane receptors for ECM proteins, and they regulate various cellular functions in the central nervous system. In hippocampal neurons, the beta3 integrin subtype is required for homeostatic synaptic scaling of AMPA receptors (AMPARs) induced by chronic activity deprivation. The surface level of beta3 integrin in postsynaptic neurons directly correlates with synaptic strength and the abundance of synaptic GluA2 AMPAR subunit. Although these observations suggest a functional link between beta3 integrin and AMPAR, little is known about the mechanistic basis for the connection. Here we investigate the nature of beta3 integrin and AMPAR interaction underlying the beta3 integrin-dependent control of synaptic AMPAR expression and thus synaptic strength. We show that beta3 integrin and GluA2 subunit form a complex in mouse brain that involves the direct binding between their cytoplasmic domains. In contrast, beta3 integrin associates with GluA1 AMPAR subunit only weakly, and, in a heterologous expression system, the interaction requires the coexpression of GluA2. Surprisingly, in hippocampal pyramidal neurons, expressing beta3 integrin mutants with either increased or decreased affinity for extracellular ligands has no differential effects in elevating excitatory synaptic currents and surface GluA2 levels compared with WT beta3 integrin. Our findings identify an integrin family member, beta3, as a direct interactor of an AMPAR subunit and provide molecular insights into how this cell-adhesion protein regulates the composition of cell-surface AMPARs.

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Critical role for phosphoinositide 3-kinase gamma in parasite invasion and disease progression of cutaneous leishmaniasis.

Publication Date: 2012 Jan 9 PMID: 22232690
Authors: Cummings, H. E. - Barbi, J. - Reville, P. - Oghumu, S. - Zorko, N. - Sarkar, A. - Keiser, T. L. - Lu, B. - Ruckle, T. - Varikuti, S. - Lezama-Davila, C. - Wewers, M. D. - Whitacre, C. - Radzioch, D. - Rommel, C. - Seveau, S. - Satoskar, A. R.
Journal: Proc Natl Acad Sci U S A

Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survival and replication. Phosphoinositide 3-kinase gamma (PI3Kgamma), a member of the class I PI3Ks that is highly expressed by leukocytes, controls cell migration by initiating actin polymerization and cytoskeletal reorganization, which are processes also critical for phagocytosis. In this study, we demonstrate that class IB PI3K, PI3Kgamma, plays a critical role in pathogenesis of chronic cutaneous leishmaniasis caused by L. mexicana. Using the isoform-selective PI3Kgamma inhibitor, AS-605240 and PI3Kgamma gene-deficient mice, we show that selective blockade or deficiency of PI3Kgamma significantly enhances resistance against L. mexicana that is associated with a significant suppression of parasite entry into phagocytes and reduction in recruitment of host phagocytes as well as regulatory T cells to the site of infection. Furthermore, we demonstrate that AS-605240 is as effective as the standard antileishmanial drug sodium stibogluconate in treatment of cutaneous leishmaniasis caused by L. mexicana. These findings reveal a unique role for PI3Kgamma in Leishmania invasion and establishment of chronic infection, and demonstrate that therapeutic targeting of host pathways involved in establishment of infection may be a viable strategy for treating infections caused by obligate intracellular pathogens such as Leishmania.

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Homeostatic plasticity mechanisms are required for juvenile, but not adult, ocular dominance plasticity.

Publication Date: 2012 Jan 9 PMID: 22232689
Authors: Ranson, A. - Cheetham, C. E. - Fox, K. - Sengpiel, F.
Journal: Proc Natl Acad Sci U S A

Ocular dominance (OD) plasticity in the visual cortex is a classic model system for understanding developmental plasticity, but the visual cortex also shows plasticity in adulthood. Whether the plasticity mechanisms are similar or different at the two ages is not clear. Several plasticity mechanisms operate during development, including homeostatic plasticity, which acts to maintain the total excitatory drive to a neuron. In agreement with this idea, we found that an often-studied substrain of C57BL/6 mice, C57BL/6JOlaHsd (6JOla), lacks both the homeostatic component of OD plasticity as assessed by intrinsic signal imaging and synaptic scaling of mEPSC amplitudes after a short period of dark exposure during the critical period, whereas another substrain, C57BL/6J (6J), exhibits both plasticity processes. However, in adult mice, OD plasticity was identical in the 6JOla and 6J substrains, suggesting that adult plasticity occurs by a different mechanism. Consistent with this interpretation, adult OD plasticity was normal in TNFalpha knockout mice, which are known to lack juvenile synaptic scaling and the homeostatic component of OD plasticity, but was absent in adult alpha-calcium/calmodulin-dependent protein kinase II;T286A (alphaCaMKII(T286A)) mice, which have a point mutation that prevents autophosphorylation of alphaCaMKII. We conclude that increased responsiveness to open-eye stimulation after monocular deprivation during the critical period is a homeostatic process that depends mechanistically on synaptic scaling during the critical period, whereas in adult mice it is mediated by a different mechanism that requires alphaCaMKII autophosphorylation. Thus, our study reveals a transition between homeostatic and long-term potentiation-like plasticity mechanisms with increasing age.

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A morphologically specialized soldier caste improves colony defense in a neotropical eusocial bee.

Publication Date: 2012 Jan 9 PMID: 22232688
Authors: Gruter, C. - Menezes, C. - Imperatriz-Fonseca, V. L. - Ratnieks, F. L.
Journal: Proc Natl Acad Sci U S A

Division of labor among workers is common in insect societies and is thought to be important in their ecological success. In most species, division of labor is based on age (temporal castes), but workers in some ants and termites show morphological specialization for particular tasks (physical castes). Large-headed soldier ants and termites are well-known examples of this specialization. However, until now there has been no equivalent example of physical worker subcastes in social bees or wasps. Here we provide evidence for a physical soldier subcaste in a bee. In the neotropical stingless bee Tetragonisca angustula, nest defense is performed by two groups of guards, one hovering near the nest entrance and the other standing on the wax entrance tube. We show that both types of guards are 30% heavier than foragers and of different shape; foragers have relatively larger heads, whereas guards have larger legs. Low variation within each subcaste results in negligible size overlap between guards and foragers, further indicating that they are distinct physical castes. In addition, workers that remove garbage from the nest are of intermediate size, suggesting that they might represent another unrecognized caste. Guards or soldiers are reared in low but sufficient numbers (1-2% of emerging workers), considering that <1% usually perform this task. When challenged by the obligate robber bee Lestrimelitta limao, an important natural enemy, larger workers were able to fight for longer before being defeated by the much larger robber. This discovery opens up opportunities for the comparative study of physical castes in social insects, including the question of why soldiers appear to be so much rarer in bees than in ants or termites.

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Underground leaves of Philcoxia trap and digest nematodes.

Publication Date: 2012 Jan 9 PMID: 22232687
Authors: Pereira, C. G. - Almenara, D. P. - Winter, C. E. - Fritsch, P. W. - Lambers, H. - Oliveira, R. S.
Journal: Proc Natl Acad Sci U S A

The recently described genus Philcoxia comprises three species restricted to well lit and low-nutrient soils in the Brazilian Cerrado. The morphological and habitat similarities of Philcoxia to those of some carnivorous plants, along with recent observations of nematodes over its subterranean leaves, prompted the suggestion that the genus is carnivorous. Here we report compelling evidence of carnivory in Philcoxia of the Plantaginaceae, a family in which no carnivorous members are otherwise known. We also document both a unique capturing strategy for carnivorous plants and a case of a plant that traps and digests nematodes with underground adhesive leaves. Our findings illustrate how much can still be discovered about the origin, distribution, and frequency of the carnivorous syndrome in angiosperms and, more generally, about the diversity of nutrient-acquisition mechanisms that have evolved in plants growing in severely nutrient-impoverished environments such as the Brazilian Cerrado, one of the world's 34 biodiversity hotspots.

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How Staphylococcus aureus biofilms develop their characteristic structure.

Publication Date: 2012 Jan 9 PMID: 22232686
Authors: Periasamy, S. - Joo, H. S. - Duong, A. C. - Bach, T. H. - Tan, V. Y. - Chatterjee, S. S. - Cheung, G. Y. - Otto, M.
Journal: Proc Natl Acad Sci U S A

Biofilms cause significant problems in the environment and during the treatment of infections. However, the molecular mechanisms underlying biofilm formation are poorly understood. There is a particular lack of knowledge about biofilm maturation processes, such as biofilm structuring and detachment, which are deemed crucial for the maintenance of biofilm viability and the dissemination of cells from a biofilm. Here, we identify the phenol-soluble modulin (PSM) surfactant peptides as key biofilm structuring factors in the premier biofilm-forming pathogen Staphylococcus aureus. We provide evidence that all known PSM classes participate in structuring and detachment processes. Specifically, absence of PSMs in isogenic S. aureus psm deletion mutants led to strongly impaired formation of biofilm channels, abolishment of the characteristic waves of biofilm detachment and regrowth, and loss of control of biofilm expansion. In contrast, induced expression of psm loci in preformed biofilms promoted those processes. Furthermore, PSMs facilitated dissemination from an infected catheter in a mouse model of biofilm-associated infection. Moreover, formation of the biofilm structure was linked to strongly variable, quorum sensing-controlled PSM expression in biofilm microenvironments, whereas overall PSM production remained constant to ascertain biofilm homeostasis. Our study describes a mechanism of biofilm structuring in molecular detail, and the general principle (i.e., quorum-sensing controlled expression of surfactants) seems to be conserved in several bacteria, despite the divergence of the respective biofilm-structuring surfactants. These findings provide a deeper understanding of biofilm development processes, which represents an important basis for strategies to interfere with biofilm formation in the environment and human disease.

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An rhs gene of Pseudomonas aeruginosa encodes a virulence protein that activates the inflammasome.

Publication Date: 2012 Jan 9 PMID: 22232685
Authors: Kung, V. L. - Khare, S. - Stehlik, C. - Bacon, E. M. - Hughes, A. J. - Hauser, A. R.
Journal: Proc Natl Acad Sci U S A

The rhs genes are a family of enigmatic composite genes, widespread among Gram-negative bacteria. In this study, we characterized rhsT, a Pseudomonas aeruginosa rhs gene that encodes a toxic protein. Expression of rhsT was induced upon contact with phagocytic cells. The RhsT protein was exposed on the bacterial surface and translocated into phagocytic cells; these cells subsequently underwent inflammasome-mediated death. Moreover, RhsT enhanced host secretion of the potent proinflammatory cytokines IL-1beta and IL-18 in an inflammasome-dependent manner. In a mouse model of acute pneumonia, infection with a P. aeruginosa strain lacking rhsT was associated with less IL-18 production, fewer recruited leukocytes, reduced pulmonary bacterial load, and enhanced animal survival. Thus, rhsT encodes a virulence determinant that activates the inflammasome.

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High-yield maize with large net energy yield and small global warming intensity.

Publication Date: 2012 Jan 9 PMID: 22232684
Authors: Grassini, P. - Cassman, K. G.
Journal: Proc Natl Acad Sci U S A

Addressing concerns about future food supply and climate change requires management practices that maximize productivity per unit of arable land while reducing negative environmental impact. On-farm data were evaluated to assess energy balance and greenhouse gas (GHG) emissions of irrigated maize in Nebraska that received large nitrogen (N) fertilizer (183 kg of Nha(-1)) and irrigation water inputs (272 mm or 2,720 m(3) ha(-1)). Although energy inputs (30 GJha(-1)) were larger than those reported for US maize systems in previous studies, irrigated maize in central Nebraska achieved higher grain and net energy yields (13.2 Mgha(-1) and 159 GJha(-1), respectively) and lower GHG-emission intensity (231 kg of CO(2)eMg(-1) of grain). Greater input-use efficiencies, especially for N fertilizer, were responsible for better performance of these irrigated systems, compared with much lower-yielding, mostly rainfed maize systems in previous studies. Large variation in energy inputs and GHG emissions across irrigated fields in the present study resulted from differences in applied irrigation water amount and imbalances between applied N inputs and crop N demand, indicating potential to further improve environmental performance through better management of these inputs. Observed variation in N-use efficiency, at any level of applied N inputs, suggests that an N-balance approach may be more appropriate for estimating soil N(2)O emissions than the Intergovernmental Panel on Climate Change approach based on a fixed proportion of applied N. Negative correlation between GHG-emission intensity and net energy yield supports the proposition that achieving high yields, large positive energy balance, and low GHG emissions in intensive cropping systems are not conflicting goals.

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Metabolic click-labeling with a fucose analog reveals pectin delivery, architecture, and dynamics in Arabidopsis cell walls.

Publication Date: 2012 Jan 9 PMID: 22232683
Authors: Anderson, C. T. - Wallace, I. S. - Somerville, C. R.
Journal: Proc Natl Acad Sci U S A

Polysaccharide-rich cell walls are a defining feature of plants that influence cell division and growth, but many details of cell-wall organization and dynamics are unknown because of a lack of suitable chemical probes. Metabolic labeling using sugar analogs compatible with click chemistry has the potential to provide new insights into cell-wall structure and dynamics. Using this approach, we found that an alkynylated fucose analog (FucAl) is metabolically incorporated into the cell walls of Arabidopsis thaliana roots and that a significant fraction of the incorporated FucAl is present in pectic rhamnogalacturonan-I (RG-I). Time-course experiments revealed that FucAl-containing RG-I first localizes in cell walls as uniformly distributed punctae that likely mark the sites of vesicle-mediated delivery of new polysaccharides to growing cell walls. In addition, we found that the pattern of incorporated FucAl differs markedly along the developmental gradient of the root. Using pulse-chase experiments, we also discovered that the pectin network is reoriented in elongating root epidermal cells. These results reveal previously undescribed details of polysaccharide delivery, organization, and dynamics in cell walls.

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CD14 cooperates with complement receptor 3 to mediate MyD88-independent phagocytosis of Borrelia burgdorferi.

Publication Date: 2012 Jan 9 PMID: 22232682
Authors: Hawley, K. L. - Olson, C. M. Jr - Iglesias-Pedraz, J. M. - Navasa, N. - Cervantes, J. L. - Caimano, M. J. - Izadi, H. - Ingalls, R. R. - Pal, U. - Salazar, J. C. - Radolf, J. D. - Anguita, J.
Journal: Proc Natl Acad Sci U S A

Phagocytosis of Borrelia burgdorferi, the causative agent of Lyme disease, is a poorly understood process, despite its importance during the host immune response to infection. B. burgdorferi has been shown to bind to different receptors on the surface of phagocytic cells, including the beta(2) integrin, complement receptor 3 (CR3). However, whether these receptors mediate the phagocytosis of the spirochete remains unknown. We now demonstrate that CR3 mediates the phagocytosis of the spirochete by murine macrophages and human monocytes. Interaction of B. burgdorferi with the integrin is not sufficient, however, to internalize the spirochete; phagocytosis requires the interaction of CR3 with the GPI-anchored protein, CD14, independently of TLR/MyD88-induced or inside-out signals. Interestingly, the absence of CR3 leads to marked increases in the production of TNF in vitro and in vivo, despite reduced spirochetal uptake. Furthermore, the absence of CR3 during infection with B. burgdorferi results in the inefficient control of bacterial burdens in the heart and increased Lyme carditis. Overall, our data identify CR3 as a MyD88-independent phagocytic receptor for B. burgdorferi that also participates in the modulation of the proinflammatory output of macrophages. These data also establish a unique mechanism of CR3-mediated phagocytosis that requires the direct cooperation of GPI-anchored proteins.

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Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway.

Publication Date: 2012 Jan 9 PMID: 22232681
Authors: Morikawa, T. - Kajimura, M. - Nakamura, T. - Hishiki, T. - Nakanishi, T. - Yukutake, Y. - Nagahata, Y. - Ishikawa, M. - Hattori, K. - Takenouchi, T. - Takahashi, T. - Ishii, I. - Matsubara, K. - Kabe, Y. - Uchiyama, S. - Nagata, E. - Gadalla, M. M. - Snyder, S. H. - Suematsu, M.
Journal: Proc Natl Acad Sci U S A

Enhancement of cerebral blood flow by hypoxia is critical for brain function, but signaling systems underlying its regulation have been unclear. We report a pathway mediating hypoxia-induced cerebral vasodilation in studies monitoring vascular disposition in cerebellar slices and in intact mouse brains using two-photon intravital laser scanning microscopy. In this cascade, hypoxia elicits cerebral vasodilation via the coordinate actions of H(2)S formed by cystathionine beta-synthase (CBS) and CO generated by heme oxygenase (HO)-2. Hypoxia diminishes CO generation by HO-2, an oxygen sensor. The constitutive CO physiologically inhibits CBS, and hypoxia leads to increased levels of H(2)S that mediate the vasodilation of precapillary arterioles. Mice with targeted deletion of HO-2 or CBS display impaired vascular responses to hypoxia. Thus, in intact adult brain cerebral cortex of HO-2-null mice, imaging mass spectrometry reveals an impaired ability to maintain ATP levels on hypoxia.

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Phytochrome regulates translation of mRNA in the cytosol.

Publication Date: 2012 Jan 9 PMID: 22232680
Authors: Paik, I. - Yang, S. - Choi, G.
Journal: Proc Natl Acad Sci U S A

An array of photoreceptors including cryptochromes, phototropin, and phytochromes regulates various light responses in plants. Among these photoreceptors, phytochromes perceive red and far-red light by switching between two interconvertible spectral forms (Pr and Pfr). The Pfr form promotes light responses partly by destabilizing negatively acting, phytochrome-interacting basic helix-loop-helix transcription factors (PIFs), thus modulating transcription in the nucleus. The Pfr form is also present in the cytosol. However, the role of phytochromes in the cytosol is not well understood. Here we show that the Pfr form interacts with the cytosolic protein PENTA1 (PNT1) and inhibits the translation of protochlorophyllide reductase (PORA) mRNA. PNT1 possesses five C3H-type zinc finger domains and displays similarity to various RNA binding proteins including Tristetraprolin, which regulates stabilities of mRNAs such as TNF-alpha mRNA in humans. Consistent with its function as an RNA binding protein, PNT1 directly binds to mRNA of a key chlorophyll biosynthetic gene, protochlorophyllide reductase in vivo and inhibits the translation of PORA mRNA in the presence of phytochromes. The present results demonstrate that phytochromes transmit light signals to regulate not only transcription in the nucleus through PIFs, but also translation in the cytosol through PNT1.

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Chlamydophila pneumoniae phospholipase D (CpPLD) drives Th17 inflammation in human atherosclerosis.

Publication Date: 2012 Jan 9 PMID: 22232679
Authors: Benagiano, M. - Munari, F. - Ciervo, A. - Amedei, A. - Paccani, S. R. - Mancini, F. - Ferrari, M. - Della Bella, C. - Ulivi, C. - D'Elios, S. - Baldari, C. T. - Prisco, D. - de Bernard, M. - D'Elios, M. M.
Journal: Proc Natl Acad Sci U S A

Phospholipases are produced from bacterial pathogens causing very different diseases. One of the most intriguing aspects of phospholipases is their potential to interfere with cellular signaling cascades and to modulate the host-immune response. Here, we investigated the role of the innate and acquired immune responses elicited by Chlamydophila pneumoniae phospholipase D (CpPLD) in the pathogenesis of atherosclerosis. We evaluated the cytokine and chemokine production induced by CpPLD in healthy donors' monocytes and in vivo activated T cells specific for CpPLD that infiltrate atherosclerotic lesions of patients with C. pneumoniae antibodies. We also examined the helper function of CpPLD-specific T cells for monocyte matrix metalloproteinase (MMP)-9 and tissue factor (TF) production as well as the CpPLD-induced chemokine expression by human venular endothelial cells (HUVECs). We report here that CpPLD is a TLR4 agonist able to induce the expression of IL-23, IL-6, IL-1beta, TGF-beta, and CCL-20 in monocytes, as well as CXCL-9, CCL-20, CCL-4, CCL-2, ICAM-1, and VCAM-1 in HUVECs. Plaque-derived T cells produce IL-17 in response to CpPLD. Moreover, CpPLD-specific CD4(+) T lymphocytes display helper function for monocyte MMP-9 and TF production. CpPLD promotes Th17 cell migration through the induction of chemokine secretion and adhesion molecule expression on endothelial cells. These findings indicate that CpPLD is able to drive the expression of IL-23, IL-6, IL-1beta, TGF-beta, and CCL-20 by monocytes and to elicit a Th17 immune response that plays a key role in the genesis of atherosclerosis.

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Digoxin inhibits development of hypoxic pulmonary hypertension in mice.

Publication Date: 2012 Jan 9 PMID: 22232678
Authors: Abud, E. M. - Maylor, J. - Undem, C. - Punjabi, A. - Zaiman, A. L. - Myers, A. C. - Sylvester, J. T. - Semenza, G. L. - Shimoda, L. A.
Journal: Proc Natl Acad Sci U S A

Chronic hypoxia is an inciting factor for the development of pulmonary arterial hypertension. The mechanisms involved in the development of hypoxic pulmonary hypertension (HPH) include hypoxia-inducible factor 1 (HIF-1)-dependent transactivation of genes controlling pulmonary arterial smooth muscle cell (PASMC) intracellular calcium concentration ([Ca(2+)](i)) and pH. Recently, digoxin was shown to inhibit HIF-1 transcriptional activity. In this study, we tested the hypothesis that digoxin could prevent and reverse the development of HPH. Mice were injected daily with saline or digoxin and exposed to room air or ambient hypoxia for 3 wk. Treatment with digoxin attenuated the development of right ventricle (RV) hypertrophy and prevented the pulmonary vascular remodeling and increases in PASMC [Ca(2+)](i), pH, and RV pressure that occur in mice exposed to chronic hypoxia. When started after pulmonary hypertension was established, digoxin attenuated the hypoxia-induced increases in RV pressure and PASMC pH and [Ca(2+)](i). These preclinical data support a role for HIF-1 inhibitors in the treatment of HPH.

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Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1.

Publication Date: 2012 Jan 9 PMID: 22232677
Authors: Kanehiro, Y. - Todo, K. - Negishi, M. - Fukuoka, J. - Gan, W. - Hikasa, T. - Kaga, Y. - Takemoto, M. - Magari, M. - Li, X. - Manley, J. L. - Ohmori, H. - Kanayama, N.
Journal: Proc Natl Acad Sci U S A

Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.

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Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes.

Publication Date: 2012 Jan 9 PMID: 22232676
Authors: Shiroguchi, K. - Jia, T. Z. - Sims, P. A. - Xie, X. S.
Journal: Proc Natl Acad Sci U S A

RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling, but is hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic to exponential PCR amplification. We developed a simple strategy for mitigating these complications, allowing truly digital RNA-Seq. Following reverse transcription, a large set of barcode sequences is added in excess, and nearly every cDNA molecule is uniquely labeled by random attachment of barcode sequences to both ends. After PCR, we applied paired-end deep sequencing to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance is measured based on the number of unique barcode sequences observed for a given cDNA sequence. We optimized the barcodes to be unambiguously identifiable, even in the presence of multiple sequencing errors. This method allows counting with single-copy resolution despite sequence-dependent bias and PCR-amplification noise, and is analogous to digital PCR but amendable to quantifying a whole transcriptome. We demonstrated transcriptome profiling of Escherichia coli with more accurate and reproducible quantification than conventional RNA-Seq.

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BDNF and glucocorticoids regulate corticotrophin-releasing hormone (CRH) homeostasis in the hypothalamus.

Publication Date: 2012 Jan 9 PMID: 22232675
Authors: Jeanneteau, F. D. - Lambert, W. M. - Ismaili, N. - Bath, K. G. - Lee, F. S. - Garabedian, M. J. - Chao, M. V.
Journal: Proc Natl Acad Sci U S A

Regulation of the hypothalamic-pituitary-adrenal (HPA) axis is critical for adaptation to environmental changes. The principle regulator of the HPA axis is corticotrophin-releasing hormone (CRH), which is made in the parventricular nucleus and is an important target of negative feedback by glucocorticoids. However, the molecular mechanisms that regulate CRH are not fully understood. Disruption of normal HPA axis activity is a major risk factor of neuropsychiatric disorders in which decreased expression of the glucocorticoid receptor (GR) has been documented. To investigate the role of the GR in CRH neurons, we have targeted the deletion of the GR, specifically in the parventricular nucleus. Impairment of GR function in the parventricular nucleus resulted in an enhancement of CRH expression and an up-regulation of hypothalamic levels of BDNF and disinhibition of the HPA axis. BDNF is a stress and activity-dependent factor involved in many activities modulated by the HPA axis. Significantly, ectopic expression of BDNF in vivo increased CRH, whereas reduced expression of BDNF, or its receptor TrkB, decreased CRH expression and normal HPA functions. We find the differential regulation of CRH relies upon the cAMP response-element binding protein coactivator CRTC2, which serves as a switch for BDNF and glucocorticoids to direct the expression of CRH.

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Epigenetic regulation of hypoxic sensing disrupts cardiorespiratory homeostasis.

Publication Date: 2012 Jan 9 PMID: 22232674
Authors: Nanduri, J. - Makarenko, V. - Reddy, V. D. - Yuan, G. - Pawar, A. - Wang, N. - Khan, S. A. - Zhang, X. - Kinsman, B. - Peng, Y. J. - Kumar, G. K. - Fox, A. P. - Godley, L. A. - Semenza, G. L. - Prabhakar, N. R.
Journal: Proc Natl Acad Sci U S A

Recurrent apnea with intermittent hypoxia is a major clinical problem in preterm infants. Recent studies, although limited, showed that adults who were born preterm exhibit increased incidence of sleep-disordered breathing and hypertension, suggesting that apnea of prematurity predisposes to autonomic dysfunction in adulthood. Here, we demonstrate that adult rats that were exposed to intermittent hypoxia as neonates exhibit exaggerated responses to hypoxia by the carotid body and adrenal chromaffin cells, which regulate cardio-respiratory function, resulting in irregular breathing with apneas and hypertension. The enhanced hypoxic sensitivity was associated with elevated oxidative stress, decreased expression of genes encoding antioxidant enzymes, and increased expression of pro-oxidant enzymes. Decreased expression of the Sod2 gene, which encodes the antioxidant enzyme superoxide dismutase 2, was associated with DNA hypermethylation of a single CpG dinucleotide close to the transcription start site. Treating neonatal rats with decitabine, an inhibitor of DNA methylation, during intermittent hypoxia exposure prevented oxidative stress, enhanced hypoxic sensitivity, and autonomic dysfunction. These findings implicate a hitherto uncharacterized role for DNA methylation in mediating neonatal programming of hypoxic sensitivity and the ensuing autonomic dysfunction in adulthood.

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Divergence of duplicate genes in exon-intron structure.

Publication Date: 2012 Jan 9 PMID: 22232673
Authors: Xu, G. - Guo, C. - Shan, H. - Kong, H.
Journal: Proc Natl Acad Sci U S A

Gene duplication plays key roles in organismal evolution. Duplicate genes, if they survive, tend to diverge in regulatory and coding regions. Divergences in coding regions, especially those that can change the function of the gene, can be caused by amino acid-altering substitutions and/or alterations in exon-intron structure. Much has been learned about the mode, tempo, and consequences of nucleotide substitutions, yet relatively little is known about structural divergences. In this study, by analyzing 612 pairs of sibling paralogs from seven representative gene families and 300 pairs of one-to-one orthologs from different species, we investigated the occurrence and relative importance of structural divergences during the evolution of duplicate and nonduplicate genes. We found that structural divergences have been very prevalent in duplicate genes and, in many cases, have led to the generation of functionally distinct paralogs. Comparisons of the genomic sequences of these genes further indicated that the differences in exon-intron structure were actually accomplished by three main types of mechanisms (exon/intron gain/loss, exonization/pseudoexonization, and insertion/deletion), each of which contributed differently to structural divergence. Like nucleotide substitutions, insertion/deletion and exonization/pseudoexonization occurred more or less randomly, with the number of observable mutational events per gene pair being largely proportional to evolutionary time. Notably, however, compared with paralogs with similar evolutionary times, orthologs have accumulated significantly fewer structural changes, whereas the amounts of amino acid replacements accumulated did not show clear differences. This finding suggests that structural divergences have played a more important role during the evolution of duplicate than nonduplicate genes.

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Systematic identification of interactions between host cell proteins and E7 oncoproteins from diverse human papillomaviruses.

Publication Date: 2012 Jan 9 PMID: 22232672
Authors: White, E. A. - Sowa, M. E. - Tan, M. J. - Jeudy, S. - Hayes, S. D. - Santha, S. - Munger, K. - Harper, J. W. - Howley, P. M.
Journal: Proc Natl Acad Sci U S A

More than 120 human papillomaviruses (HPVs) have now been identified and have been associated with a variety of clinical lesions. To understand the molecular differences among these viruses that result in lesions with distinct pathologies, we have begun a MS-based proteomic analysis of HPV-host cellular protein interactions and have created the plasmid and cell line libraries required for these studies. To validate our system, we have characterized the host cellular proteins that bind to the E7 proteins expressed from 17 different HPV types. These studies reveal a number of interactions, some of which are conserved across HPV types and others that are unique to a single HPV species or HPV genus. Binding of E7 to UBR4/p600 is conserved across all virus types, whereas the cellular protein ENC1 binds specifically to the E7s from HPV18 and HPV45, both members of genus alpha, species 7. We identify a specific interaction of HPV16 E7 with ZER1, a substrate specificity factor for a cullin 2 (CUL2)-RING ubiquitin ligase, and show that ZER1 is required for the binding of HPV16 E7 to CUL2. We further show that ZER1 is required for the destabilization of the retinoblastoma tumor suppressor RB1 in HPV16 E7-expressing cells and propose that a CUL2-ZER1 complex functions to target RB1 for degradation in HPV16 E7-expressing cells. These studies refine the current understanding of HPV E7 functions and establish a platform for the rapid identification of virus-host interactions.

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Telomere length in early life predicts lifespan.

Publication Date: 2012 Jan 9 PMID: 22232671
Authors: Heidinger, B. J. - Blount, J. D. - Boner, W. - Griffiths, K. - Metcalfe, N. B. - Monaghan, P.
Journal: Proc Natl Acad Sci U S A

The attrition of telomeres, the ends of eukaryote chromosomes, is thought to play an important role in cell deterioration with advancing age. The observed variation in telomere length among individuals of the same age is therefore thought to be related to variation in potential longevity. Studies of this relationship are hampered by the time scale over which individuals need to be followed, particularly in long-lived species where lifespan variation is greatest. So far, data are based either on simple comparisons of telomere length among different age classes or on individuals whose telomere length is measured at most twice and whose subsequent survival is monitored for only a short proportion of the typical lifespan. Both approaches are subject to bias. Key studies, in which telomere length is tracked from early in life, and actual lifespan recorded, have been lacking. We measured telomere length in zebra finches (n = 99) from the nestling stage and at various points thereafter, and recorded their natural lifespan (which varied from less than 1 to almost 9 y). We found telomere length at 25 d to be a very strong predictor of realized lifespan (P < 0.001); those individuals living longest had relatively long telomeres at all points at which they were measured. Reproduction increased adult telomere loss, but this effect appeared transient and did not influence survival. Our results provide the strongest evidence available of the relationship between telomere length and lifespan and emphasize the importance of understanding factors that determine early life telomere length.

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Uric acid stones in the urinary bladder of aryl hydrocarbon receptor (AhR) knockout mice.

Publication Date: 2012 Jan 9 PMID: 22232670
Authors: Butler, R. - Inzunza, J. - Suzuki, H. - Fujii-Kuriyama, Y. - Warner, M. - Gustafsson, J. A.
Journal: Proc Natl Acad Sci U S A

The aryl hydrocarbon receptor (AhR) knockout mice raised in the laboratory of Fujii-Kuriyama have been under investigation for several years because of the presence in their urinary bladder of large, yellowish stones. The stones are composed of uric acid and become apparent in the bladders as tiny stones when mice are 10 wk of age. By the time the mice are 6 mo of age, there are usually two or three stones with diameters of 3-4 mm. The urate concentration in the serum was normal but in the urine the concentration was 40-50 mg/dL, which is 10 times higher than that in the WT littermates. There were no apparent histological pathologies in the kidney or joints and the levels of enzymes involved in elimination of purines were normal. The source of the uric acid was therefore judged to be from degradation of nucleic acids due to a high turnover of cells in the bladder itself. The bladder was fibrotic and the luminal side of the bladder epithelium was filled with eosinophilic granules. There was loss of E-cadherin between some epithelial cells, with an enlarged submucosal area filled with immune cells and sometimes invading epithelial cells. We hypothesize that in the absence of AhR there is loss of detoxifying enzymes, which leads to accumulation of unconjugated cytotoxins and carcinogens in the bladder. The presence of bladder toxins may have led to the increased apoptosis and inflammation as well as invasion of epithelial cells in the bladders of older mice.

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Microbial diversity determines the invasion of soil by a bacterial pathogen.

Publication Date: 2012 Jan 9 PMID: 22232669
Authors: van Elsas, J. D. - Chiurazzi, M. - Mallon, C. A. - Elhottova, D. - Kristufek, V. - Salles, J. F.
Journal: Proc Natl Acad Sci U S A

Natural ecosystems show variable resistance to invasion by alien species, and this resistance can relate to the species diversity in the system. In soil, microorganisms are key components that determine life support functions, but the functional redundancy in the microbiota of most soils has long been thought to overwhelm microbial diversity-function relationships. We here show an inverse relationship between soil microbial diversity and survival of the invading species Escherichia coli O157:H7, assessed by using the marked derivative strain T. The invader's fate in soil was determined in the presence of (i) differentially constructed culturable bacterial communities, and (ii) microbial communities established using a dilution-to-extinction approach. Both approaches revealed a negative correlation between the diversity of the soil microbiota and survival of the invader. The relationship could be explained by a decrease in the competitive ability of the invader in species-rich vs. species-poor bacterial communities, reflected in the amount of resources used and the rate of their consumption. Soil microbial diversity is a key factor that controls the extent to which bacterial invaders can establish.

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Betacellulin promotes cell proliferation in the neural stem cell niche and stimulates neurogenesis.

Publication Date: 2012 Jan 9 PMID: 22232668
Authors: Gomez-Gaviro, M. V. - Scott, C. E. - Sesay, A. K. - Matheu, A. - Booth, S. - Galichet, C. - Lovell-Badge, R.
Journal: Proc Natl Acad Sci U S A

Neural stem cells (NSCs) reside in specialized niches in the adult mammalian brain, including the subventricular zone and the dentate gyrus, which act to control NSC behavior. Among other cell types within these niches, NSCs are found in close proximity to blood vessels. We carried out an analysis of the interaction between endothelial cells and NSCs, and show that betacellulin (BTC), a member of the EGF family and one of several signaling molecules made by the former, induces NSC proliferation and prevents spontaneous differentiation in culture. When infused into the lateral ventricle, BTC induces expansion of NSCs and neuroblasts, and promotes neurogenesis in the olfactory bulb and dentate gyrus, whereas specific blocking antibodies reduce the number of stem/progenitor cells. BTC-null mice are less able to regenerate neuroblast numbers compared with WT littermates following depletion of proliferating cells using cytosine-beta-D-arabinofuranoside. BTC acts via both the EGF receptor, located on NSCs, and ErbB4, located on neuroblasts, with the latter explaining why its effects are distinct from those of EGF itself. Our results suggest that BTC could be a good candidate to aid regenerative therapies.

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T helper cell- and CD40-dependent germline IgM prevents chronic virus-induced demyelinating disease.

Publication Date: 2012 Jan 9 PMID: 22232667
Authors: Gil-Cruz, C. - Perez-Shibayama, C. - Firner, S. - Waisman, A. - Bechmann, I. - Thiel, V. - Cervantes-Barragan, L. - Ludewig, B.
Journal: Proc Natl Acad Sci U S A

Generation of antiviral IgM is usually considered as a marker of a short-lived initial antibody response that is replaced by hypermutated and more-efficient IgG. However, once viruses have established a particular niche for their persistence (e.g., within the CNS), the immune system has to specifically mobilize a broad range of antimicrobial effectors to contain the pathogen in the long term. Infection of the CNS with the mouse hepatitis virus (MHV) provides a unique model situation in which the extent of inflammatory CNS disease is determined by the balance between antiviral immune control, viral replication, and immune-mediated damage. We show here that whereas antibody- or B cell-deficient mice failed to contain MHV CNS infection and developed progressive demyelinating disease, germline IgM produced in activation-induced cytidine deaminase-deficient mice (aicda(-/-)) provided long-term protection against the chronic multiple sclerosis-like disease. Furthermore, we found that appropriate B-cell activation within the CNS-draining lymph node and subsequent CXCR3-mediated migration of antiviral IgM-secreting cells to the infected CNS was dependent on CD40-mediated interaction of B cells with T helper cells. These data indicate that the CD40-mediated collaboration of T and B cells is critical to secure neuroprotective IgM responses during viral CNS infection.

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Virulence determinants in the obligate intracellular pathogen Chlamydia trachomatis revealed by forward genetic approaches.

Publication Date: 2012 Jan 9 PMID: 22232666
Authors: Nguyen, B. D. - Valdivia, R. H.
Journal: Proc Natl Acad Sci U S A

Chlamydia trachomatis, a pathogen responsible for diseases of significant clinical and public health importance, remains poorly characterized because of its intractability to routine molecular genetic manipulation. We have developed a combinatorial approach to rapidly generate a comprehensive library of genetically defined mutants. Chemical mutagenesis, coupled with whole-genome sequencing (WGS) and a system for DNA exchange within infected cells, was used to generate Chlamydia mutants with distinct phenotypes, map the underlying genetic lesions, and generate isogenic strains. As a result, we identified mutants with altered glycogen metabolism, including an attenuated strain defective for type II secretion. The coupling of chemically induced gene variation and WGS to establish genotype-phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

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Structuring economic incentives to reduce emissions from deforestation within Indonesia.

Publication Date: 2012 Jan 9 PMID: 22232665
Authors: Busch, J. - Lubowski, R. N. - Godoy, F. - Steininger, M. - Yusuf, A. A. - Austin, K. - Hewson, J. - Juhn, D. - Farid, M. - Boltz, F.
Journal: Proc Natl Acad Sci U S A

We estimate and map the impacts that alternative national and subnational economic incentive structures for reducing emissions from deforestation (REDD+) in Indonesia would have had on greenhouse gas emissions and national and local revenue if they had been in place from 2000 to 2005. The impact of carbon payments on deforestation is calibrated econometrically from the pattern of observed deforestation and spatial variation in the benefits and costs of converting land to agriculture over that time period. We estimate that at an international carbon price of $10/tCO(2)e, a "mandatory incentive structure," such as a cap-and-trade or symmetric tax-and-subsidy program, would have reduced emissions by 163-247 MtCO(2)e/y (20-31% below the without-REDD+ reference scenario), while generating a programmatic budget surplus. In contrast, a "basic voluntary incentive structure" modeled after a standard payment-for-environmental-services program would have reduced emissions nationally by only 45-76 MtCO(2)e/y (6-9%), while generating a programmatic budget shortfall. By making four policy improvements-paying for net emission reductions at the scale of an entire district rather than site-by-site; paying for reductions relative to reference levels that match business-as-usual levels; sharing a portion of district-level revenues with the national government; and sharing a portion of the national government's responsibility for costs with districts-an "improved voluntary incentive structure" would have been nearly as effective as a mandatory incentive structure, reducing emissions by 136-207 MtCO(2)e/y (17-26%) and generating a programmatic budget surplus.

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Indirect evidence that maternal microchimerism in cord blood mediates a graft-versus-leukemia effect in cord blood transplantation.

Publication Date: 2012 Jan 9 PMID: 22232664
Authors: van Rood, J. J. - Scaradavou, A. - Stevens, C. E.
Journal: Proc Natl Acad Sci U S A

During pregnancy women can develop B- and T-cell immunity against the inherited paternal antigens (IPAs) of the fetus, such as HLA, peptides of minor histocompatibilty antigens, and possibly onco-fetal antigens. The biological and pathological role of these pregnancy-induced immunological events is only understood in part. However, anti-IPA immunity in the mother persists for many decades after delivery and may reduce relapse in offspring with leukemia after HLA-haploidentical transplantation of maternal hematopoietic stem cells (HSC). We hypothesized that maternal anti-IPA immune elements cross the placenta and might confer a potent graft-versus-leukemia effect when cord blood (CB) is used in unrelated HSC transplantation. In a retrospective study of single-unit CB recipients with all grafts provided by the New York Blood Center, we show that patients with acute myeloid or lymphoblastic leukemia (n = 845) who shared one or more HLA-A, -B, or -DRB1 antigens with their CB donor's IPAs had a significant decrease in leukemic relapse posttransplantation [hazard ratio (HR) = 0.38, P < 0.001] compared with those that did not. Remarkably, relapse reduction in patients receiving CB with one HLA mismatch (HR = 0.15, P < 0.001) was not associated with an increased risk of severe acute graft-versus-host disease (HR = 1.43, P = 0.730). Our findings may explain the unexpected low relapse rate after CB transplantation, open new avenues in the study of leukemic relapse after HSC transplantation (possibly of malignancies in general), and have practical implications for CB unit selection.

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Ablation of neurons expressing agouti-related protein, but not melanin concentrating hormone, in leptin-deficient mice restores metabolic functions and fertility.

Publication Date: 2012 Jan 9 PMID: 22232663
Authors: Wu, Q. - Whiddon, B. B. - Palmiter, R. D.
Journal: Proc Natl Acad Sci U S A

Leptin-deficient (Lep(ob/ob)) mice are obese, diabetic, and infertile. Ablation of neurons that make agouti-related protein (AgRP) in moderately obese adult Lep(ob/ob) mice caused severe anorexia. The mice stopped eating for 2 wk and then gradually recovered. Their body weight fell to within a normal range for WT mice, at which point food intake and glucose tolerance were restored to that of WT mice. Remarkably, both male and female Lep(ob/ob) mice became fertile. Ablation of neurons that express melanin-concentrating hormone (MCH) in adult Lep(ob/ob) mice had no effect on food intake, body weight, or fertility, but resulted in improved glucose tolerance. We conclude that AgRP-expressing neurons play a critical role in mediating the metabolic syndrome and infertility of Lep(ob/ob) mice, whereas MCH-expressing neurons have only a minor role.

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Heterogeneity of hunting ability and nutritional status among domestic dogs in lowland Nicaragua.

Publication Date: 2012 Jan 9 PMID: 22232662
Authors: Koster, J. M. - Tankersley, K. B.
Journal: Proc Natl Acad Sci U S A

In past and modern human societies, dogs have played an important role as hunting companions. Given considerable ethnographic evidence that dogs vary in their hunting abilities, this paper addresses the effects of key demographic variables, namely age and sex, on the amount of harvested game that dogs contribute in an indigenous Nicaraguan community. Controlling for variation in the time spent potentially hunting, male dogs and older dogs are significantly associated with greater harvests. These results may account for documented preferences for males in both archaeological and ethnographic contexts. Among societies in which dogs are used both as hunting companions and sources of food, the age-related delay in peak hunting ability also suggests a tradeoff that might explain the consumption of dogs shortly after they have reached adult size. Informant rankings of two cohorts of dogs indicate that residents of the community exhibit high agreement about the relative abilities of the dogs, and the rankings indicate that dogs from the same household exhibit comparable skill. There is little evidence that talented, highly-ranked dogs are provided a more nutritious diet, as measured by nitrogen-based and carbon-based isotopic analysis of hair samples. Overall, although dogs can be quite advantageous as hunting companions, this research suggests that the heterogeneity of hunting ability combines with the high mortality of dogs to impose risks on households that depend on dogs as a source of harvested meat.

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Unveiling the biosynthetic puzzle of destruxins in Metarhizium species.

Publication Date: 2012 Jan 9 PMID: 22232661
Authors: Wang, B. - Kang, Q. - Lu, Y. - Bai, L. - Wang, C.
Journal: Proc Natl Acad Sci U S A

Insect pathogenic fungi produce a plethora of insecticidally and pharmaceutically active compounds, including 39 cyclohexadepsipeptide destruxins (dtxs). Even though dtxs were first discovered more than 50 y ago, the genes responsible for their biosynthesis were unknown until this study. Based on our comparative genomic information and targeted gene disruptions, we report the gene cluster for dtx biosynthesis in the insect pathogen Metarhizium robertsii. The nonribosomal peptide synthetase DtxS1 has six adenylation domains, two of which are capable of selecting different amino acids to synthesize dtx B and its analogs. The cytochrome P450 enzyme DtxS2 converts dtx B into other dtxs by a chain of reactions, each producing a new derivative. The aldo-keto reductase DtxS3 and aspartic acid decarboxylase DtxS4 are responsible for the conversion and provision of the first and last substrates for the dtx assembly line, respectively. Insect bioassays showed that dtxs could suppress both cellular and humoral immune responses thereby assisting fungal propagation in insects. The differing abilities of Metarhizium species to produce toxins is dependent on the presence of the dtxS1 gene. The toxigenic species are capable of killing multiple orders of insects, whereas the nontoxigenic Metarhizium spp. have narrow host ranges. Thus, the acquisition or retention of the dtx biosynthesis gene cluster in Metarhizium lineages has been coordinated with the evolution of fungal host specificity. The data from this study will facilitate the development of dtxs as bioinsecticides or pharmaceuticals.

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Brain structure in healthy adults is related to serum transferrin and the H63D polymorphism in the HFE gene.

Publication Date: 2012 Jan 9 PMID: 22232660
Authors: Jahanshad, N. - Kohannim, O. - Hibar, D. P. - Stein, J. L. - McMahon, K. L. - de Zubicaray, G. I. - Medland, S. E. - Montgomery, G. W. - Whitfield, J. B. - Martin, N. G. - Wright, M. J. - Toga, A. W. - Thompson, P. M.
Journal: Proc Natl Acad Sci U S A

Control of iron homeostasis is essential for healthy central nervous system function: iron deficiency is associated with cognitive impairment, yet iron overload is thought to promote neurodegenerative diseases. Specific genetic markers have been previously identified that influence levels of transferrin, the protein that transports iron throughout the body, in the blood and brain. Here, we discovered that transferrin levels are related to detectable differences in the macro- and microstructure of the living brain. We collected brain MRI scans from 615 healthy young adult twins and siblings, of whom 574 were also scanned with diffusion tensor imaging at 4 Tesla. Fiber integrity was assessed by using the diffusion tensor imaging-based measure of fractional anisotropy. In bivariate genetic models based on monozygotic and dizygotic twins, we discovered that partially overlapping additive genetic factors influenced transferrin levels and brain microstructure. We also examined common variants in genes associated with transferrin levels, TF and HFE, and found that a commonly carried polymorphism (H63D at rs1799945) in the hemochromatotic HFE gene was associated with white matter fiber integrity. This gene has a well documented association with iron overload. Our statistical maps reveal previously unknown influences of the same gene on brain microstructure and transferrin levels. This discovery may shed light on the neural mechanisms by which iron affects cognition, neurodevelopment, and neurodegeneration.

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Mechanism of proton/substrate coupling in the heptahelical lysosomal transporter cystinosin.

Publication Date: 2012 Jan 9 PMID: 22232659
Authors: Ruivo, R. - Bellenchi, G. C. - Chen, X. - Zifarelli, G. - Sagne, C. - Debacker, C. - Pusch, M. - Supplisson, S. - Gasnier, B.
Journal: Proc Natl Acad Sci U S A

Secondary active transporters use electrochemical gradients provided by primary ion pumps to translocate metabolites or drugs "uphill" across membranes. Here we report the ion-coupling mechanism of cystinosin, an unusual eukaryotic, proton-driven transporter distantly related to the proton pump bacteriorhodopsin. In humans, cystinosin exports the proteolysis-derived dimeric amino acid cystine from lysosomes and is impaired in cystinosis. Using voltage-dependence analysis of steady-state and transient currents elicited by cystine and neutralization-scanning mutagenesis of conserved protonatable residues, we show that cystine binding is coupled to protonation of a clinically relevant aspartate buried in the membrane. Deuterium isotope substitution experiments are consistent with an access of this aspartate from the lysosomal lumen through a deep proton channel. This aspartate lies in one of the two PQ-loop motifs shared by cystinosin with a set of eukaryotic membrane proteins of unknown function and is conserved in about half of them, thus suggesting that other PQ-loop proteins may translocate protons.

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Interplay between mismatch repair and chromatin assembly.

Publication Date: 2012 Jan 9 PMID: 22232658
Authors: Schopf, B. - Bregenhorn, S. - Quivy, J. P. - Kadyrov, F. A. - Almouzni, G. - Jiricny, J.
Journal: Proc Natl Acad Sci U S A

Single strand nicks and gaps in DNA have been reported to increase the efficiency of nucleosome loading mediated by chromatin assembly factor 1 (CAF-1). However, on mismatch-containing substrates, these strand discontinuities are utilized by the mismatch repair (MMR) system as loading sites for exonuclease 1, at which degradation of the error-containing strand commences. Because packaging of DNA into chromatin might inhibit MMR, we were interested to learn whether chromatin assembly is differentially regulated on heteroduplex and homoduplex substrates. We now show that the presence of a mismatch in a nicked plasmid substrate delays nucleosome loading in human cell extracts. Our data also suggest that, once the mismatch is removed, repair of the single-stranded gap is accompanied by efficient nucleosome loading. We postulated that the balance between MMR and chromatin assembly might be governed by proliferating cell nuclear antigen (PCNA), the processivity factor of replicative DNA polymerases, which is loaded at DNA termini and which interacts with the MSH6 subunit of the mismatch recognition factor MutSalpha, as well as with CAF-1. We now show that this regulation might be more complex; MutSalpha and CAF-1 interact not only with PCNA, but also with each other. In vivo this interaction increases during S-phase and may be controlled by the phosphorylation status of the p150 subunit of CAF-1.

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Distinct conformations of the protein complex p97-Ufd1-Npl4 revealed by electron cryomicroscopy.

Publication Date: 2012 Jan 9 PMID: 22232657
Authors: Bebeacua, C. - Forster, A. - McKeown, C. - Meyer, H. H. - Zhang, X. - Freemont, P. S.
Journal: Proc Natl Acad Sci U S A

p97 is a key regulator of numerous cellular pathways and associates with ubiquitin-binding adaptors to remodel ubiquitin-modified substrate proteins. How adaptor binding to p97 is coordinated and how adaptors contribute to substrate remodeling is unclear. Here we present the 3D electron cryomicroscopy reconstructions of the major Ufd1-Npl4 adaptor in complex with p97. Our reconstructions show that p97-Ufd1-Npl4 is highly dynamic and that Ufd1-Npl4 assumes distinct positions relative to the p97 ring upon addition of nucleotide. Our results suggest a model for substrate remodeling by p97 and also explains how p97-Ufd1-Npl4 could form other complexes in a hierarchical model of p97-cofactor assembly.

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Reconciling long-term cultural diversity and short-term collective social behavior.

Publication Date: 2012 Jan 9 PMID: 22232656
Authors: Valori, L. - Picciolo, F. - Allansdottir, A. - Garlaschelli, D.
Journal: Proc Natl Acad Sci U S A

An outstanding open problem is whether collective social phenomena occurring over short timescales can systematically reduce cultural heterogeneity in the long run, and whether offline and online human interactions contribute differently to the process. Theoretical models suggest that short-term collective behavior and long-term cultural diversity are mutually excluding, since they require very different levels of social influence. The latter jointly depends on two factors: the topology of the underlying social network and the overlap between individuals in multidimensional cultural space. However, while the empirical properties of social networks are intensively studied, little is known about the large-scale organization of real societies in cultural space, so that random input specifications are necessarily used in models. Here we use a large dataset to perform a high-dimensional analysis of the scientific beliefs of thousands of Europeans. We find that interopinion correlations determine a nontrivial ultrametric hierarchy of individuals in cultural space. When empirical data are used as inputs in models, ultrametricity has strong and counterintuitive effects. On short timescales, it facilitates a symmetry-breaking phase transition triggering coordinated social behavior. On long timescales, it suppresses cultural convergence by restricting it within disjoint groups. Moreover, ultrametricity implies that these results are surprisingly robust to modifications of the dynamical rules considered. Thus the empirical distribution of individuals in cultural space appears to systematically optimize the coexistence of short-term collective behavior and long-term cultural diversity, which can be realized simultaneously for the same moderate level of mutual influence in a diverse range of online and offline settings.

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Osmotic spreading of Bacillus subtilis biofilms driven by an extracellular matrix.

Publication Date: 2012 Jan 9 PMID: 22232655
Authors: Seminara, A. - Angelini, T. E. - Wilking, J. N. - Vlamakis, H. - Ebrahim, S. - Kolter, R. - Weitz, D. A. - Brenner, M. P.
Journal: Proc Natl Acad Sci U S A

Bacterial biofilms are organized communities of cells living in association with surfaces. The hallmark of biofilm formation is the secretion of a polymeric matrix rich in sugars and proteins in the extracellular space. In Bacillus subtilis, secretion of the exopolysaccharide (EPS) component of the extracellular matrix is genetically coupled to the inhibition of flagella-mediated motility. The onset of this switch results in slow expansion of the biofilm on a substrate. Different strains have radically different capabilities in surface colonization: Flagella-null strains spread at the same rate as wild type, while both are dramatically faster than EPS mutants. Multiple functions have been attributed to the EPS, but none of these provides a physical mechanism for generating spreading. We propose that the secretion of EPS drives surface motility by generating osmotic pressure gradients in the extracellular space. A simple mathematical model based on the physics of polymer solutions shows quantitative agreement with experimental measurements of biofilm growth, thickening, and spreading. We discuss the implications of this osmotically driven type of surface motility for nutrient uptake that may elucidate the reduced fitness of the matrix-deficient mutant strains.

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Translational inhibition by deadenylation-independent mechanisms is central to microRNA-mediated silencing in zebrafish.

Publication Date: 2012 Jan 9 PMID: 22232654
Authors: Mishima, Y. - Fukao, A. - Kishimoto, T. - Sakamoto, H. - Fujiwara, T. - Inoue, K.
Journal: Proc Natl Acad Sci U S A

MicroRNA (miRNA) is a class of small noncoding RNA approximately 22 nt in length. Animal miRNA silences complementary mRNAs via translational inhibition, deadenylation, and mRNA degradation. However, the underlying molecular mechanisms remain unclear. A key question is whether these three outputs are independently induced by miRNA through distinct mechanisms or sequentially induced within a single molecular pathway. Here, we successfully dissected these intricate outputs of miRNA-mediated repression using zebrafish embryos as a model system. Our results indicate that translational inhibition and deadenylation are independent outputs mediated by distinct domains of TNRC6A, which is an effector protein in the miRNA pathway. Translational inhibition by TNRC6A is divided into two mechanisms: PAM2 motif-mediated interference of poly(A)-binding protein (PABP), and inhibition of 5' cap- and poly(A) tail-independent step(s) by a previously undescribed P-GL motif. Consistent with these observations, we show that, in zebrafish embryos, miRNA inhibits translation of the target mRNA in a deadenylation- and PABP-independent manner at early time points. These results indicate that miRNA exerts multiple posttranscriptional outputs via physically and functionally independent mechanisms and that direct translational inhibition is central to miRNA-mediated repression.

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Quasi-liquid layers on ice crystal surfaces are made up of two different phases.

Publication Date: 2012 Jan 9 PMID: 22232653
Authors: Sazaki, G. - Zepeda, S. - Nakatsubo, S. - Yokomine, M. - Furukawa, Y.
Journal: Proc Natl Acad Sci U S A

Ice plays crucially important roles in various phenomena because of its abundance on Earth. However, revealing the dynamic behavior of quasi-liquid layers (QLLs), which governs the surface properties of ice crystals at temperatures near the melting point, remains an experimental challenge. Here we show that two types of QLL phases appear that exhibit different morphologies and dynamics. We directly visualized the two types of QLLs on ice crystal surfaces by advanced optical microscopy, which can visualize the individual 0.37-nm-thick elementary steps on ice crystal surfaces. We found that they had different stabilities and different interactions with ice crystal surfaces. The two immiscible QLL phases appeared heterogeneously, moved around, and coalesced dynamically on ice crystal surfaces. This picture of surface melting is quite different from the conventional picture in which one QLL phase appears uniformly on ice crystal surfaces.

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Structure of ice crystallized from supercooled water.

Publication Date: 2012 Jan 9 PMID: 22232652
Authors: Malkin, T. L. - Murray, B. J. - Brukhno, A. V. - Anwar, J. - Salzmann, C. G.
Journal: Proc Natl Acad Sci U S A

The freezing of water to ice is fundamentally important to fields as diverse as cloud formation to cryopreservation. At ambient conditions, ice is considered to exist in two crystalline forms: stable hexagonal ice and metastable cubic ice. Using X-ray diffraction data and Monte Carlo simulations, we show that ice that crystallizes homogeneously from supercooled water is neither of these phases. The resulting ice is disordered in one dimension and therefore possesses neither cubic nor hexagonal symmetry and is instead composed of randomly stacked layers of cubic and hexagonal sequences. We refer to this ice as stacking-disordered ice I. Stacking disorder and stacking faults have been reported earlier for metastable ice I, but only for ice crystallizing in mesopores and in samples recrystallized from high-pressure ice phases rather than in water droplets. Review of the literature reveals that almost all ice that has been identified as cubic ice in previous diffraction studies and generated in a variety of ways was most likely stacking-disordered ice I with varying degrees of stacking disorder. These findings highlight the need to reevaluate the physical and thermodynamic properties of this metastable ice as a function of the nature and extent of stacking disorder using well-characterized samples.

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Structural basis for membrane targeting by the MVB12-associated beta-prism domain of the human ESCRT-I MVB12 subunit.

Publication Date: 2012 Jan 9 PMID: 22232651
Authors: Boura, E. - Hurley, J. H.
Journal: Proc Natl Acad Sci U S A

MVB12-associated beta-prism (MABP) domains are predicted to occur in a diverse set of membrane-associated bacterial and eukaryotic proteins, but their existence, structure, and biochemical properties have not been characterized experimentally. Here, we find that the MABP domains of the MVB12A and B subunits of ESCRT-I are functional modules that bind in vitro to liposomes containing acidic lipids depending on negative charge density. The MABP domain is capable of autonomously localizing to subcellular puncta and to the plasma membrane. The 1.3-A atomic resolution crystal structure of the MVB12B MABP domain reveals a beta-prism fold, a hydrophobic membrane-anchoring loop, and an electropositive phosphoinositide-binding patch. The basic patch is open, which explains how it senses negative charge density but lacks stereoselectivity. These observations show how ESCRT-I could act as a coincidence detector for acidic phospholipids and protein ligands, enabling it to function both in protein transport at endosomes and in cytokinesis and viral budding at the plasma membrane.

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Collective and single cell behavior in epithelial contact inhibition.

Publication Date: 2012 Jan 6 PMID: 22228306
Authors: Puliafito, A. - Hufnagel, L. - Neveu, P. - Streichan, S. - Sigal, A. - Fygenson, D. K. - Shraiman, B. I.
Journal: Proc Natl Acad Sci U S A

Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing, and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as "contact inhibition." The study presented here provides a quantitative characterization of contact inhibition dynamics on tissue-wide and single cell levels. Using long-term tracking of cultured Madin-Darby canine kidney cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size, and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition.

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Full-length myosin Va exhibits altered gating during processive movement on actin.

Publication Date: 2012 Jan 6 PMID: 22228305
Authors: Armstrong, J. M. - Krementsova, E. - Michalek, A. J. - Heaslip, A. T. - Nelson, S. R. - Trybus, K. M. - Warshaw, D. M.
Journal: Proc Natl Acad Sci U S A

Myosin Va (myoV) is a processive molecular motor that transports intracellular cargo along actin tracks with each head taking multiple 72-nm hand-over-hand steps. This stepping behavior was observed with a constitutively active, truncated myoV, in which the autoinhibitory interactions between the globular tail and motor domains (i.e., heads) that regulate the full-length molecule no longer exist. Without cargo at near physiologic ionic strength (100 mM KCl), full-length myoV adopts a folded (approximately 15 S), enzymatically-inhibited state that unfolds to an extended (approximately 11 S), active conformation at higher salt (250 mM). Under conditions favoring the folded, inhibited state, we show that Quantum-dot-labeled myoV exhibits two types of interaction with actin in the presence of MgATP. Most motors bind to actin and remain stationary, but surprisingly, approximately 20% are processive. The moving motors transition between a strictly gated and hand-over-hand stepping pattern typical of a constitutively active motor, and a new mode with a highly variable stepping pattern suggestive of altered gating. Each head of this partially inhibited motor takes longer-lived, short forward (35 nm) and backward (28 nm) steps, presumably due to globular tail-head interactions that modify the gating of the individual heads. This unique mechanical state may be an intermediate in the pathway between the inhibited and active states of the motor.

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Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism.

Publication Date: 2012 Jan 6 PMID: 22228304
Authors: Cassago, A. - Ferreira, A. P. - Ferreira, I. M. - Fornezari, C. - Gomes, E. R. - Greene, K. S. - Pereira, H. M. - Garratt, R. C. - Dias, S. M. - Ambrosio, A. L.
Journal: Proc Natl Acad Sci U S A

Glutamine is an essential nutrient for cancer cell proliferation, especially in the context of citric acid cycle anaplerosis. In this manuscript we present results that collectively demonstrate that, of the three major mammalian glutaminases identified to date, the lesser studied splice variant of the gene gls, known as Glutaminase C (GAC), is important for tumor metabolism. We show that, although levels of both the kidney-type isoforms are elevated in tumor vs. normal tissues, GAC is distinctly mitochondrial. GAC is also most responsive to the activator inorganic phosphate, the content of which is supposedly higher in mitochondria subject to hypoxia. Analysis of X-ray crystal structures of GAC in different bound states suggests a mechanism that introduces the tetramerization-induced lifting of a "gating loop" as essential for the phosphate-dependent activation process. Surprisingly, phosphate binds inside the catalytic pocket rather than at the oligomerization interface. Phosphate also mediates substrate entry by competing with glutamate. A greater tendency to oligomerize differentiates GAC from its alternatively spliced isoform and the cycling of phosphate in and out of the active site distinguishes it from the liver-type isozyme, which is known to be less dependent on this ion.

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p63-microRNA feedback in keratinocyte senescence.

Publication Date: 2012 Jan 6 PMID: 22228303
Authors: Cervo, P. R. - Lena, A. M. - Nicoloso, M. - Rossi, S. - Mancini, M. - Zhou, H. - Saintigny, G. - Dellambra, E. - Odorisio, T. - Mahe, C. - Calin, G. A. - Candi, E. - Melino, G.
Journal: Proc Natl Acad Sci U S A

We investigated the expression of microRNAs (miRNAs) associated with replicative senescence in human primary keratinocytes. A cohort of miRNAs up-regulated in senescence was identified by genome-wide miRNA profiling, and their change in expression was validated in proliferative versus senescent cells. Among these, miRNA (miR)-138, -181a, -181b, and -130b expression increased with serial passages. miR-138, -181a, and -181b, but not miR-130b, overexpression in proliferating cells was sufficient per se to induce senescence, as evaluated by inhibition of BrdU incorporation and quantification of senescence-activated beta-galactosidase staining. We identified Sirt1 as a direct target of miR-138, -181a, and -181b, whereas DeltaNp63 expression was inhibited by miR-130b. We also found that DeltaNp63alpha inhibits miR-138, -181a, -181b, and -130b expression by binding directly to p63-responsive elements located in close proximity to the genomic loci of these miRNAs in primary keratinocytes. These findings suggest that changes in miRNA expression, by modulating the levels of regulatory proteins such as p63 and Sirt1, strongly contribute to induction of senescence in primary human keratinocytes, thus linking these two proteins. Our data also indicate that suppression of miR-138, -181a, -181b, and -130b expression is part of a growth-promoting strategy of DeltaNp63alpha in epidermal proliferating cells.

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Drosophila RNA polymerase III repressor Maf1 controls body size and developmental timing by modulating tRNAiMet synthesis and systemic insulin signaling.

Publication Date: 2012 Jan 6 PMID: 22228302
Authors: Rideout, E. J. - Marshall, L. - Grewal, S. S.
Journal: Proc Natl Acad Sci U S A

The target-of-rapamycin pathway couples nutrient availability with tissue and organismal growth in metazoans. The key effectors underlying this growth are, however, unclear. Here we show that Maf1, a repressor of RNA polymerase III-dependent tRNA transcription, is an important mediator of nutrient-dependent growth in Drosophila. We find nutrients promote tRNA synthesis during larval development by inhibiting Maf1. Genetic inhibition of Maf1 accelerates development and increases body size. These phenotypes are due to a non-cell-autonomous effect of Maf1 inhibition in the fat body, the main larval endocrine organ. Inhibiting Maf1 in the fat body increases growth by promoting the expression of brain-derived insulin-like peptides and consequently enhanced systemic insulin signaling. Remarkably, the effects of Maf1 inhibition are reproduced in flies carrying one extra copy of the initiator methionine tRNA, tRNA(i)(Met). These findings suggest the stimulation of tRNA(i)(Met) synthesis via inhibition of dMaf1 is limiting for nutrition-dependent growth during development.

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Extensive chromosomal variation in a recently formed natural allopolyploid species, Tragopogon miscellus (Asteraceae).

Publication Date: 2012 Jan 6 PMID: 22228301
Authors: Chester, M. - Gallagher, J. P. - Symonds, V. V. - Cruz da Silva, A. V. - Mavrodiev, E. V. - Leitch, A. R. - Soltis, P. S. - Soltis, D. E.
Journal: Proc Natl Acad Sci U S A

Polyploidy, or whole genome duplication, has played a major role in the evolution of many eukaryotic lineages. Although the prevalence of polyploidy in plants is well documented, the molecular and cytological consequences are understood largely from newly formed polyploids (neopolyploids) that have been grown experimentally. Classical cytological and molecular cytogenetic studies both have shown that experimental neoallopolyploids often have meiotic irregularities, producing chromosomally variable gametes and progeny; however, little is known about the extent or duration of chromosomal variation in natural neoallopolyploid populations. We report the results of a molecular cytogenetic study on natural populations of a neoallopolyploid, Tragopogon miscellus, which formed multiple times in the past 80 y. Using genomic and fluorescence in situ hybridization, we uncovered massive and repeated patterns of chromosomal variation in all populations. No population was fixed for a particular karyotype; 76% of the individuals showed intergenomic translocations, and 69% were aneuploid for one or more chromosomes. Importantly, 85% of plants exhibiting aneuploidy still had the expected chromosome number, mostly through reciprocal monosomy-trisomy of homeologous chromosomes (1:3 copies) or nullisomy-tetrasomy (0:4 copies). The extensive chromosomal variation still present after ca. 40 generations in this biennial species suggests that substantial and prolonged chromosomal instability might be common in natural populations after whole genome duplication. A protracted period of genome instability in neoallopolyploids may increase opportunities for alterations to genome structure, losses of coding and noncoding DNA, and changes in gene expression.

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Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f microRNA and Wnt/beta-catenin signaling.

Publication Date: 2012 Jan 5 PMID: 22223664
Authors: Egea, V. - Zahler, S. - Rieth, N. - Neth, P. - Popp, T. - Kehe, K. - Jochum, M. - Ries, C.
Journal: Proc Natl Acad Sci U S A

Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a matrix metalloproteinase (MMP)-independent regulator of growth and apoptosis in various cell types. The receptors and signaling pathways that are involved in the growth factor activities of TIMP-1, however, remain controversial. RNA interference of TIMP-1 has revealed that endogenous TIMP-1 suppresses the proliferation, metabolic activity, and osteogenic differentiation capacity of human mesenchymal stem cells (hMSCs). The knockdown of TIMP-1 in hMSCs activated the Wnt/beta-catenin signaling pathway as indicated by the increased stability and nuclear localization of beta-catenin in TIMP-1-deficient hMSCs. Moreover, TIMP-1 knockdown cells exhibited enhanced beta-catenin transcriptional activity, determined by Wnt/beta-catenin target gene expression analysis and a luciferase-based beta-catenin-activated reporter assay. An analysis of a mutant form of TIMP-1 that cannot inhibit MMP indicated that the effect of TIMP-1 on beta-catenin signaling is MMP independent. Furthermore, the binding of CD63 to TIMP-1 on the surface of hMSCs is essential for the TIMP-1-mediated effects on Wnt/beta-catenin signaling. An array analysis of microRNAs (miRNAs) and transfection studies with specific miRNA inhibitors and mimics showed that let-7f miRNA is crucial for the regulation of beta-catenin activity and osteogenic differentiation by TIMP-1. Let-7f was up-regulated in TIMP-1-depleted hMSCs and demonstrably reduced axin 2, an antagonist of beta-catenin stability. Our results demonstrate that TIMP-1 is a direct regulator of hMSC functions and reveal a regulatory network in which let-7f modulates Wnt/beta-catenin activity.

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Compensatory functions of histone deacetylase 1 (HDAC1) and HDAC2 regulate transcription and apoptosis during mouse oocyte development.

Publication Date: 2012 Jan 5 PMID: 22223663
Authors: Ma, P. - Pan, H. - Montgomery, R. L. - Olson, E. N. - Schultz, R. M.
Journal: Proc Natl Acad Sci U S A

Dramatic changes in chromatin structure and histone modification occur during oocyte growth, as well as a global cessation of transcription. The role of histone modifications in these processes is poorly understood. We report the effect of conditionally deleting Hdac1 and Hdac2 on oocyte development. Deleting either gene has little or no effect on oocyte development, whereas deleting both genes results in follicle development arrest at the secondary follicle stage. This developmental arrest is accompanied by substantial perturbation of the transcriptome and a global reduction in transcription even though histone acetylation is markedly increased. There is no apparent change in histone repressive marks, but there is a pronounced decrease in histone H3K4 methylation, an activating mark. The decrease in H3K4 methylation is likely a result of increased expression of Kdm5b because RNAi-mediated targeting of Kdm5b in double-mutant oocytes results in an increase in H3K4 methylation. An increase in TRP53 acetylation also occurs in mutant oocytes and may contribute to the observed increased incidence of apoptosis. Taken together, these results suggest seminal roles of acetylation of histone and nonhistone proteins in oocyte development.

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The mystery of missing heritability: Genetic interactions create phantom heritability.

Publication Date: 2012 Jan 5 PMID: 22223662
Authors: Zuk, O. - Hechter, E. - Sunyaev, S. R. - Lander, E. S.
Journal: Proc Natl Acad Sci U S A

Human genetics has been haunted by the mystery of "missing heritability" of common traits. Although studies have discovered >1,200 variants associated with common diseases and traits, these variants typically appear to explain only a minority of the heritability. The proportion of heritability explained by a set of variants is the ratio of (i) the heritability due to these variants (numerator), estimated directly from their observed effects, to (ii) the total heritability (denominator), inferred indirectly from population data. The prevailing view has been that the explanation for missing heritability lies in the numerator-that is, in as-yet undiscovered variants. While many variants surely remain to be found, we show here that a substantial portion of missing heritability could arise from overestimation of the denominator, creating "phantom heritability." Specifically, (i) estimates of total heritability implicitly assume the trait involves no genetic interactions (epistasis) among loci; (ii) this assumption is not justified, because models with interactions are also consistent with observable data; and (iii) under such models, the total heritability may be much smaller and thus the proportion of heritability explained much larger. For example, 80% of the currently missing heritability for Crohn's disease could be due to genetic interactions, if the disease involves interaction among three pathways. In short, missing heritability need not directly correspond to missing variants, because current estimates of total heritability may be significantly inflated by genetic interactions. Finally, we describe a method for estimating heritability from isolated populations that is not inflated by genetic interactions.

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Distinct influences of peptide-MHC quality and quantity on in vivo T-cell responses.

Publication Date: 2012 Jan 5 PMID: 22223661
Authors: Gottschalk, R. A. - Hathorn, M. M. - Beuneu, H. - Corse, E. - Dustin, M. L. - Altan-Bonnet, G. - Allison, J. P.
Journal: Proc Natl Acad Sci U S A

The strength of T-cell receptor (TCR) stimulation and subsequent T-cell response depend on a combination of peptide-major histocompatibility complex (pMHC) density and potency. By comparing two different pMHC at doses yielding similar proliferation in vivo, we have highlighted unexpected differences in the qualitative and quantitative effects of TCR ligand. Measurements of cytokine sensitivity and two-photon imaging of T cell-dendritic cell (T-DC) interactions reveal discrimination between comparably weak stimuli resulting from either decreased pMHC potency or pMHC density. In addition, TCR-induced genes in broad gene expression profiles segregate into two groups: one that responds to cumulative TCR signal and another that responds to pMHC quality, independent of quantity. These observations suggest that models of TCR ligand discrimination must account for disparate sensitivity of downstream responses to specific influences of pMHC potency.

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14-3-3 fusion oncogenes in high-grade endometrial stromal sarcoma.

Publication Date: 2012 Jan 5 PMID: 22223660
Authors: Lee, C. H. - Ou, W. B. - Marino-Enriquez, A. - Zhu, M. - Mayeda, M. - Wang, Y. - Guo, X. - Brunner, A. L. - Amant, F. - French, C. A. - West, R. B. - McAlpine, J. N. - Gilks, C. B. - Yaffe, M. B. - Prentice, L. M. - McPherson, A. - Jones, S. J. - Marra, M. A. - Shah, S. P. - van de Rijn, M. - Huntsman, D. G. - Dal Cin, P. - Debiec-Rychter, M. - Nucci, M. R. - Fletcher, J. A.
Journal: Proc Natl Acad Sci U S A

14-3-3 proteins are ubiquitously expressed regulators of various cellular functions, including proliferation, metabolism, and differentiation, and altered 14-3-3 expression is associated with development and progression of cancer. We report a transforming 14-3-3 oncoprotein, which we identified through conventional cytogenetics and whole-transcriptome sequencing analysis as a highly recurrent genetic mechanism in a clinically aggressive form of uterine sarcoma: high-grade endometrial stromal sarcoma (ESS). The 14-3-3 oncoprotein results from a t(10;17) genomic rearrangement, leading to fusion between 14-3-3epsilon (YWHAE) and either of two nearly identical FAM22 family members (FAM22A or FAM22B). Expression of YWHAE-FAM22 fusion oncoproteins was demonstrated by immunoblot in t(10;17)-bearing frozen tumor and cell line samples. YWHAE-FAM22 fusion gene knockdowns were performed with shRNAs and siRNAs targeting various FAM22A exons in an t(10;17)-bearing ESS cell line (ESS1): Fusion protein expression was inhibited, with corresponding reduction in cell growth and migration. YWHAE-FAM22 maintains a structurally and functionally intact 14-3-3epsilon (YWHAE) protein-binding domain, which is directed to the nucleus by a FAM22 nuclear localization sequence. In contrast to classic ESS, harboring JAZF1 genetic fusions, YWHAE-FAM22 ESS display high-grade histologic features, a distinct gene-expression profile, and a more aggressive clinical course. Fluorescence in situ hybridization analysis demonstrated absolute specificity of YWHAE-FAM22A/B genetic rearrangement for high-grade ESS, with no fusions detected in other uterine and nonuterine mesenchymal tumors (55 tumor types, n = 827). These discoveries reveal diagnostically and therapeutically relevant models for characterizing aberrant 14-3-3 oncogenic functions.

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Forkhead transcription factor FoxA1 regulates sweat secretion through Bestrophin 2 anion channel and Na-K-Cl cotransporter 1.

Publication Date: 2012 Jan 5 PMID: 22223659
Authors: Cui, C. Y. - Childress, V. - Piao, Y. - Michel, M. - Johnson, A. A. - Kunisada, M. - Ko, M. S. - Kaestner, K. H. - Marmorstein, A. D. - Schlessinger, D.
Journal: Proc Natl Acad Sci U S A

Body temperature is maintained in a narrow range in mammals, primarily controlled by sweating. In humans, the dynamic thermoregulatory organ, comprised of 2-4 million sweat glands distributed over the body, can secrete up to 4 L of sweat per day, thereby making it possible to withstand high temperatures and endure prolonged physical stress (e.g., long-distance running). The genetic basis for sweat gland function, however, is largely unknown. We find that the forkhead transcription factor, FoxA1, is required to generate mouse sweating capacity. Despite continued sweat gland morphogenesis, ablation of FoxA1 in mice results in absolute anihidrosis (lack of sweating). This inability to sweat is accompanied by down-regulation of the Na-K-Cl cotransporter 1 (Nkcc1) and the Ca(2+)-activated anion channel Bestrophin 2 (Best2), as well as glycoprotein accumulation in gland lumens and ducts. Furthermore, Best2-deficient mice display comparable anhidrosis and glycoprotein accumulation. These findings link earlier observations that both sodium/potassium/chloride exchange and Ca(2+) are required for sweat production. FoxA1 is inferred to regulate two corresponding features of sweat secretion. One feature, via Best2, catalyzes a bicarbonate gradient that could help to drive calcium-associated ionic transport; the other, requiring Nkcc1, facilitates monovalent ion exchange into sweat. These mechanistic components can be pharmaceutical targets to defend against hyperthermia and alleviate defective thermoregulation in the elderly, and may provide a model relevant to more complex secretory processes.

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Correction for Kogan et al., Thin-slicing study of the oxytocin receptor (OXTR) gene and the evaluation and expression of the prosocial disposition.

Publication Date: 2012 Jan 5 PMID: 22223658
Authors:
Journal: Proc Natl Acad Sci U S A

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Correction for Zhang et al., Meiotic double-strand breaks occur once per pair of (sister) chromatids and, viaMec1/ATRand Tel1/ATM, once per quartet of chromatids.

Publication Date: 2012 Jan 5 PMID: 22223657
Authors:
Journal: Proc Natl Acad Sci U S A

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Crystal structures of (Mg1-x,Fex)SiO3postperovskite at high pressures.

Publication Date: 2012 Jan 5 PMID: 22223656
Authors: Yamanaka, T. - Hirose, K. - Mao, W. L. - Meng, Y. - Ganesh, P. - Shulenburger, L. - Shen, G. - Hemley, R. J.
Journal: Proc Natl Acad Sci U S A

X-ray diffraction experiments on postperovskite (ppv) with compositions (Mg(0.9)Fe(0.1))SiO(3) and (Mg(0.6)Fe(0.4))SiO(3) at Earth core-mantle boundary pressures reveal different crystal structures. The former adopts the CaIrO(3)-type structure with space group Cmcm, whereas the latter crystallizes in a structure with the Pmcm (Pmma) space group. The latter has a significantly higher density (rho = 6.119(1) g/cm(3)) than the former (rho = 5.694(8) g/cm(3)) due to both the larger amount of iron and the smaller ionic radius of Fe(2+) as a result of an electronic spin transition observed by X-ray emission spectroscopy (XES). The smaller ionic radius for low-spin compared to high-spin Fe(2+) also leads to an ordered cation distribution in the M1 and M2 crystallographic sites of the higher density ppv structure. Rietveld structure refinement indicates that approximately 70% of the total Fe(2+) in that phase occupies the M2 site. XES results indicate a loss of 70% of the unpaired electronic spins consistent with a low spin M2 site and high spin M1 site. First-principles calculations of the magnetic ordering confirm that Pmcm with a two-site model is energetically more favorable at high pressure, and predict that the ordered structure is anisotropic in its electrical and elastic properties. These results suggest that interpretations of seismic structure in the deep mantle need to treat a broader range of mineral structures than previously considered.

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NaV1.1 channels are critical for intercellular communication in the suprachiasmatic nucleus and for normal circadian rhythms.

Publication Date: 2012 Jan 5 PMID: 22223655
Authors: Han, S. - Yu, F. H. - Schwartz, M. D. - Linton, J. D. - Bosma, M. M. - Hurley, J. B. - Catterall, W. A. - de la Iglesia, H. O.
Journal: Proc Natl Acad Sci U S A

Na(V)1.1 is the primary voltage-gated Na(+) channel in several classes of GABAergic interneurons, and its reduced activity leads to reduced excitability and decreased GABAergic tone. Here, we show that Na(V)1.1 channels are expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus. Mice carrying a heterozygous loss of function mutation in the Scn1a gene (Scn1a(+/-)), which encodes the pore-forming alpha-subunit of the Na(V)1.1 channel, have longer circadian period than WT mice and lack light-induced phase shifts. In contrast, Scn1a(+/-) mice have exaggerated light-induced negative-masking behavior and normal electroretinogram, suggesting an intact retina light response. Scn1a(+/-) mice show normal light induction of c-Fos and mPer1 mRNA in ventral SCN but impaired gene expression responses in dorsal SCN. Electrical stimulation of the optic chiasm elicits reduced calcium transients and impaired ventro-dorsal communication in SCN neurons from Scn1a(+/-) mice, and this communication is barely detectable in the homozygous gene KO (Scn1a(-/-)). Enhancement of GABAergic transmission with tiagabine plus clonazepam partially rescues the effects of deletion of Na(V)1.1 on circadian period and phase shifting. Our report demonstrates that a specific voltage-gated Na(+) channel and its associated impairment of SCN interneuronal communication lead to major deficits in the function of the master circadian pacemaker. Heterozygous loss of Na(V)1.1 channels is the underlying cause for severe myoclonic epilepsy of infancy; the circadian deficits that we report may contribute to sleep disorders in severe myoclonic epilepsy of infancy patients.

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Correction for Zhang et al., Duplication and partitioning in evolution and function of homoeologous Q loci governing domestication characters in polyploid wheat.

Publication Date: 2012 Jan 5 PMID: 22223654
Authors:
Journal: Proc Natl Acad Sci U S A

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Culture does account for variation in game behavior.

Publication Date: 2012 Jan 10 PMID: 22219355
Authors: Henrich, J. - Boyd, R. - McElreath, R. - Gurven, M. - Richerson, P. J. - Ensminger, J. - Alvard, M. - Barr, A. - Barrett, C. - Bolyanatz, A. - Camerer, C. F. - Cardenas, J. C. - Fehr, E. - Gintis, H. M. - Gil-White, F. - Gwako, E. L. - Henrich, N. - Hill, K. - Lesorogol, C. - Patton, J. Q. - Marlowe, F. W. - Tracer, D. P. - Ziker, J.
Journal: Proc Natl Acad Sci U S A

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Double-strand break motions shift radiation risk notions?

Publication Date: 2012 Jan 10 PMID: 22210116
Authors: Hlatky, L.
Journal: Proc Natl Acad Sci U S A

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More with less Xist.

Publication Date: 2012 Jan 10 PMID: 22205767
Authors: Wells, K. D.
Journal: Proc Natl Acad Sci U S A

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Air quality implications of the Deepwater Horizon oil spill.

Publication Date: 2012 Jan 5 PMID: 22205764
Authors: Middlebrook, A. M. - Murphy, D. M. - Ahmadov, R. - Atlas, E. L. - Bahreini, R. - Blake, D. R. - Brioude, J. - de Gouw, J. A. - Fehsenfeld, F. C. - Frost, G. J. - Holloway, J. S. - Lack, D. A. - Langridge, J. M. - Lueb, R. A. - McKeen, S. A. - Meagher, J. F. - Meinardi, S. - Neuman, J. A. - Nowak, J. B. - Parrish, D. D. - Peischl, J. - Perring, A. E. - Pollack, I. B. - Roberts, J. M. - Ryerson, T. B. - Schwarz, J. P. - Spackman, J. R. - Warneke, C. - Ravishankara, A. R.
Journal: Proc Natl Acad Sci U S A

During the Deepwater Horizon (DWH) oil spill, a wide range of gas and aerosol species were measured from an aircraft around, downwind, and away from the DWH site. Additional hydrocarbon measurements were made from ships in the vicinity. Aerosol particles of respirable sizes were on occasions a significant air quality issue for populated areas along the Gulf Coast. Yields of organic aerosol particles and emission factors for other atmospheric pollutants were derived for the sources from the spill, recovery, and cleanup efforts. Evaporation and subsequent secondary chemistry produced organic particulate matter with a mass yield of 8 +/- 4% of the oil mixture reaching the water surface. Approximately 4% by mass of oil burned on the surface was emitted as soot particles. These yields can be used to estimate the effects on air quality for similar events as well as for this spill at other times without these data. Whereas emission of soot from burning surface oil was large during the episodic burns, the mass flux of secondary organic aerosol to the atmosphere was substantially larger overall. We use a regional air quality model to show that some observed enhancements in organic aerosol concentration along the Gulf Coast were likely due to the DWH spill. In the presence of evaporating hydrocarbons from the oil, NO(x) emissions from the recovery and cleanup operations produced ozone.

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Rate of meristem maturation determines inflorescence architecture in tomato.

Publication Date: 2012 Jan 10 PMID: 22203998
Authors: Park, S. J. - Jiang, K. - Schatz, M. C. - Lippman, Z. B.
Journal: Proc Natl Acad Sci U S A

Flower production and crop yields are highly influenced by the architectures of inflorescences. In the compound inflorescences of tomato and related nightshades (Solanaceae), new lateral inflorescence branches develop on the flanks of older branches that have terminated in flowers through a program of plant growth known as "sympodial." Variability in the number and organization of sympodial branches produces a remarkable array of inflorescence architectures, but little is known about the mechanisms underlying sympodial growth and branching diversity. One hypothesis is that the rate of termination modulates branching. By performing deep sequencing of transcriptomes, we have captured gene expression dynamics from individual shoot meristems in tomato as they gradually transition from a vegetative state to a terminal flower. Surprisingly, we find thousands of age-dependent expression changes, even when there is little change in meristem morphology. From these data, we reveal that meristem maturation is an extremely gradual process defined molecularly by a "meristem maturation clock." Using hundreds of stage-enriched marker genes that compose this clock, we show that extreme branching, conditioned by loss of expression of the COMPOUND INFLORESCENCE gene, is driven by delaying the maturation of both apical and lateral meristems. In contrast, we find that wild tomato species display a delayed maturation only in apical meristems, which leads to modest branching. Our systems genetics approach reveals that the program for inflorescence branching is initiated surprisingly early during meristem maturation and that evolutionary diversity in inflorescence architecture is modulated by heterochronic shifts in the acquisition of floral fate.

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Lupus-associated causal mutation in neutrophil cytosolic factor 2 (NCF2) brings unique insights to the structure and function of NADPH oxidase.

Publication Date: 2012 Jan 10 PMID: 22203994
Authors: Jacob, C. O. - Eisenstein, M. - Dinauer, M. C. - Ming, W. - Liu, Q. - John, S. - Quismorio, F. P. Jr - Reiff, A. - Myones, B. L. - Kaufman, K. M. - McCurdy, D. - Harley, J. B. - Silverman, E. - Kimberly, R. P. - Vyse, T. J. - Gaffney, P. M. - Moser, K. L. - Klein-Gitelman, M. - Wagner-Weiner, L. - Langefeld, C. D. - Armstrong, D. L. - Zidovetzki, R.
Journal: Proc Natl Acad Sci U S A

Systemic lupus erythematosus (SLE), the prototypic systemic autoimmune disease, is a debilitating multisystem autoimmune disorder characterized by chronic inflammation and extensive immune dysregulation in multiple organ systems, resulting in significant morbidity and mortality. Here, we present a multidisciplinary approach resulting in the identification of neutrophil cytosolic factor 2 (NCF2) as an important risk factor for SLE and the detailed characterization of its causal variant. We show that NCF2 is strongly associated with increased SLE risk in two independent populations: childhood-onset SLE and adult-onset SLE. The association between NCF2 and SLE can be attributed to a single nonsynonymous coding mutation in exon 12, the effect of which is the substitution of histidine-389 with glutamine (H389Q) in the PB1 domain of the NCF2 protein, with glutamine being the risk allele. Computational modeling suggests that the NCF2 H389Q mutation reduces the binding efficiency of NCF2 with the guanine nucleotide exchange factor Vav1. The model predicts that NCF2/H389 residue interacts with Vav1 residues E509, N510, E556, and G559 in the ZF domain of Vav1. Furthermore, replacing H389 with Q results in 1.5 kcal/mol weaker binding. To examine the effect of the NCF2 H389Q mutation on NADPH oxidase function, site-specific mutations at the 389 position in NCF2 were tested. Results show that an H389Q mutation causes a twofold decrease in reactive oxygen species production induced by the activation of the Vav-dependent Fcgamma receptor-elicited NADPH oxidase activity. Our study completes the chain of evidence from genetic association to specific molecular function.

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AMP-activated protein kinase is physiologically regulated by inositol polyphosphate multikinase.

Publication Date: 2012 Jan 10 PMID: 22203993
Authors: Bang, S. - Kim, S. - Dailey, M. J. - Chen, Y. - Moran, T. H. - Snyder, S. H. - Kim, S. F.
Journal: Proc Natl Acad Sci U S A

The AMP-activated kinase (AMPK) senses the energy status of cells and regulates fuel availability, whereas hypothalamic AMPK regulates food intake. We report that inositol polyphosphate multikinase (IPMK) regulates glucose signaling to AMPK in a pathway whereby glucose activates phosphorylation of IPMK at tyrosine 174 enabling the enzyme to bind to AMPK and regulate its activation. Thus, refeeding fasted mice rapidly and markedly stimulates transcriptional enhancement of IPMK expression while down-regulating AMPK. Also, AMPK is up-regulated in mice with genetic depletion of hypothalamic IPMK. IPMK physiologically binds AMPK, with binding enhanced by glucose treatment. Regulation by glucose of phospho-AMPK in hypothalamic cell lines is prevented by blocking AMPK-IPMK binding. These findings imply that IPMK inhibitors will be beneficial in treating obesity and diabetes.

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Extensive genetic diversity and substructuring among zebrafish strains revealed through copy number variant analysis.

Publication Date: 2012 Jan 10 PMID: 22203992
Authors: Brown, K. H. - Dobrinski, K. P. - Lee, A. S. - Gokcumen, O. - Mills, R. E. - Shi, X. - Chong, W. W. - Chen, J. Y. - Yoo, P. - David, S. - Peterson, S. M. - Raj, T. - Choy, K. W. - Stranger, B. E. - Williamson, R. E. - Zon, L. I. - Freeman, J. L. - Lee, C.
Journal: Proc Natl Acad Sci U S A

Copy number variants (CNVs) represent a substantial source of genomic variation in vertebrates and have been associated with numerous human diseases. Despite this, the extent of CNVs in the zebrafish, an important model for human disease, remains unknown. Using 80 zebrafish genomes, representing three commonly used laboratory strains and one native population, we constructed a genome-wide, high-resolution CNV map for the zebrafish comprising 6,080 CNV elements and encompassing 14.6% of the zebrafish reference genome. This amount of copy number variation is four times that previously observed in other vertebrates, including humans. Moreover, 69% of the CNV elements exhibited strain specificity, with the highest number observed for Tubingen. This variation likely arose, in part, from Tubingen's large founding size and composite population origin. Additional population genetic studies also provided important insight into the origins and substructure of these commonly used laboratory strains. This extensive variation among and within zebrafish strains may have functional effects that impact phenotype and, if not properly addressed, such extensive levels of germ-line variation and population substructure in this commonly used model organism can potentially confound studies intended for translation to human diseases.

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Growth, metabolic partitioning, and the size of microorganisms.

Publication Date: 2012 Jan 10 PMID: 22203990
Authors: Kempes, C. P. - Dutkiewicz, S. - Follows, M. J.
Journal: Proc Natl Acad Sci U S A

Population growth rate is a fundamental ecological and evolutionary characteristic of living organisms, but individuals must balance the metabolism devoted to biosynthesis and reproduction against the maintenance of existing structure and other functionality. Here we present a mathematical model that relates metabolic partitioning to the form of growth. The model captures the observed growth trajectory of single cells and individuals for a variety of species and taxa spanning prokaryotes, unicellular eukaryotes, and small multicellular eukaryotes. Our analysis suggests that the per-unit costs of biosynthesis and maintenance are conserved across prokaryotes and eukaryotes. However, the relative metabolic expenditure on growth and maintenance of whole organisms clearly differentiates taxa: prokaryotes spend an increasing fraction of their entire metabolism on growth with increasing cell size, whereas eukaryotes devote a diminishing fraction. These differences allow us to predict the minimum and maximum size for each taxonomic group, anticipating observed evolutionary life-history transitions. The framework provides energetic insights into taxonomic tradeoffs related to growth and metabolism and constrains traits that are important for size-structured modeling of microbial communities and their ecological and biogeochemical effects.

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Unexpectedly high mortality in Pacific herring embryos exposed to the 2007 Cosco Busan oil spill in San Francisco Bay.

Publication Date: 2012 Jan 10 PMID: 22203989
Authors: Incardona, J. P. - Vines, C. A. - Anulacion, B. F. - Baldwin, D. H. - Day, H. L. - French, B. L. - Labenia, J. S. - Linbo, T. L. - Myers, M. S. - Olson, O. P. - Sloan, C. A. - Sol, S. - Griffin, F. J. - Menard, K. - Morgan, S. G. - West, J. E. - Collier, T. K. - Ylitalo, G. M. - Cherr, G. N. - Scholz, N. L.
Journal: Proc Natl Acad Sci U S A

In November 2007, the container ship Cosco Busan released 54,000 gallons of bunker fuel oil into San Francisco Bay. The accident oiled shoreline near spawning habitats for the largest population of Pacific herring on the west coast of the continental United States. We assessed the health and viability of herring embryos from oiled and unoiled locations that were either deposited by natural spawning or incubated in subtidal cages. Three months after the spill, caged embryos at oiled sites showed sublethal cardiac toxicity, as expected from exposure to oil-derived polycyclic aromatic compounds (PACs). By contrast, embryos from the adjacent and shallower intertidal zone showed unexpectedly high rates of tissue necrosis and lethality unrelated to cardiotoxicity. No toxicity was observed in embryos from unoiled sites. Patterns of PACs at oiled sites were consistent with oil exposure against a background of urban sources, although tissue concentrations were lower than expected to cause lethality. Embryos sampled 2 y later from oiled sites showed modest sublethal cardiotoxicity but no elevated necrosis or mortality. Bunker oil contains the chemically uncharacterized remains of crude oil refinement, and one or more of these unidentified chemicals likely interacted with natural sunlight in the intertidal zone to kill herring embryos. This reveals an important discrepancy between the resolving power of current forensic analytical chemistry and biological responses of keystone ecological species in oiled habitats. Nevertheless, we successfully delineated the biological impacts of an oil spill in an urbanized coastal estuary with an overlapping backdrop of atmospheric, vessel, and land-based sources of PAC pollution.

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Activation of growth hormone releasing hormone (GHRH) receptor stimulates cardiac reverse remodeling after myocardial infarction (MI).

Publication Date: 2012 Jan 10 PMID: 22203988
Authors: Kanashiro-Takeuchi, R. M. - Takeuchi, L. M. - Rick, F. G. - Dulce, R. - Treuer, A. V. - Florea, V. - Rodrigues, C. O. - Paulino, E. C. - Hatzistergos, K. E. - Selem, S. M. - Gonzalez, D. R. - Block, N. L. - Schally, A. V. - Hare, J. M.
Journal: Proc Natl Acad Sci U S A

Both cardiac myocytes and cardiac stem cells (CSCs) express the receptor of growth hormone releasing hormone (GHRH), activation of which improves injury responses after myocardial infarction (MI). Here we show that a GHRH-agonist (GHRH-A; JI-38) reverses ventricular remodeling and enhances functional recovery in the setting of chronic MI. This response is mediated entirely by activation of GHRH receptor (GHRHR), as demonstrated by the use of a highly selective GHRH antagonist (MIA-602). One month after MI, animals were randomly assigned to receive: placebo, GHRH-A (JI-38), rat recombinant GH, MIA-602, or a combination of GHRH-A and MIA-602, for a 4-wk period. We assessed cardiac performance and hemodynamics by using echocardiography and micromanometry derived pressure-volume loops. Morphometric measurements were carried out to determine MI size and capillary density, and the expression of GHRHR was assessed by immunofluorescence and quantitative RT-PCR. GHRH-A markedly improved cardiac function as shown by echocardiographic and hemodynamic parameters. MI size was substantially reduced, whereas myocyte and nonmyocyte mitosis was markedly increased by GHRH-A. These effects occurred without increases in circulating levels of growth hormone and insulin-like growth factor I and were, at least partially, nullified by GHRH antagonism, confirming a receptor-mediated mechanism. GHRH-A stimulated CSCs proliferation ex vivo, in a manner offset by MIA-602. Collectively, our findings reveal the importance of the GHRH signaling pathway within the heart. Therapy with GHRH-A although initiated 1 mo after MI substantially improved cardiac performance and reduced infarct size, suggesting a regenerative process. Therefore, activation of GHRHR provides a unique therapeutic approach to reverse remodeling after MI.

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Human cytomegalovirus activation of ERK and myeloid cell leukemia-1 protein correlates with survival of latently infected cells.

Publication Date: 2012 Jan 10 PMID: 22203987
Authors: Reeves, M. B. - Breidenstein, A. - Compton, T.
Journal: Proc Natl Acad Sci U S A

The ability of human CMV (HCMV) to enter and establish a latent infection in myeloid cells is crucial for survival and transmission in the human population. Initial pathogen binding and entry triggers a number of antiviral responses, including the activation of proapoptotic cell death pathways, which must be countered during latency establishment. However, mechanisms responsible for a prosurvival state in myeloid cells upon latent HCMV infection remain completely undefined. We hypothesized that the cellular antiapoptotic machinery must be initially activated by HCMV to promote early survival events upon entry. Here we show that HCMV transiently protects nonpermissive myeloid cells from chemical and virus entry induced cell death by up-regulating a key myeloid cell survival gene, myeloid cell leukemia (MCL)-1 protein. The induction of MCL-1 expression was independent of viral gene expression but dependent on activation of the ERK-MAPK pathway by viral glycoprotein B. Inhibition of ERK-MAPK signaling, inhibition of HCMV fusion, antibody-mediated neutralization of glycoprotein B signaling or expression of a shRNA against MCL-1 all correlated with increased cell death in response to virus infection or chemical stimulation. Finally we show that activation of ERK-MAPK signaling impacts on long-term latency and reactivation in hematopoietic cells. Thus, HCMV primes myeloid cells for from the initial virus-cell encounter. Given the importance of ERK and MCL-1 for myeloid cell survival, the successful establishment of HCMV latency in myeloid progenitors begins at the point of virus entry.

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D-Amino acid oxidase controls motoneuron degeneration through D-serine.

Publication Date: 2012 Jan 10 PMID: 22203986
Authors: Sasabe, J. - Miyoshi, Y. - Suzuki, M. - Mita, M. - Konno, R. - Matsuoka, M. - Hamase, K. - Aiso, S.
Journal: Proc Natl Acad Sci U S A

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder involving an extensive loss of motoneurons. Aberrant excitability of motoneurons has been implicated in the pathogenesis of selective motoneuronal death in ALS. d-Serine, an endogenous coagonist of N-methyl-d-aspartate receptors, exacerbates motoneuronal death and is increased both in patients with sporadic/familial ALS and in a G93A-SOD1 mouse model of ALS (mSOD1 mouse). More recently, a unique mutation in the d-amino acid oxidase (DAO) gene, encoding a d-serine degrading enzyme, was reported to be associated with classical familial ALS. However, whether DAO affects the motoneuronal phenotype and d-serine increase in ALS remains uncertain. Here, we show that genetic inactivation of DAO in mice reduces the number and size of lower motoneurons with axonal degeneration, and that suppressed DAO activity in reactive astrocytes in the reticulospinal tract, one of the major inputs to the lower motoneurons, predominantly contributes to the d-serine increase in the mSOD1 mouse. The DAO inactivity resulted from expressional down-regulation, which was reversed by inhibitors of a glutamate receptor and MEK, but not by those of inflammatory stimuli. Our findings provide evidence that DAO has a pivotal role in motoneuron degeneration through d-serine regulation and that inactivity of DAO is a common feature between the mSOD1 ALS mouse model and the mutant DAO-associated familial ALS. The therapeutic benefit of reducing d-serine or controlling DAO activity in ALS should be tested in future studies.

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Hepatocyte growth factor (HGF) autocrine activation predicts sensitivity to MET inhibition in glioblastoma.

Publication Date: 2012 Jan 10 PMID: 22203985
Authors: Xie, Q. - Bradley, R. - Kang, L. - Koeman, J. - Ascierto, M. L. - Worschech, A. - De Giorgi, V. - Wang, E. - Kefene, L. - Su, Y. - Essenburg, C. - Kaufman, D. W. - Dekoning, T. - Enter, M. A. - O'Rourke, T. J. - Marincola, F. M. - Vande Woude, G. F.
Journal: Proc Natl Acad Sci U S A

Because oncogene MET and EGF receptor (EGFR) inhibitors are in clinical development against several types of cancer, including glioblastoma, it is important to identify predictive markers that indicate patient subgroups suitable for such therapies. We investigated in vivo glioblastoma models characterized by hepatocyte growth factor (HGF) autocrine or paracrine activation, or by MET or EGFR amplification, for their susceptibility to MET inhibitors. HGF autocrine expression correlated with high phospho-MET levels in HGF autocrine cell lines, and these lines showed high sensitivity to MET inhibition in vivo. An HGF paracrine environment may enhance glioblastoma growth in vivo but did not indicate sensitivity to MET inhibition. EGFRvIII amplification predicted sensitivity to EGFR inhibition, but in the same tumor, increased copies of MET from gains of chromosome 7 did not result in increased MET activity and did not predict sensitivity to MET inhibitors. Thus, HGF autocrine glioblastoma bears an activated MET signaling pathway that may predict sensitivity to MET inhibitors. Moreover, serum HGF levels may serve as a biomarker for the presence of autocrine tumors and their responsiveness to MET therapeutics.

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Alpha-1-antitrypsin monotherapy reduces graft-versus-host disease after experimental allogeneic bone marrow transplantation.

Publication Date: 2012 Jan 10 PMID: 22203983
Authors: Tawara, I. - Sun, Y. - Lewis, E. C. - Toubai, T. - Evers, R. - Nieves, E. - Azam, T. - Dinarello, C. A. - Reddy, P.
Journal: Proc Natl Acad Sci U S A

Acute graft-versus-host disease (GvHD) is a major complication that prevents successful outcomes after allogeneic bone marrow transplantation (BMT), an effective therapy for hematological malignancies. Several studies demonstrate that donor T cells and host antigen-presenting cells along with several proinflammatory cytokines are required for the induction of GvHD and contribute to its severity. Increasing evidence demonstrates that human serum-derived alphaalpha-1- anti-trypsin (AAT) reduces production of proinflammatory cytokines, induces anti-inflammatory cytokines, and interferes with maturation of dendritic cells. Using well-characterized mouse models of BMT, we have studied the effects of AAT on GvHD severity. Administration of AAT early after BMT decreased mortality in three models of GvHD and reduced serum levels of proinflammatory cytokines in the allogeneic recipients compared with vehicle (albumin) treated animals. AAT treatment reduced the expansion of alloreactive T effector cells but enhanced the recovery of T regulatory T cells, (Tregs) thus altering the ratio of donor T effector to T regulatory cells in favor of reducing the pathological process. However, despite altering the ratio in vivo, AAT had no direct effects on either the donor T effector cells or T regulatory cells Tregs in vitro. In contrast, AAT suppressed LPS-induced in vitro secretion of proinflammatory cytokines such as TNF-alpha and IL-1beta, enhanced the production of the anti-inflammatory cytokine IL-10, and impaired NF-kappaB translocation in the host dendritic cells. In light of its long history of safety in humans, these findings suggest that administration of AAT represents a novel unique and viable strategy to mitigate clinical GvHD.

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Genome and physiology of a model Epsilonproteobacterium responsible for sulfide detoxification in marine oxygen depletion zones.

Publication Date: 2012 Jan 10 PMID: 22203982
Authors: Grote, J. - Schott, T. - Bruckner, C. G. - Glockner, F. O. - Jost, G. - Teeling, H. - Labrenz, M. - Jurgens, K.
Journal: Proc Natl Acad Sci U S A

Eutrophication and global climate change lead to expansion of hypoxia in the ocean, often accompanied by the production of hydrogen sulfide, which is toxic to higher organisms. Chemoautotrophic bacteria are thought to buffer against increased sulfide concentrations by oxidizing hydrogen sulfide before its diffusion to oxygenated surface waters. Model organisms from such environments have not been readily available, which has contributed to a poor understanding of these microbes. We present here a detailed study of "Sulfurimonas gotlandica" str. GD1, an Epsilonproteobacterium isolated from the Baltic Sea oxic-anoxic interface, where it plays a key role in nitrogen and sulfur cycling. Whole-genome analysis and laboratory experiments revealed a high metabolic flexibility, suggesting a considerable capacity for adaptation to variable redox conditions. S. gotlandica str. GD1 was shown to grow chemolithoautotrophically by coupling denitrification with oxidation of reduced sulfur compounds and dark CO(2) fixation. Metabolic versatility was further suggested by the use of a range of different electron donors and acceptors and organic carbon sources. The number of genes involved in signal transduction and metabolic pathways exceeds those of other Epsilonproteobacteria. Oxygen tolerance and environmental-sensing systems combined with chemotactic responses enable this organism to thrive successfully in marine oxygen-depletion zones. We propose that S. gotlandica str. GD1 will serve as a model organism in investigations that will lead to a better understanding how members of the Epsilonproteobacteria are able to cope with water column anoxia and the role these microorganisms play in the detoxification of sulfidic waters.

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Role for the molecular chaperones Zuo1 and Ssz1 in quorum sensing via activation of the transcription factor Pdr1.

Publication Date: 2012 Jan 10 PMID: 22203981
Authors: Prunuske, A. J. - Waltner, J. K. - Kuhn, P. - Gu, B. - Craig, E. A.
Journal: Proc Natl Acad Sci U S A

Zuo1 functions as a J-protein cochaperone of its partner Hsp70. In addition, the C terminus of Zuo1 and the N terminus of Ssz1, with which Zuo1 forms a heterodimer, can independently activate the Saccharomyces cerevisiae transcription factor pleiotropic drug resistance 1 (Pdr1). Here we report that activation of Pdr1 by Zuo1 or Ssz1 causes premature growth arrest of cells during the diauxic shift, as they adapt to the changing environmental conditions. Conversely, cells lacking Zuo1 or Ssz1 overgrow, arresting at a higher cell density, an effect overcome by activation of Pdr1. Cells lacking the genes encoding plasma membrane transporters Pdr5 and Snq2, two targets of Pdr1, also overgrow at the diauxic shift. Adding conditioned medium harvested from cultures of wild-type cells attenuated the overgrowth of both zuo1Deltassz1Delta and pdr5Deltasnq2Delta cells, suggesting the extracellular presence of molecules that signal growth arrest. In addition, our yeast two-hybrid analysis revealed an interaction between Pdr1 and both Zuo1 and Ssz1. Together, our results support a model in which (i) membrane transporters, encoded by Pdr1 target genes act to promote cell-cell communication by exporting quorum sensing molecules, in addition to playing a role in pleiotropic drug resistance; and (ii) molecular chaperones function at promoters to regulate this intercellular communication through their activation of the transcription factor Pdr1.

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Cheating monkeys undermine group strength in enemy territory.

Publication Date: 2012 Jan 10 PMID: 22203978
Authors: Crofoot, M. C. - Gilby, I. C.
Journal: Proc Natl Acad Sci U S A

In many social animals, group-mates cooperate to defend their range against intrusion by neighboring groups. Because group size tends to be highly variable, such conflicts are often asymmetric. Although numerical superiority is assumed to provide a competitive advantage, small groups can generally defend their ranges, even when greatly outnumbered. The prevailing explanation for this puzzling phenomenon is that individuals in relatively large groups experience a greater temptation to flee from conflicts, in effect leveling the balance of power. Using playback experiments simulating territorial intrusions by wild capuchin monkey (Cebus capucinus) groups, we show that such a collective action problem does indeed undermine the competitive ability of large groups. Focal capuchins were more likely to run away from territorial intrusions when their group had a numeric advantage; each one-individual increase in relative group size raised the odds of flight by 25%. However, interaction location had a more important impact on individuals' reactions, creating a strong home-field advantage. After controlling for relative group size, the odds that a focal animal fled were 91% lower in experiments that occurred in the center compared with on the edge of its group's range, whereas the odds that it rushed toward the speaker were more than sixfold higher. These location-dependent patterns of defection and cooperation create a competitive advantage for residents over intruders across a wide range of relative group sizes, which may stabilize range boundaries and provide a general explanation for how groups of widely divergent sizes can coexist, even in the face of intense intergroup competition.

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Thrombocytopenia and erythrocytosis in mice with a mutation in the gene encoding the hemoglobin beta minor chain.

Publication Date: 2012 Jan 10 PMID: 22203977
Authors: Kauppi, M. - Hilton, A. A. - Metcalf, D. - Ng, A. P. - Hyland, C. D. - Collinge, J. E. - Kile, B. T. - Hilton, D. J. - Alexander, W. S.
Journal: Proc Natl Acad Sci U S A

Diverse mutations in the genes encoding hemoglobin (Hb) have been characterized in human disease. We describe here a mutation in the mouse Hbb-b2 gene, denoted Plt12, that precisely mimics the human hemoglobin Hotel Dieu variant. The mutation results in increased affinity of Hb for oxygen and Plt12 mutant mice exhibited reduced partial pressure of O(2) in the blood, accompanied by erythrocytosis characterized by elevated erythropoietin levels and splenomegaly with excess erythropoiesis. Most homozygous Hbb-b2(Plt12/Plt12) mice succumbed to early lethality associated with emphysema, cardiac abnormalities, and liver degeneration. Survivors displayed a marked thrombocytopenia without significant deficiencies in the numbers of megakaryocytes or megakaryocyte progenitor cells. The lifespan of platelets in the circulation of Hbb-b2(Plt12/Plt12) mice was normal, and splenectomy did not correct the thrombocytopenia, suggesting that increased sequestration was unlikely to be a major contributor. These data, together with the observation that megakaryocytes in Hbb-b2(Plt12/Plt12) mice appeared smaller and deficient in cytoplasm, support a model in which hypoxia causes thrombocytopenia as a consequence of an inability of megakaryocytes, once formed, to properly mature and produce sufficient platelets. The Plt12 mouse is a model of high O(2)-affinity hemoglobinopathy and provides insights into hematopoiesis under conditions of chronic hypoxia.

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Type 1 ryanodine receptor knock-in mutation causing central core disease of skeletal muscle also displays a neuronal phenotype.

Publication Date: 2012 Jan 10 PMID: 22203976
Authors: De Crescenzo, V. - Fogarty, K. E. - Lefkowitz, J. J. - Bellve, K. D. - Zvaritch, E. - Maclennan, D. H. - Walsh, J. V. Jr
Journal: Proc Natl Acad Sci U S A

The type 1 ryanodine receptor (RyR1) is expressed widely in the brain, with high levels in the cerebellum, hippocampus, and hypothalamus. We have shown that L-type Ca(2+) channels in terminals of hypothalamic magnocellular neurons are coupled to RyRs, as they are in skeletal muscle, allowing voltage-induced Ca(2+) release (VICaR) from internal Ca(2+) stores without Ca(2+) influx. Here we demonstrate that RyR1 plays a role in VICaR in nerve terminals. Furthermore, in heterozygotes from the Ryr1(I4895T/WT) (IT/+) mouse line, carrying a knock-in mutation corresponding to one that causes a severe form of human central core disease, VICaR is absent, demonstrating that type 1 RyR mediates VICaR and that these mice have a neuronal phenotype. The absence of VICaR was shown in two ways: first, depolarization in the absence of Ca(2+) influx elicited Ca(2+)syntillas (scintilla, spark, in a nerve terminal, a SYNaptic structure) in WT, but not in mutant terminals; second, in the presence of extracellular Ca(2+), IT/+ terminals showed a twofold decrease in global Ca(2+) transients, with no change in plasmalemmal Ca(2+) current. From these studies we draw two conclusions: (i) RyR1 plays a role in VICaR in hypothalamic nerve terminals; and (ii) a neuronal alteration accompanies the myopathy in IT/+ mice, and, possibly in humans carrying the corresponding RyR1 mutation.

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Multiple independent introductions of Plasmodium falciparum in South America.

Publication Date: 2012 Jan 10 PMID: 22203975
Authors: Yalcindag, E. - Elguero, E. - Arnathau, C. - Durand, P. - Akiana, J. - Anderson, T. J. - Aubouy, A. - Balloux, F. - Besnard, P. - Bogreau, H. - Carnevale, P. - D'Alessandro, U. - Fontenille, D. - Gamboa, D. - Jombart, T. - Le Mire, J. - Leroy, E. - Maestre, A. - Mayxay, M. - Menard, D. - Musset, L. - Newton, P. N. - Nkoghe, D. - Noya, O. - Ollomo, B. - Rogier, C. - Veron, V. - Wide, A. - Zakeri, S. - Carme, B. - Legrand, E. - Chevillon, C. - Ayala, F. J. - Renaud, F. - Prugnolle, F.
Journal: Proc Natl Acad Sci U S A

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence-archeological and genetic-suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

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Evolutionary constraints on visual cortex architecture from the dynamics of hallucinations.

Publication Date: 2012 Jan 10 PMID: 22203969
Authors: Butler, T. C. - Benayoun, M. - Wallace, E. - van Drongelen, W. - Goldenfeld, N. - Cowan, J.
Journal: Proc Natl Acad Sci U S A

In the cat or primate primary visual cortex (V1), normal vision corresponds to a state where neural excitation patterns are driven by external visual stimuli. A spectacular failure mode of V1 occurs when such patterns are overwhelmed by spontaneously generated spatially self-organized patterns of neural excitation. These are experienced as geometric visual hallucinations. The problem of identifying the mechanisms by which V1 avoids this failure is made acute by recent advances in the statistical mechanics of pattern formation, which suggest that the hallucinatory state should be very robust. Here, we report how incorporating physiologically realistic long-range connections between inhibitory neurons changes the behavior of a model of V1. We find that the sparsity of long-range inhibition in V1 plays a previously unrecognized but key functional role in preserving the normal vision state. Surprisingly, it also contributes to the observed regularity of geometric visual hallucinations. Our results provide an explanation for the observed sparsity of long-range inhibition in V1-this generic architectural feature is an evolutionary adaptation that tunes V1 to the normal vision state. In addition, it has been shown that exactly the same long-range connections play a key role in the development of orientation preference maps. Thus V1's most striking long-range features-patchy excitatory connections and sparse inhibitory connections-are strongly constrained by two requirements: the need for the visual state to be robust and the developmental requirements of the orientational preference map.

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Non-coalescence of oppositely charged droplets in pH-sensitive emulsions.

Publication Date: 2012 Jan 10 PMID: 22203968
Authors: Liu, T. - Seiffert, S. - Thiele, J. - Abate, A. R. - Weitz, D. A. - Richtering, W.
Journal: Proc Natl Acad Sci U S A

Like charges stabilize emulsions, whereas opposite charges break emulsions. This is the fundamental principle for many industrial and practical processes. Using micrometer-sized pH-sensitive polymeric hydrogel particles as emulsion stabilizers, we prepare emulsions that consist of oppositely charged droplets, which do not coalesce. We observe noncoalescence of oppositely charged droplets in bulk emulsification as well as in microfluidic devices, where oppositely charged droplets are forced to collide within channel junctions. The results demonstrate that electrostatic interactions between droplets do not determine their stability and reveal the unique pH-dependent properties of emulsions stabilized by soft microgel particles. The noncoalescence can be switched to coalescence by neutralizing the microgels, and the emulsion can be broken on demand. This unusual feature of the microgel-stabilized emulsions offers fascinating opportunities for future applications of these systems.

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High-resolution dose-response screening using droplet-based microfluidics.

Publication Date: 2012 Jan 10 PMID: 22203966
Authors: Miller, O. J. - Harrak, A. E. - Mangeat, T. - Baret, J. C. - Frenz, L. - Debs, B. E. - Mayot, E. - Samuels, M. L. - Rooney, E. K. - Dieu, P. - Galvan, M. - Link, D. R. - Griffiths, A. D.
Journal: Proc Natl Acad Sci U S A

A critical early step in drug discovery is the screening of a chemical library. Typically, promising compounds are identified in a primary screen and then more fully characterized in a dose-response analysis with 7-10 data points per compound. Here, we describe a robust microfluidic approach that increases the number of data points to approximately 10,000 per compound. The system exploits Taylor-Aris dispersion to create concentration gradients, which are then segmented into picoliter microreactors by droplet-based microfluidics. The large number of data points results in IC(50) values that are highly precise (+/- 2.40% at 95% confidence) and highly reproducible (CV = 2.45%, n = 16). In addition, the high resolution of the data reveals complex dose-response relationships unambiguously. We used this system to screen a chemical library of 704 compounds against protein tyrosine phosphatase 1B, a diabetes, obesity, and cancer target. We identified a number of novel inhibitors, the most potent being sodium cefsulodine, which has an IC(50) of 27 +/- 0.83 muM.

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Revealing conformational substates of lipidated N-Ras protein by pressure modulation.

Publication Date: 2012 Jan 10 PMID: 22203965
Authors: Kapoor, S. - Triola, G. - Vetter, I. R. - Erlkamp, M. - Waldmann, H. - Winter, R.
Journal: Proc Natl Acad Sci U S A

Regulation of protein function is often linked to a conformational switch triggered by chemical or physical signals. To evaluate such conformational changes and to elucidate the underlying molecular mechanisms of subsequent protein function, experimental identification of conformational substates and characterization of conformational equilibria are mandatory. We apply pressure modulation in combination with FTIR spectroscopy to reveal equilibria between spectroscopically resolved substates of the lipidated signaling protein N-Ras. Pressure has the advantage that its thermodynamic conjugate is volume, a parameter that is directly related to structure. The conformational dynamics of N-Ras in its different nucleotide binding states in the absence and presence of a model biomembrane was probed by pressure perturbation. We show that not only nucleotide binding but also the presence of the membrane has a drastic effect on the conformational dynamics and selection of conformational substates of the protein, and a new substate appearing upon membrane binding could be uncovered. Population of this new substate is accompanied by structural reorientations of the G domain, as also indicated by complementary ATR-FTIR and IRRAS measurements. These findings thus illustrate that the membrane controls signaling conformations by acting as an effective interaction partner, which has consequences for the G-domain orientation of membrane-associated N-Ras, which in turn is known to be critical for its effector and modulator interactions. Finally, these results provide insights into the influence of pressure on Ras-controlled signaling events in organisms living under extreme environmental conditions as they are encountered in the deep sea where pressures reach the kbar range.

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Regulation of oxidative DNA damage repair by DNA polymerase lambda and MutYH by cross-talk of phosphorylation and ubiquitination.

Publication Date: 2012 Jan 10 PMID: 22203964
Authors: Markkanen, E. - van Loon, B. - Ferrari, E. - Parsons, J. L. - Dianov, G. L. - Hubscher, U.
Journal: Proc Natl Acad Sci U S A

It is of pivotal importance for genome stability that repair DNA polymerases (Pols), such as Pols lambda and beta, which all exhibit considerably reduced fidelity when replicating undamaged DNA, are tightly regulated, because their misregulation could lead to mutagenesis. Recently, we found that the correct repair of the abundant and highly miscoding oxidative DNA lesion 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-G) is performed by an accurate repair pathway that is coordinated by the MutY glycosylase homologue (MutYH) and Pol lambda in vitro and in vivo. Pol lambda is phosphorylated by Cdk2/cyclinA in late S and G2 phases of the cell cycle, promoting Pol lambda stability by preventing it from being targeted for proteasomal degradation by ubiquitination. However, it has remained a mystery how the levels of Pol lambda are controlled, how phosphorylation promotes its stability, and how the engagement of Pol lambda in active repair complexes is coordinated. Here, we show that the E3 ligase Mule mediates the degradation of Pol lambda and that the control of Pol lambda levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. Furthermore, we demonstrate that phosphorylation of Pol lambda by Cdk2/cyclinA counteracts its Mule-mediated degradation by promoting recruitment of Pol lambda to chromatin into active 8-oxo-G repair complexes through an increase in Pol lambda's affinity to chromatin-bound MutYH. Finally, MutYH appears to promote the stability of Pol lambda by binding it to chromatin. In contrast, Pol lambda not engaged in active repair on chromatin is subject for proteasomal degradation.

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Disordered form of the scaffold protein IscU is the substrate for iron-sulfur cluster assembly on cysteine desulfurase.

Publication Date: 2012 Jan 10 PMID: 22203963
Authors: Kim, J. H. - Tonelli, M. - Markley, J. L.
Journal: Proc Natl Acad Sci U S A

The scaffold protein for iron-sulfur cluster assembly, apo-IscU, populates two interconverting conformational states, one disordered (D) and one structured (S) as revealed by extensive NMR assignments. At pH 8 and 25 degrees C, approximately 70% of the protein is S, and the lifetimes of the states are 1.3 s (S) and 0.50 s (D). Zn(II) and Fe(II) each bind and stabilize structured (S-like) states. Single amino acid substitutions at conserved residues were found that shift the equilibrium toward either the S or the D state. Cluster assembly takes place in the complex between IscU and the cysteine desulfurase, IscS, and our NMR studies demonstrate that IscS binds preferentially the D form of apo-IscU. The addition of 10% IscS to IscU was found to greatly increase H/D exchange at protected amides of IscU, to increase the rate of the S --> D reaction, and to decrease the rate of the D --> S reaction. In the saturated IscU:IscS complex, IscU is largely disordered. In vitro cluster assembly reactions provided evidence for the functional importance of the S&lrarr2;D equilibrium. IscU variants that favor the S state were found to undergo a lag phase, not observed with the wild type, that delayed cluster assembly; variants that favor the D state were found to assemble less stable clusters at an intermediate rate without the lag. It appears that IscU has evolved to exist in a disordered conformational state that is the initial substrate for the desulfurase and to convert to a structured state that stabilizes the cluster once it is assembled.

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Understanding fast macroscale fracture from microcrack post mortem patterns.

Publication Date: 2012 Jan 10 PMID: 22203962
Authors: Guerra, C. - Scheibert, J. - Bonamy, D. - Dalmas, D.
Journal: Proc Natl Acad Sci U S A

Dynamic crack propagation drives catastrophic solid failures. In many amorphous brittle materials, sufficiently fast crack growth involves small-scale, high-frequency microcracking damage localized near the crack tip. The ultrafast dynamics of microcrack nucleation, growth, and coalescence is inaccessible experimentally and fast crack propagation was therefore studied only as a macroscale average. Here, we overcome this limitation in polymethylmethacrylate, the archetype of brittle amorphous materials: We reconstruct the complete spatiotemporal microcracking dynamics, with micrometer/nanosecond resolution, through post mortem analysis of the fracture surfaces. We find that all individual microcracks propagate at the same low, load-independent velocity. Collectively, the main effect of microcracks is not to slow down fracture by increasing the energy required for crack propagation, as commonly believed, but on the contrary to boost the macroscale velocity through an acceleration factor selected on geometric grounds. Our results emphasize the key role of damage-related internal variables in the selection of macroscale fracture dynamics.

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Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells.

Publication Date: 2012 Jan 10 PMID: 22203961
Authors: Shi, Q. - Qin, L. - Wei, W. - Geng, F. - Fan, R. - Shik Shin, Y. - Guo, D. - Hood, L. - Mischel, P. S. - Heath, J. R.
Journal: Proc Natl Acad Sci U S A

We describe a microchip designed to quantify the levels of a dozen cytoplasmic and membrane proteins from single cells. We use the platform to assess protein-protein interactions associated with the EGF-receptor-mediated PI3K signaling pathway. Single-cell sensitivity is achieved by isolating a defined number of cells (n = 0-5) in 2 nL volume chambers, each of which is patterned with two copies of a miniature antibody array. The cells are lysed on-chip, and the levels of released proteins are assayed using the antibody arrays. We investigate three isogenic cell lines representing the cancer glioblastoma multiforme, at the basal level, under EGF stimulation, and under erlotinib inhibition plus EGF stimulation. The measured protein abundances are consistent with previous work, and single-cell analysis uniquely reveals single-cell heterogeneity, and different types and strengths of protein-protein interactions. This platform helps provide a comprehensive picture of altered signal transduction networks in tumor cells and provides insight into the effect of targeted therapies on protein signaling networks.

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Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells.

Publication Date: 2012 Jan 10 PMID: 22203960
Authors: Giraud, M. - Yoshida, H. - Abramson, J. - Rahl, P. B. - Young, R. A. - Mathis, D. - Benoist, C.
Journal: Proc Natl Acad Sci U S A

Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analysis with mRNA-spanning probes. This analysis revealed initiation of Aire-activated genes to be comparable in Aire-deficient and wild-type MECs, but with a block to elongation after 50-100 bp in the absence of Aire, suggesting activation by release of stalled polymerases by Aire. In contrast, patterns of activation by transcription factors such as Klf4 were consistent with regulation of initiation. Mapping of Aire and RNA polymerase-II (Pol-II) by ChIP and high-throughput sequencing (ChIP-seq) revealed that Aire bound all Pol-II-rich transcriptional start sites (TSS), irrespective of its eventual effect. However, the genes it preferentially activated were characterized by a relative surfeit of stalled polymerases at the TSS, which resolved once Aire was introduced into cells. Thus, transcript mapping and ChIP-seq data indicate that Aire activates ectopic transcription not through specific recognition of PTA gene promoters but by releasing stalled polymerases.

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Legume pectate lyase required for root infection by rhizobia.

Publication Date: 2012 Jan 10 PMID: 22203959
Authors: Xie, F. - Murray, J. D. - Kim, J. - Heckmann, A. B. - Edwards, A. - Oldroyd, G. E. - Downie, J. A.
Journal: Proc Natl Acad Sci U S A

To allow rhizobial infection of legume roots, plant cell walls must be locally degraded for plant-made infection threads (ITs) to be formed. Here we identify a Lotus japonicus nodulation pectate lyase gene (LjNPL), which is induced in roots and root hairs by rhizobial nodulation (Nod) factors via activation of the nodulation signaling pathway and the NIN transcription factor. Two Ljnpl mutants produced uninfected nodules and most infections arrested as infection foci in root hairs or roots. The few partially infected nodules that did form contained large abnormal infections. The purified LjNPL protein had pectate lyase activity, demonstrating that this activity is required for rhizobia to penetrate the cell wall and initiate formation of plant-made infection threads. Therefore, we conclude that legume-determined degradation of plant cell walls is required for root infection during initiation of the symbiotic interaction between rhizobia and legumes.

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Vertebrate-like regeneration in the invertebrate chordate amphioxus.

Publication Date: 2012 Jan 10 PMID: 22203957
Authors: Somorjai, I. M. - Somorjai, R. L. - Garcia-Fernandez, J. - Escriva, H.
Journal: Proc Natl Acad Sci U S A

An important question in biology is why some animals are able to regenerate, whereas others are not. The basal chordate amphioxus is uniquely positioned to address the evolution of regeneration. We report here the high regeneration potential of the European amphioxus Branchiostoma lanceolatum. Adults regenerate both anterior and posterior structures, including neural tube, notochord, fin, and muscle. Development of a classifier based on tail regeneration profiles predicts the assignment of young and old adults to their own class with >94% accuracy. The process involves loss of differentiated characteristics, formation of an msx-expressing blastema, and neurogenesis. Moreover, regeneration is linked to the activation of satellite-like Pax3/7 progenitor cells, the extent of which declines with size and age. Our results provide a framework for understanding the evolution and diversity of regeneration mechanisms in vertebrates.

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Secreted Wingless-interacting molecule (Swim) promotes long-range signaling by maintaining Wingless solubility.

Publication Date: 2012 Jan 10 PMID: 22203956
Authors: Mulligan, K. A. - Fuerer, C. - Ching, W. - Fish, M. - Willert, K. - Nusse, R.
Journal: Proc Natl Acad Sci U S A

Lipid-modified Wnt/Wingless (Wg) proteins can signal to their target cells in a short- or long-range manner. How these hydrophobic proteins travel through the extracellular environment remains an outstanding question. Here, we report on a Wg binding protein, Secreted Wg-interacting molecule (Swim), that facilitates Wg diffusion through the extracellular matrix. Swim, a putative member of the Lipocalin family of extracellular transport proteins, binds to Wg with nanomolar affinity in a lipid-dependent manner. In quantitative signaling assays, Swim is sufficient to maintain the solubility and activity of purified Wg. In Drosophila, swim RNAi phenotypes resemble wg loss-of-function phenotypes in long-range signaling. We propose that Swim is a cofactor that promotes long-range Wg signaling in vivo by maintaining the solubility of Wg.

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Gastrin-releasing peptide receptor (GRPR) mediates chemotaxis in neutrophils.

Publication Date: 2012 Jan 10 PMID: 22203955
Authors: Czepielewski, R. S. - Porto, B. N. - Rizzo, L. B. - Roesler, R. - Abujamra, A. L. - Pinto, L. G. - Schwartsmann, G. - Cunha Fde, Q. - Bonorino, C.
Journal: Proc Natl Acad Sci U S A

Neutrophil migration to inflamed sites is crucial for both the initiation of inflammation and resolution of infection, yet these cells are involved in perpetuation of different chronic inflammatory diseases. Gastrin-releasing peptide (GRP) is a neuropeptide that acts through G protein coupled receptors (GPCRs) involved in signal transmission in both central and peripheral nervous systems. Its receptor, gastrin-releasing peptide receptor (GRPR), is expressed by various cell types, and it is overexpressed in cancer cells. RC-3095 is a selective GRPR antagonist, recently found to have antiinflammatory properties in arthritis and sepsis models. Here we demonstrate that i.p. injection of GRP attracts neutrophils in 4 h, and attraction is blocked by RC-3095. Macrophage depletion or neutralization of TNF abrogates GRP-induced neutrophil recruitment to the peritoneum. In vitro, GRP-induced neutrophil migration was dependent on PLC-beta2, PI3K, ERK, p38 and independent of Galphai protein, and neutrophil migration toward synovial fluid of arthritis patients was inhibited by treatment with RC-3095. We propose that GRPR is an alternative chemotactic receptor that may play a role in the pathogenesis of inflammatory disorders.

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Targeting protein-trafficking pathways alters melanoma treatment sensitivity.

Publication Date: 2012 Jan 10 PMID: 22203954
Authors: Huang, Z. M. - Chinen, M. - Chang, P. J. - Xie, T. - Zhong, L. - Demetriou, S. - Patel, M. P. - Scherzer, R. - Sviderskaya, E. V. - Bennett, D. C. - Millhauser, G. L. - Oh, D. H. - Cleaver, J. E. - Wei, M. L.
Journal: Proc Natl Acad Sci U S A

Protein-trafficking pathways are targeted here in human melanoma cells using methods independent of oncogene mutational status, and the ability to up-regulate and down-regulate tumor treatment sensitivity is demonstrated. Sensitivity of melanoma cells to cis-diaminedichloroplatinum II (cDDP, cis-platin), carboplatin, dacarbazine, or temozolomide together with velaparib, an inhibitor of poly (ADP ribose) polymerase 1, is increased by up to 10-fold by targeting genes that regulate both protein trafficking and the formation of melanosomes, intracellular organelles unique to melanocytes and melanoma cells. Melanoma cells depleted of either of the protein-trafficking regulators vacuolar protein sorting 33A protein (VPS33A) or cappuccino protein (CNO) have increased nuclear localization of cDDP, increased nuclear DNA damage by platination, and increased apoptosis, resulting in increased treatment sensitivity. Depleted cells also exhibit a decreased proportion of intracellular, mature melanosomes compared with undepleted cells. Modulation of protein trafficking via cell-surface signaling by binding the melanocortin 1 receptor with the antagonist agouti-signaling protein decreased the proportion of mature melanosomes formed and increased cDDP sensitivity, whereas receptor binding with the agonist melanocyte-stimulating hormone resulted in an increased proportion of mature melanosomes formed and in decreased sensitivity (i.e., increased resistance) to cDDP. Mutation of the protein-trafficking gene Hps6, known to impair the formation of mature melanosomes, also increased cDDP sensitivity. Together, these results indicate that targeting protein-trafficking molecules markedly increases melanoma treatment sensitivity and influences the degree of melanosomes available for sequestration of therapeutic agents.

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Sperm-borne microRNA-34c is required for the first cleavage division in mouse.

Publication Date: 2012 Jan 10 PMID: 22203953
Authors: Liu, W. M. - Pang, R. T. - Chiu, P. C. - Wong, B. P. - Lao, K. - Lee, K. F. - Yeung, W. S.
Journal: Proc Natl Acad Sci U S A

In mammals, the sperm deliver mRNA of unknown function into the oocytes during fertilization. The role of sperm microRNAs (miRNAs) in preimplantation development is unknown. miRNA profiling identified six miRNAs expressed in the sperm and the zygotes but not in the oocytes or preimplantation embryos. Sperm contained both the precursor and the mature form of one of these miRNAs, miR-34c. The absence of an increased level of miR-34c in zygotes derived from alpha-amanitin-treated oocytes and in parthenogenetic oocytes supported a sperm origin of zygotic miR-34c. Injection of miR-34c inhibitor into zygotes inhibited DNA synthesis and significantly suppressed first cleavage division. A 3' UTR luciferase assay and Western blotting demonstrated that miR-34c regulates B-cell leukemia/lymphoma 2 (Bcl-2) expression in the zygotes. Coinjection of anti-Bcl-2 antibody in zygotes partially reversed but injection of Bcl-2 protein mimicked the effect of miR-34c inhibition. Oocyte activation is essential for the miR-34c action in zygotes, as demonstrated by a decrease in 3'UTR luciferase reporter activity and Bcl-2 expression after injection of precursor miR-34c into parthenogenetic oocytes. Our findings provide evidence that sperm-borne miR-34c is important for the first cell division via modulation of Bcl-2 expression.

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Schooling in adolescence raises IQ scores.

Publication Date: 2012 Jan 10 PMID: 22203952
Authors: Brinch, C. N. - Galloway, T. A.
Journal: Proc Natl Acad Sci U S A

Although some scholars maintain that education has little effect on intelligence quotient (IQ) scores, others claim that IQ scores are indeed malleable, primarily through intervention in early childhood. The causal effect of education on IQ at later ages is often difficult to uncover because analyses based on observational data are plagued by problems of reverse causation and self-selection into further education. We exploit a reform that increased compulsory schooling from 7 to 9 y in Norway in the 1960s to estimate the effect of education on IQ. We find that this schooling reform, which primarily affected education in the middle teenage years, had a substantial effect on IQ scores measured at the age of 19 y.

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Formation of buckminsterfullerene (C60) in interstellar space.

Publication Date: 2012 Jan 10 PMID: 22198841
Authors: Berne, O. - Tielens, A. G.
Journal: Proc Natl Acad Sci U S A

Buckminsterfullerene (C(60)) was recently confirmed as the largest molecule identified in space. However, it remains unclear how and where this molecule is formed. It is generally believed that C(60) is formed from the buildup of small carbonaceous compounds in the hot and dense envelopes of evolved stars. Analyzing infrared observations, obtained by Spitzer and Herschel, we found that C(60) is efficiently formed in the tenuous and cold environment of an interstellar cloud illuminated by strong ultraviolet (UV) radiation fields. This implies that another formation pathway, efficient at low densities, must exist. Based on recent laboratory and theoretical studies, we argue that polycyclic aromatic hydrocarbons are converted into graphene, and subsequently C(60), under UV irradiation from massive stars. This shows that alternative-top-down-routes are key to understanding the organic inventory in space.

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Nonplanar peptide bonds in proteins are common and conserved but not biased toward active sites.

Publication Date: 2012 Jan 10 PMID: 22198840
Authors: Berkholz, D. S. - Driggers, C. M. - Shapovalov, M. V. - Dunbrack, R. L. Jr - Karplus, P. A.
Journal: Proc Natl Acad Sci U S A

The planarity of peptide bonds is an assumption that underlies decades of theoretical modeling of proteins. Peptide bonds strongly deviating from planarity are considered very rare features of protein structure that occur for functional reasons. Here, empirical analyses of atomic-resolution protein structures reveal that trans peptide groups can vary by more than 25 degrees from planarity and that the true extent of nonplanarity is underestimated even in 1.2 A resolution structures. Analyses as a function of the varphi,psi-backbone dihedral angles show that the expected value deviates by +/- 8 degrees from planar as a systematic function of conformation, but that the large majority of variation in planarity depends on tertiary effects. Furthermore, we show that those peptide bonds in proteins that are most nonplanar, deviating by over 20 degrees from planarity, are not strongly associated with active sites. Instead, highly nonplanar peptides are simply integral components of protein structure related to local and tertiary structural features that tend to be conserved among homologs. To account for the systematic varphi,psi-dependent component of nonplanarity, we present a conformation-dependent library that can be used in crystallographic refinement and predictive protein modeling.

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Database independent proteomics analysis of the ostrich and human proteome.

Publication Date: 2012 Jan 10 PMID: 22198768
Authors: Altelaar, A. F. - Navarro, D. - Boekhorst, J. - van Breukelen, B. - Snel, B. - Mohammed, S. - Heck, A. J.
Journal: Proc Natl Acad Sci U S A

Mass spectrometry (MS)-based proteome analysis relies heavily on the presence of complete protein databases. Such a strategy is extremely powerful, albeit not adequate in the analysis of unpredicted postgenome events, such as posttranslational modifications, which exponentially increase the search space. Therefore, it is of interest to explore "database-free" approaches. Here, we sampled the ostrich and human proteomes with a method facilitating de novo sequencing, utilizing the protease Lys-N in combination with electron transfer dissociation. By implementing several validation steps, including the combined use of collision-induced dissociation/electron transfer dissociation data and a cross-validation with conventional database search strategies, we identified approximately 2,500 unique de novo peptide sequences from the ostrich sample with over 900 peptides generating full backbone sequence coverage. This dataset allowed the appropriate positioning of ostrich in the evolutionary tree. The described database-free sequencing approach is generically applicable and has great potential in important proteomics applications such as in the analysis of variable parts of endogenous antibodies or proteins modified by a plethora of complex posttranslational modifications.

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A single conformational transglutaminase 2 epitope contributed by three domains is critical for celiac antibody binding and effects.

Publication Date: 2012 Jan 10 PMID: 22198767
Authors: Simon-Vecsei, Z. - Kiraly, R. - Bagossi, P. - Toth, B. - Dahlbom, I. - Caja, S. - Csosz, E. - Lindfors, K. - Sblattero, D. - Nemes, E. - Maki, M. - Fesus, L. - Korponay-Szabo, I. R.
Journal: Proc Natl Acad Sci U S A

The multifunctional, protein cross-linking transglutaminase 2 (TG2) is the main autoantigen in celiac disease, an autoimmune disorder with defined etiology. Glutamine-rich gliadin peptides from ingested cereals, after their deamidation by TG2, induce T-lymphocyte activation accompanied by autoantibody production against TG2 in 1-2% of the population. The pathogenic role and exact binding properties of these antibodies to TG2 are still unclear. Here we show that antibodies from different celiac patients target the same conformational TG2 epitope formed by spatially close amino acids of adjacent domains. Glu153 and 154 on the first alpha-helix of the core domain and Arg19 on first alpha-helix of the N-terminal domain determine the celiac epitope that is accessible both in the closed and open conformation of TG2 and dependent on the relative position of these helices. Met659 on the C-terminal domain also can cooperate in antibody binding. This composite epitope is disease-specific, recognized by antibodies derived from celiac tissues and associated with biological effects when passively transferred from celiac mothers into their newborns. These findings suggest that celiac antibodies are produced in a surface-specific way for which certain homology of the central glutamic acid residues of the TG2 epitope with deamidated gliadin peptides could be a structural basis. Monoclonal mouse antibodies with partially overlapping epitope specificity released celiac antibodies from patient tissues and antagonized their harmful effects in cell culture experiments. Such antibodies or similar specific competitors will be useful in further functional studies and in exploring whether interference with celiac antibody actions leads to therapeutic benefits.

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Bayesian analysis of the astrobiological implications of life's early emergence on Earth.

Publication Date: 2012 Jan 10 PMID: 22198766
Authors: Spiegel, D. S. - Turner, E. L.
Journal: Proc Natl Acad Sci U S A

Life arose on Earth sometime in the first few hundred million years after the young planet had cooled to the point that it could support water-based organisms on its surface. The early emergence of life on Earth has been taken as evidence that the probability of abiogenesis is high, if starting from young Earth-like conditions. We revisit this argument quantitatively in a Bayesian statistical framework. By constructing a simple model of the probability of abiogenesis, we calculate a Bayesian estimate of its posterior probability, given the data that life emerged fairly early in Earth's history and that, billions of years later, curious creatures noted this fact and considered its implications. We find that, given only this very limited empirical information, the choice of Bayesian prior for the abiogenesis probability parameter has a dominant influence on the computed posterior probability. Although terrestrial life's early emergence provides evidence that life might be abundant in the universe if early-Earth-like conditions are common, the evidence is inconclusive and indeed is consistent with an arbitrarily low intrinsic probability of abiogenesis for plausible uninformative priors. Finding a single case of life arising independently of our lineage (on Earth, elsewhere in the solar system, or on an extrasolar planet) would provide much stronger evidence that abiogenesis is not extremely rare in the universe.

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Tumor suppression by cell competition through regulation of the Hippo pathway.

Publication Date: 2012 Jan 10 PMID: 22190496
Authors: Chen, C. L. - Schroeder, M. C. - Kango-Singh, M. - Tao, C. - Halder, G.
Journal: Proc Natl Acad Sci U S A

Homeostatic mechanisms can eliminate abnormal cells to prevent diseases such as cancer. However, the underlying mechanisms of this surveillance are poorly understood. Here we investigated how clones of cells mutant for the neoplastic tumor suppressor gene scribble (scrib) are eliminated from Drosophila imaginal discs. When all cells in imaginal discs are mutant for scrib, they hyperactivate the Hippo pathway effector Yorkie (Yki), which drives growth of the discs into large neoplastic masses. Strikingly, when discs also contain normal cells, the scrib(-) cells do not overproliferate and eventually undergo apoptosis through JNK-dependent mechanisms. However, induction of apoptosis does not explain how scrib(-) cells are prevented from overproliferating. We report that cell competition between scrib(-) and wild-type cells prevents hyperproliferation by suppressing Yki activity in scrib(-) cells. Suppressing Yki activation is critical for scrib(-) clone elimination by cell competition, and experimental elevation of Yki activity in scrib(-) cells is sufficient to fuel their neoplastic growth. Thus, cell competition acts as a tumor-suppressing mechanism by regulating the Hippo pathway in scrib(-) cells.

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A role for neuronal cAMP responsive-element binding (CREB)-1 in brain responses to calorie restriction.

Publication Date: 2012 Jan 10 PMID: 22190495
Authors: Fusco, S. - Ripoli, C. - Podda, M. V. - Ranieri, S. C. - Leone, L. - Toietta, G. - McBurney, M. W. - Schutz, G. - Riccio, A. - Grassi, C. - Galeotti, T. - Pani, G.
Journal: Proc Natl Acad Sci U S A

Calorie restriction delays brain senescence and prevents neurodegeneration, but critical regulators of these beneficial responses other than the NAD(+)-dependent histone deacetylase Sirtuin-1 (Sirt-1) are unknown. We report that effects of calorie restriction on neuronal plasticity, memory and social behavior are abolished in mice lacking cAMP responsive-element binding (CREB)-1 in the forebrain. Moreover, CREB deficiency drastically reduces the expression of Sirt-1 and the induction of genes relevant to neuronal metabolism and survival in the cortex and hippocampus of dietary-restricted animals. Biochemical studies reveal a complex interplay between CREB and Sirt-1: CREB directly regulates the transcription of the sirtuin in neuronal cells by binding to Sirt-1 chromatin; Sirt-1, in turn, is recruited by CREB to DNA and promotes CREB-dependent expression of target gene peroxisome proliferator-activated receptor-gamma coactivator-1alpha and neuronal NO Synthase. Accordingly, expression of these CREB targets is markedly reduced in the brain of Sirt KO mice that are, like CREB-deficient mice, poorly responsive to calorie restriction. Thus, the above circuitry, modulated by nutrient availability, links energy metabolism with neurotrophin signaling, participates in brain adaptation to nutrient restriction, and is potentially relevant to accelerated brain aging by overnutrition and diabetes.

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The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations.

Publication Date: 2012 Jan 10 PMID: 22190486
Authors: Yan, K. S. - Chia, L. A. - Li, X. - Ootani, A. - Su, J. - Lee, J. Y. - Su, N. - Luo, Y. - Heilshorn, S. C. - Amieva, M. R. - Sangiorgi, E. - Capecchi, M. R. - Kuo, C. J.
Journal: Proc Natl Acad Sci U S A

The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1(+) ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1(+) ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5(+) ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.

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Signal transducer and activator of transcription 3 (STAT3) and survivin induction by varicella-zoster virus promote replication and skin pathogenesis.

Publication Date: 2012 Jan 10 PMID: 22190485
Authors: Sen, N. - Che, X. - Rajamani, J. - Zerboni, L. - Sung, P. - Ptacek, J. - Arvin, A. M.
Journal: Proc Natl Acad Sci U S A

Varicella-zoster virus (VZV) is a human alpha-herpesvirus that causes varicella (chickenpox) during primary infection and zoster (shingles) upon reactivation. Like other viruses, VZV must subvert the intrinsic antiviral defenses of differentiated human cells to produce progeny virions. Accordingly, VZV inhibits the activation of the cellular transcription factors IFN regulatory factor 3 (IRF3) and signal transducers and activators of transcription 1 (STAT1), thereby downregulating antiviral factors, including IFNs. Conversely, in this study, we found that VZV triggers STAT3 phosphorylation in cells infected in vitro and in human skin xenografts in SCID mice in vivo and that STAT3 activation induces the anti-apoptotic protein survivin. Small-molecule inhibitors of STAT3 phosphorylation and survivin restrict VZV replication in vitro, and VZV infection of skin xenografts in vivo is markedly impaired by the administration of the phospho-STAT3 inhibitor S3I-201. STAT3 and survivin are required for malignant transformation caused by gamma-herpesviruses, such as Kaposi's sarcoma virus. We show that STAT3 activation is also critical for VZV, a nononcogenic herpesvirus, via a survivin-dependent mechanism. Furthermore, STAT3 activation is critical for the life cycle of the virus because VZV skin infection is necessary for viral transmission and persistence in the human population. Therefore, we conclude that takeover of this major cell-signaling pathway is necessary, independent of cell transformation, for herpesvirus pathogenesis and that STAT3 activation and up-regulation of survivin is a common mechanism important for the pathogenesis of lytic as well as tumorigenic herpesviruses.

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DNA methyltransferase 3a limits the expression of interleukin-13 in T helper 2 cells and allergic airway inflammation.

Publication Date: 2012 Jan 10 PMID: 22190484
Authors: Yu, Q. - Zhou, B. - Zhang, Y. - Nguyen, E. T. - Du, J. - Glosson, N. L. - Kaplan, M. H.
Journal: Proc Natl Acad Sci U S A

The inverse correlation between DNA methylation and lineage-specific gene expression during T helper cell development is well documented. However, the specific functions of the de novo methyltransferases Dnmt3a and Dnmt3b in cytokine gene regulation have not been defined. We demonstrate that the expression of Dnmt3a and Dnmt3b are induced to a greater extent in T helper 2 (Th2) cells than in T helper 1 cells during polarization. Using conditional mutant mice, we determined that Dnmt3a, but not Dnmt3b, regulated expression of T helper cell cytokine genes, with the Il13 gene most prominently affected. Dnmt3a deficiency was accompanied by decreases in DNA methylation and changes in the H3K27 acetylation/methylation status at the Il13 locus. Dnmt3a-dependent regulation of Il13 also occurred in vivo because Dnmt3a(fl/fl)Cd4cre mice exhibited increased lung inflammation in a murine asthma model, compared with littermate controls. Based on these observations, we conclude that Dnmt3a is required for controlling normal Il13 gene expression and functions as a rate-limiting factor to restrict T helper 2-mediated inflammation.

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Gene order and chromosome dynamics coordinate spatiotemporal gene expression during the bacterial growth cycle.

Publication Date: 2012 Jan 10 PMID: 22184251
Authors: Sobetzko, P. - Travers, A. - Muskhelishvili, G.
Journal: Proc Natl Acad Sci U S A

In Escherichia coli crosstalk between DNA supercoiling, nucleoid-associated proteins and major RNA polymerase sigma initiation factors regulates growth phase-dependent gene transcription. We show that the highly conserved spatial ordering of relevant genes along the chromosomal replichores largely corresponds both to their temporal expression patterns during growth and to an inferred gradient of DNA superhelical density from the origin to the terminus. Genes implicated in similar functions are related mainly in trans across the chromosomal replichores, whereas DNA-binding transcriptional regulators interact predominantly with targets in cis along the replichores. We also demonstrate that macrodomains (the individual structural partitions of the chromosome) are regulated differently. We infer that spatial and temporal variation of DNA superhelicity during the growth cycle coordinates oxygen and nutrient availability with global chromosome structure, thus providing a mechanistic insight into how the organization of a complete bacterial chromosome encodes a spatiotemporal program integrating DNA replication and global gene expression.

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The nuclear receptor REV-ERBalpha mediates circadian regulation of innate immunity through selective regulation of inflammatory cytokines.

Publication Date: 2012 Jan 10 PMID: 22184247
Authors: Gibbs, J. E. - Blaikley, J. - Beesley, S. - Matthews, L. - Simpson, K. D. - Boyce, S. H. - Farrow, S. N. - Else, K. J. - Singh, D. - Ray, D. W. - Loudon, A. S.
Journal: Proc Natl Acad Sci U S A

Diurnal variation in inflammatory and immune function is evident in the physiology and pathology of humans and animals, but molecular mechanisms and mediating cell types that provide this gating remain unknown. By screening cytokine responses in mice to endotoxin challenge at different times of day, we reveal that the magnitude of response exhibited pronounced temporal dependence, yet only within a subset of proinflammatory cytokines. Disruption of the circadian clockwork in macrophages (primary effector cells of the innate immune system) by conditional targeting of a key clock gene (bmal1) removed all temporal gating of endotoxin-induced cytokine response in cultured cells and in vivo. Loss of circadian gating was coincident with suppressed rev-erbalpha expression, implicating this nuclear receptor as a potential link between the clock and inflammatory pathways. This finding was confirmed in vivo and in vitro through genetic and pharmacological modulation of REV-ERBalpha activity. Circadian gating of endotoxin response was lost in rev-erbalpha(-/-) mice and in cultured macrophages from these animals, despite maintenance of circadian rhythmicity within these cells. Using human macrophages, which show circadian clock gene oscillations and rhythmic endotoxin responses, we demonstrate that administration of a synthetic REV-ERB ligand, or genetic knockdown of rev-erbalpha expression, is effective at modulating the production and release of the proinflammatory cytokine IL-6. This work demonstrates that the macrophage clockwork provides temporal gating of systemic responses to endotoxin, and identifies REV-ERBalpha as the key link between the clock and immune function. REV-ERBalpha may therefore represent a unique therapeutic target in human inflammatory disease.

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Metagenomic systems biology of the human gut microbiome reveals topological shifts associated with obesity and inflammatory bowel disease.

Publication Date: 2012 Jan 10 PMID: 22184244
Authors: Greenblum, S. - Turnbaugh, P. J. - Borenstein, E.
Journal: Proc Natl Acad Sci U S A

The human microbiome plays a key role in a wide range of host-related processes and has a profound effect on human health. Comparative analyses of the human microbiome have revealed substantial variation in species and gene composition associated with a variety of disease states but may fall short of providing a comprehensive understanding of the impact of this variation on the community and on the host. Here, we introduce a metagenomic systems biology computational framework, integrating metagenomic data with an in silico systems-level analysis of metabolic networks. Focusing on the gut microbiome, we analyze fecal metagenomic data from 124 unrelated individuals, as well as six monozygotic twin pairs and their mothers, and generate community-level metabolic networks of the microbiome. Placing variations in gene abundance in the context of these networks, we identify both gene-level and network-level topological differences associated with obesity and inflammatory bowel disease (IBD). We show that genes associated with either of these host states tend to be located at the periphery of the metabolic network and are enriched for topologically derived metabolic "inputs." These findings may indicate that lean and obese microbiomes differ primarily in their interface with the host and in the way they interact with host metabolism. We further demonstrate that obese microbiomes are less modular, a hallmark of adaptation to low-diversity environments. We additionally link these topological variations to community species composition. The system-level approach presented here lays the foundation for a unique framework for studying the human microbiome, its organization, and its impact on human health.

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Safe storage and effective monitoring of CO2 in depleted gas fields.

Publication Date: 2012 Jan 10 PMID: 22184225
Authors: Jenkins, C. R. - Cook, P. J. - Ennis-King, J. - Undershultz, J. - Boreham, C. - Dance, T. - de Caritat, P. - Etheridge, D. M. - Freifeld, B. M. - Hortle, A. - Kirste, D. - Paterson, L. - Pevzner, R. - Schacht, U. - Sharma, S. - Stalker, L. - Urosevic, M.
Journal: Proc Natl Acad Sci U S A

Carbon capture and storage (CCS) is vital to reduce CO(2) emissions to the atmosphere, potentially providing 20% of the needed reductions in global emissions. Research and demonstration projects are important to increase scientific understanding of CCS, and making processes and results widely available helps to reduce public concerns, which may otherwise block this technology. The Otway Project has provided verification of the underlying science of CO(2) storage in a depleted gas field, and shows that the support of all stakeholders can be earned and retained. Quantitative verification of long-term storage has been demonstrated. A direct measurement of storage efficiency has been made, confirming that CO(2) storage in depleted gas fields can be safe and effective, and that these structures could store globally significant amounts of CO(2).

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Evidence for formation of DNA repair centers and dose-response nonlinearity in human cells.

Publication Date: 2012 Jan 10 PMID: 22184222
Authors: Neumaier, T. - Swenson, J. - Pham, C. - Polyzos, A. - Lo, A. T. - Yang, P. - Dyball, J. - Asaithamby, A. - Chen, D. J. - Bissell, M. J. - Thalhammer, S. - Costes, S. V.
Journal: Proc Natl Acad Sci U S A

The concept of DNA "repair centers" and the meaning of radiation-induced foci (RIF) in human cells have remained controversial. RIFs are characterized by the local recruitment of DNA damage sensing proteins such as p53 binding protein (53BP1). Here, we provide strong evidence for the existence of repair centers. We used live imaging and mathematical fitting of RIF kinetics to show that RIF induction rate increases with increasing radiation dose, whereas the rate at which RIFs disappear decreases. We show that multiple DNA double-strand breaks (DSBs) 1 to 2 mum apart can rapidly cluster into repair centers. Correcting mathematically for the dose dependence of induction/resolution rates, we observe an absolute RIF yield that is surprisingly much smaller at higher doses: 15 RIF/Gy after 2 Gy exposure compared to approximately 64 RIF/Gy after 0.1 Gy. Cumulative RIF counts from time lapse of 53BP1-GFP in human breast cells confirmed these results. The standard model currently in use applies a linear scale, extrapolating cancer risk from high doses to low doses of ionizing radiation. However, our discovery of DSB clustering over such large distances casts considerable doubts on the general assumption that risk to ionizing radiation is proportional to dose, and instead provides a mechanism that could more accurately address risk dose dependency of ionizing radiation.

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Modularity of a carbon-fixing protein organelle.

Publication Date: 2012 Jan 10 PMID: 22184212
Authors: Bonacci, W. - Teng, P. K. - Afonso, B. - Niederholtmeyer, H. - Grob, P. - Silver, P. A. - Savage, D. F.
Journal: Proc Natl Acad Sci U S A

Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO(2)-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO(2). Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO(2) in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures.

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Correction for Ribas et al., Myeloid-specific estrogen receptor alpha deficiency impairs metabolic homeostasis and accelerates atherosclerotic lesion development.

Publication Date: 2012 Jan 10 PMID: 22178755
Authors:
Journal: Proc Natl Acad Sci U S A

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Candida albicans Dicer (CaDcr1) is required for efficient ribosomal and spliceosomal RNA maturation.

Publication Date: 2012 Jan 10 PMID: 22173636
Authors: Bernstein, D. A. - Vyas, V. K. - Weinberg, D. E. - Drinnenberg, I. A. - Bartel, D. P. - Fink, G. R.
Journal: Proc Natl Acad Sci U S A

The generation of mature functional RNAs from nascent transcripts requires the precise and coordinated action of numerous RNAs and proteins. One such protein family, the ribonuclease III (RNase III) endonucleases, includes Rnt1, which functions in fungal ribosome and spliceosome biogenesis, and Dicer, which generates the siRNAs of the RNAi pathway. The recent discovery of small RNAs in Candida albicans led us to investigate the function of C. albicans Dicer (CaDcr1). CaDcr1 is capable of generating siRNAs in vitro and is required for siRNA generation in vivo. In addition, CaDCR1 complements a Dicer knockout in Saccharomyces castellii, restoring RNAi-mediated gene repression. Unexpectedly, deletion of the C. albicans CaDCR1 results in a severe slow-growth phenotype, whereas deletion of another core component of the RNAi pathway (CaAGO1) has little effect on growth, suggesting that CaDCR1 may have an essential function in addition to producing siRNAs. Indeed CaDcr1, the sole functional RNase III enzyme in C. albicans, has additional functions: it is required for cleavage of the 3' external transcribed spacer from unprocessed pre-rRNA and for processing the 3' tail of snRNA U4. Our results suggest two models whereby the RNase III enzymes of a fungal ancestor, containing both a canonical Dicer and Rnt1, evolved through a series of gene-duplication and gene-loss events to generate the variety of RNase III enzymes found in modern-day budding yeasts.

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Correction for Chen et al., Tomosyn-dependent regulation of synaptic transmission is required for a late phase of associative odor memory.

Publication Date: 2012 Jan 10 PMID: 22171013
Authors:
Journal: Proc Natl Acad Sci U S A

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QnAs with John A. Rogers.

Publication Date: 2012 Jan 10 PMID: 22160717
Authors: Nair, P.
Journal: Proc Natl Acad Sci U S A

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Structural basis for gating charge movement in the voltage sensor of a sodium channel.

Publication Date: 2012 Jan 10 PMID: 22160714
Authors: Yarov-Yarovoy, V. - Decaen, P. G. - Westenbroek, R. E. - Pan, C. Y. - Scheuer, T. - Baker, D. - Catterall, W. A.
Journal: Proc Natl Acad Sci U S A

Voltage-dependent gating of ion channels is essential for electrical signaling in excitable cells, but the structural basis for voltage sensor function is unknown. We constructed high-resolution structural models of resting, intermediate, and activated states of the voltage-sensing domain of the bacterial sodium channel NaChBac using the Rosetta modeling method, crystal structures of related channels, and experimental data showing state-dependent interactions between the gating charge-carrying arginines in the S4 segment and negatively charged residues in neighboring transmembrane segments. The resulting structural models illustrate a network of ionic and hydrogen-bonding interactions that are made sequentially by the gating charges as they move out under the influence of the electric field. The S4 segment slides 6-8 A outward through a narrow groove formed by the S1, S2, and S3 segments, rotates approximately 30 degrees , and tilts sideways at a pivot point formed by a highly conserved hydrophobic region near the middle of the voltage sensor. The S4 segment has a 3(10)-helical conformation in the narrow inner gating pore, which allows linear movement of the gating charges across the inner one-half of the membrane. Conformational changes of the intracellular one-half of S4 during activation are rigidly coupled to lateral movement of the S4-S5 linker, which could induce movement of the S5 and S6 segments and open the intracellular gate of the pore. We confirmed the validity of these structural models by comparing with a high-resolution structure of a NaChBac homolog and showing predicted molecular interactions of hydrophobic residues in the S4 segment in disulfide-locking studies.

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Specific neural substrate linking respiration to locomotion.

Publication Date: 2012 Jan 10 PMID: 22160700
Authors: Gariepy, J. F. - Missaghi, K. - Chevallier, S. - Chartre, S. - Robert, M. - Auclair, F. - Lund, J. P. - Dubuc, R.
Journal: Proc Natl Acad Sci U S A

When animals move, respiration increases to adapt for increased energy demands; the underlying mechanisms are still not understood. We investigated the neural substrates underlying the respiratory changes in relation to movement in lampreys. We showed that respiration increases following stimulation of the mesencephalic locomotor region (MLR) in an in vitro isolated preparation, an effect that persists in the absence of the spinal cord and caudal brainstem. By using electrophysiological and anatomical techniques, including whole-cell patch recordings, we identified a subset of neurons located in the dorsal MLR that send direct inputs to neurons in the respiratory generator. In semi-intact preparations, blockade of this region with 6-cyano-7-nitroquinoxaline-2,3-dione and (2R)-amino-5-phosphonovaleric acid greatly reduced the respiratory increases without affecting the locomotor movements. These results show that neurons in the respiratory generator receive direct glutamatergic connections from the MLR and that a subpopulation of MLR neurons plays a key role in the respiratory changes linked to movement.

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Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin.

Publication Date: 2012 Jan 10 PMID: 22160674
Authors: Liu, J. - Xu, Y. - Stoleru, D. - Salic, A.
Journal: Proc Natl Acad Sci U S A

Synthesis of many proteins is tightly controlled at the level of translation, and plays an essential role in fundamental processes such as cell growth and proliferation, signaling, differentiation, or death. Methods that allow imaging and identification of nascent proteins are critical for dissecting regulation of translation, both spatially and temporally, particularly in whole organisms. We introduce a simple and robust chemical method to image and affinity-purify nascent proteins in cells and in animals, based on an alkyne analog of puromycin, O-propargyl-puromycin (OP-puro). OP-puro forms covalent conjugates with nascent polypeptide chains, which are rapidly turned over by the proteasome and can be visualized or captured by copper(I)-catalyzed azide-alkyne cycloaddition. Unlike methionine analogs, OP-puro does not require methionine-free conditions and, uniquely, can be used to label and assay nascent proteins in whole organisms. This strategy should have broad applicability for imaging protein synthesis and for identifying proteins synthesized under various physiological and pathological conditions in vivo.

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An antinociceptive role for substance P in acid-induced chronic muscle pain.

Publication Date: 2012 Jan 10 PMID: 22084095
Authors: Lin, C. C. - Chen, W. N. - Chen, C. J. - Lin, Y. W. - Zimmer, A. - Chen, C. C.
Journal: Proc Natl Acad Sci U S A

Release of substance P (SP) from nociceptive nerve fibers and activation of its receptor neurokinin 1 (NK1) are important effectors in the transmission of pain signals. Nonetheless, the role of SP in muscle pain remains unknown. Here we show that a single i.m. acid injection in mice lacking SP signaling by deletion of the tachykinin precursor 1 (Tac1) gene or coadministration of NK1 receptor antagonists produces long-lasting hyperalgesia rather than the transient hyperalgesia seen in control animals. The inhibitory effect of SP was found exclusively in neurons expressing acid-sensing ion channel 3, where SP enhances M-channel-like potassium currents through the NK1 receptor in a G protein-independent but tyrosine kinase-dependent manner. Furthermore, the SP signaling could alter action potential thresholds and modulate the expression of TTX-resistant sodium currents in medium-sized muscle nociceptors. Thus, i.m. SP mediates an unconventional NK1 receptor signal pathway to inhibit acid activation in muscle nociceptors, resulting in an unexpected antinociceptive effect against chronic mechanical hyperalgesia, here induced by repeated i.m. acid injection.

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Wip1 promotes RUNX2-dependent apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents.

Publication Date: 2012 Jan 10 PMID: 22065775
Authors: Goloudina, A. R. - Tanoue, K. - Hammann, A. - Fourmaux, E. - Le Guezennec, X. - Bulavin, D. V. - Mazur, S. J. - Appella, E. - Garrido, C. - Demidov, O. N.
Journal: Proc Natl Acad Sci U S A

The inactivation of the p53 tumor suppressor pathway in many cancers often increases their resistance to anticancer therapy. Here we show that a previously proposed strategy directed to Wip1 inhibition could be ineffective in tumors lacking p53. On the contrary, Wip1 overexpression sensitized these tumors to chemotherapeutic agents. This effect was mediated through interaction between Wip1 and RUNX2 that resulted, in response to anticancer treatment, in RUNX2-dependent transcriptional induction of the proapoptotic Bax protein. The potentiating effects of Wip1 overexpression on chemotherapeutic agents were directed only to tumor cells lacking p53. The overexpression of Wip1 in normal tissues provided protection from cisplatin-induced apoptosis through decreased strength of upstream signaling to p53. Thus, Wip1 phosphatase promotes apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents.

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Making short-term climate forecasts useful: Linking science and action.

Publication Date: 2012 Jan 12 PMID: 20133668
Authors: Buizer, J. - Jacobs, K. - Cash, D.
Journal: Proc Natl Acad Sci U S A

This paper discusses the evolution of scientific and social understanding that has led to the development of knowledge systems supporting the application of El Nino-Southern Oscillation (ENSO) forecasts, including the development of successful efforts to connect climate predictions with sectoral applications and actions "on the ground". The evolution of "boundary-spanning" activities to connect science and decisionmaking is then discussed, setting the stage for a report of outcomes from an international workshop comprised of producers, translators, and users of climate predictions. The workshop, which focused on identifying critical boundary-spanning features of successful boundary organizations, included participants from Australia, Hawaii, and the Pacific Islands, the US Pacific Northwest, and the state of Ceara in northwestern Brazil. Workshop participants agreed that boundary organizations have multiple roles including those of information broker, convenor of forums for engagement, translator of scientific information, arbiter of access to knowledge, and exemplar of adaptive behavior. Through these roles, boundary organizations will ensure the stability of the knowledge system in a changing political, economic, and climatic context. The international examples reviewed in this workshop demonstrated an interesting case of convergent evolution, where organizations that were very different in origin evolved toward similar structures and individuals engaged in them had similar experiences to share. These examples provide evidence that boundary organizations and boundary-spanners fill some social/institutional roles that are independent of culture.

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