PNAS

Reply to Wernick et al.: Global scale quantification of forest change.

Publication Date: 2010 Aug 30 PMID: 20805517
Authors: Hansen, M. C. - Stehman, S. V. - Potapov, P. V.
Journal: Proc Natl Acad Sci U S A

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Quantifying forest change.

Publication Date: 2010 Aug 30 PMID: 20805516
Authors: Wernick, I. K. - Waggoner, P. E. - Kauppi, P. E. - Sedjo, R. A. - Ausubel, J. H.
Journal: Proc Natl Acad Sci U S A

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Catalytic and chaperone-like functions in an intrinsically disordered protein associated with desiccation tolerance.

Publication Date: 2010 Aug 30 PMID: 20805515
Authors: Chakrabortee, S. - Meersman, F. - Kaminski Schierle, G. S. - Bertoncini, C. W. - McGee, B. - Kaminski, C. F. - Tunnacliffe, A.
Journal: Proc Natl Acad Sci U S A

Intrinsically disordered proteins (IDPs) lack well-defined structure but are widely represented in eukaryotic proteomes. Although the functions of most IDPs are not understood, some have been shown to have molecular recognition and/or regulatory roles where their disordered nature might be advantageous. Anhydrin is an uncharacterized IDP induced by dehydration in an anhydrobiotic nematode, Aphelenchus avenae. We show here that anhydrin is a moonlighting protein with two novel, independent functions relating to desiccation tolerance. First, it has a chaperone-like activity that can reduce desiccation-induced enzyme aggregation and inactivation in vitro. When expressed in a human cell line, anhydrin localizes to the nucleus and reduces the propensity of a polyalanine expansion protein associated with oculopharyngeal muscular dystrophy to form aggregates. This in vivo activity is distinguished by a loose association of anhydrin with its client protein, consistent with a role as a molecular shield. In addition, anhydrin exhibits a second function as an endonuclease whose substrates include supercoiled, linear, and chromatin linker DNA. This nuclease activity could be involved in either repair of desiccation-induced DNA damage incurred during anhydrobiosis or in apoptotic or necrotic processes, for example, but it is particularly unexpected for anhydrin because IDP functions defined to date anticorrelate with enzyme activity. Enzymes usually require precise three-dimensional positioning of residues at the active site, but our results suggest this need not be the case. Anhydrin therefore extends the range of IDP functional categories to include catalysis and highlights the potential for the discovery of new functions in disordered proteomes.

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From the Cover: An aberrant island-dwelling theropod dinosaur from the Late Cretaceous of Romania.

Publication Date: 2010 Aug 31 PMID: 20805514
Authors: Csiki, Z. - Vremir, M. - Brusatte, S. L. - Norell, M. A.
Journal: Proc Natl Acad Sci U S A

Islands are noted for the occurrence of aberrant, endemic, and dwarfed taxa (the "island effect"). Late Cretaceous vertebrate assemblages of Romania and elsewhere in Europe are classic examples of island faunas in the fossil record, and are characterized by dwarfed herbivorous dinosaurs and other endemic taxa that are noticeably primitive relative to their mainland contemporaries. Fossils of the predators inhabiting the European paleoislands, however, are exceptionally rare and fragmentary. We describe a new dromaeosaurid theropod, based on an articulated skeleton from the Maastrichtian of Romania, which represents the most complete predatory dinosaur from the middle to Late Cretaceous of Europe. This taxon is characterized by a peculiar body plan, most notably extensive fusion in the hand and distal hindlimb, a highly retroverted pelvis with enlarged femoral muscle attachments, and a pair of hyperextensive pedal claws. However, unlike the island-dwelling herbivorous dinosaurs, its closest relatives are contemporary similar-sized Laurasian taxa, indicating faunal connections between Asia and the European islands late into the Cretaceous. This theropod provides support for the aberrant nature of the Late Cretaceous European island-dwelling dinosaurs, but indicates that predators on these islands were not necessarily small, geographically endemic, or primitive.

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Nonuniversal power law scaling in the probability distribution of scientific citations.

Publication Date: 2010 Aug 30 PMID: 20805513
Authors: Peterson, G. J. - Presse, S. - Dill, K. A.
Journal: Proc Natl Acad Sci U S A

We develop a model for the distribution of scientific citations. The model involves a dual mechanism: in the direct mechanism, the author of a new paper finds an old paper A and cites it. In the indirect mechanism, the author of a new paper finds an old paper A only via the reference list of a newer intermediary paper B, which has previously cited A. By comparison to citation databases, we find that papers having few citations are cited mainly by the direct mechanism. Papers already having many citations ("classics") are cited mainly by the indirect mechanism. The indirect mechanism gives a power-law tail. The "tipping point" at which a paper becomes a classic is about 25 citations for papers published in the Institute for Scientific Information (ISI) Web of Science database in 1981, 31 for Physical Review D papers published from 1975-1994, and 37 for all publications from a list of high h-index chemists assembled in 2007. The power-law exponent is not universal. Individuals who are highly cited have a systematically smaller exponent than individuals who are less cited.

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The liquid-liquid phase transition in silicon revealed by snapshots of valence electrons.

Publication Date: 2010 Aug 30 PMID: 20805512
Authors: Beye, M. - Sorgenfrei, F. - Schlotter, W. F. - Wurth, W. - Fohlisch, A.
Journal: Proc Natl Acad Sci U S A

The basis for the anomalies of water is still mysterious. Quite generally tetrahedrally coordinated systems, also silicon, show similar thermodynamic behavior but lack-like water-a thorough explanation. Proposed models-controversially discussed-explain the anomalies as a remainder of a first-order phase transition between high and low density liquid phases, buried deeply in the "no man's land"-a part of the supercooled liquid region where rapid crystallization prohibits any experimental access. Other explanations doubt the existence of the phase transition and its first-order nature. Here, we provide experimental evidence for the first-order-phase transition in silicon. With ultrashort optical pulses of femtosecond duration we instantaneously heat the electronic system of silicon while the atomic structure as defined by the much heavier nuclear system remains initially unchanged. Only on a picosecond time scale the energy is transferred into the atomic lattice providing the energy to drive the phase transitions. With femtosecond X-ray pulses from FLASH, the free-electron laser at Hamburg, we follow the evolution of the valence electronic structure during this process. As the relevant phases are easily distinguishable in their electronic structure, we track how silicon melts into the low-density-liquid phase while a second phase transition into the high-density-liquid phase only occurs after the latent heat for the first-order phase transition has been transferred to the atomic structure. Proving the existence of the liquid-liquid phase transition in silicon, the hypothesized liquid-liquid scenario for water is strongly supported.

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No evidence of nanodiamonds in Younger-Dryas sediments to support an impact event.

Publication Date: 2010 Aug 30 PMID: 20805511
Authors: Daulton, T. L. - Pinter, N. - Scott, A. C.
Journal: Proc Natl Acad Sci U S A

The causes of the late Pleistocene megafaunal extinctions in North America, disappearance of Clovis paleoindian lithic technology, and abrupt Younger-Dryas (YD) climate reversal of the last deglacial warming in the Northern Hemisphere remain an enigma. A controversial hypothesis proposes that one or more cometary airbursts/impacts barraged North America approximately 12,900 cal yr B.P. and caused these events. Most evidence supporting this hypothesis has been discredited except for reports of nanodiamonds (including the rare hexagonal polytype) in Bolling-Allerod-YD-boundary sediments. The hexagonal polytype of diamond, lonsdaleite, is of particular interest because it is often associated with shock pressures related to impacts where it has been found to occur naturally. Unfortunately, previous reports of YD-boundary nanodiamonds have left many unanswered questions regarding the nature and occurrence of the nanodiamonds. Therefore, we examined carbon-rich materials isolated from sediments dated 15,818 cal yr B.P. to present (including the Bolling-Allerod-YD boundary). No nanodiamonds were found in our study. Instead, graphene- and graphene/graphane-oxide aggregates are ubiquitous in all specimens examined. We demonstrate that previous studies misidentified graphene/graphane-oxide aggregates as hexagonal diamond and likely misidentified graphene as cubic diamond. Our results cast doubt upon one of the last widely discussed pieces of evidence supporting the YD impact hypothesis.

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Early evidence (ca. 12,000 B.P.) for feasting at a burial cave in Israel.

Publication Date: 2010 Aug 31 PMID: 20805510
Authors: Munro, N. D. - Grosman, L.
Journal: Proc Natl Acad Sci U S A

Feasting is one of humanity's most universal and unique social behaviors. Although evidence for feasting is common in the early agricultural societies of the Neolithic, evidence in pre-Neolithic contexts is more elusive. We found clear evidence for feasting on wild cattle and tortoises at Hilazon Tachtit cave, a Late Epipaleolithic (12,000 calibrated years B.P.) burial site in Israel. This includes unusually high densities of butchered tortoise and wild cattle remains in two structures, the unique location of the feasting activity in a burial cave, and the manufacture of two structures for burial and related feasting activities. The results indicate that community members coalesced at Hilazon to engage in special rituals to commemorate the burial of the dead and that feasts were central elements in these important events. Feasts likely served important roles in the negotiation and solidification of social relationships, the integration of communities, and the mitigation of scalar stress. These and other social changes in the Natufian period mark significant changes in human social complexity that continued into the Neolithic period. Together, social and economic change signal the very beginning of the agricultural transition.

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CCAAT/enhancer binding protein delta (C/EBP{delta}, CEBPD)-mediated nuclear import of FANCD2 by IPO4 augments cellular response to DNA damage.

Publication Date: 2010 Aug 30 PMID: 20805509
Authors: Wang, J. - Sarkar, T. R. - Zhou, M. - Sharan, S. - Ritt, D. A. - Veenstra, T. D. - Morrison, D. K. - Huang, A. M. - Sterneck, E.
Journal: Proc Natl Acad Sci U S A

Maintenance of genomic integrity is an essential cellular function. We previously reported that the transcription factor and tumor suppressor CCAAT/enhancer binding protein delta (C/EBPdelta, CEBPD; also known as "NFIL-6beta") promotes genomic stability. However, the molecular mechanism was not known. Here, we show that C/EBPdelta is a DNA damage-induced gene, which supports survival of mouse bone marrow cells, mouse embryo fibroblasts (MEF), human fibroblasts, and breast tumor cells in response to the DNA cross-linking agent mitomycin C (MMC). Using gene knockout, protein depletion, and overexpression studies, we found that C/EBPdelta promotes monoubiquitination of the Fanconi anemia complementation group D2 protein (FANCD2), which is necessary for its function in replication-associated DNA repair. C/EBPdelta interacts with FANCD2 and importin 4 (IPO4, also known as "Imp4" and "RanBP4") via separate domains, mediating FANCD2-IPO4 association and augmenting nuclear import of FANCD2, a prerequisite for its monoubiquitination. This study identifies a transcription-independent activity of C/EBPdelta in the DNA damage response that may in part underlie its tumor suppressor function. Furthermore, we report a function of IPO4 and nuclear import in the Fanconi anemia pathway of DNA repair.

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Mechanism of Trypanosoma brucei gambiense (group 1) resistance to human trypanosome lytic factor.

Publication Date: 2010 Aug 30 PMID: 20805508
Authors: Kieft, R. - Capewell, P. - Turner, C. M. - Veitch, N. J. - Macleod, A. - Hajduk, S.
Journal: Proc Natl Acad Sci U S A

Human innate immunity against most African trypanosomes, including Trypanosoma brucei brucei, is mediated by a minor subclass of toxic serum HDL, called trypanosome lytic factor-1 (TLF-1). This HDL contains two primate specific proteins, apolipoprotein L-1 and haptoglobin (Hp)-related protein, as well as apolipoprotein A-1. These assembled proteins provide a powerful defense against trypanosome infection. Trypanosoma brucei rhodesiense causes human African sleeping sickness because it has evolved an inhibitor of TLF-1, serum resistance-associated (SRA) protein. Trypanosoma brucei gambiense lacks the SRA gene, yet it infects humans. As transfection of T. b. gambiense (group 1) is not possible, we initially used in vitro-selected TLF-1-resistant T. b. brucei to examine SRA-independent mechanisms of TLF-1 resistance. Here we show that TLF-1 resistance in T. b. brucei is caused by reduced expression of the Hp/Hb receptor gene (TbbHpHbR). Importantly, T. b. gambiense (group 1) also showed a marked reduction in uptake of TLF-1 and a corresponding decrease in expression of T. b. gambiense Hp/Hb receptor (TbgHpHbR). Ectopic expression of TbbHpHbR in TLF-1-resistant T. b. brucei rescued TLF-1 uptake, demonstrating that decreased TbbHpHbR expression conferred TLF-1 resistance. Ectopic expression of TbgHpHbR in TLF-1-resistant T. b. brucei failed to rescue TLF-1 killing, suggesting that coding sequence changes altered Hp/Hb receptor binding affinity for TLF-1. We propose that the combination of coding sequence mutations and decreased expression of TbgHpHbR directly contribute to parasite evasion of human innate immunity and infectivity of group 1 T. b. gambiense.

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Alterations in choice behavior by manipulations of world model.

Publication Date: 2010 Aug 30 PMID: 20805507
Authors: Green, C. S. - Benson, C. - Kersten, D. - Schrater, P.
Journal: Proc Natl Acad Sci U S A

How to compute initially unknown reward values makes up one of the key problems in reinforcement learning theory, with two basic approaches being used. Model-free algorithms rely on the accumulation of substantial amounts of experience to compute the value of actions, whereas in model-based learning, the agent seeks to learn the generative process for outcomes from which the value of actions can be predicted. Here we show that (i) "probability matching"-a consistent example of suboptimal choice behavior seen in humans-occurs in an optimal Bayesian model-based learner using a max decision rule that is initialized with ecologically plausible, but incorrect beliefs about the generative process for outcomes and (ii) human behavior can be strongly and predictably altered by the presence of cues suggestive of various generative processes, despite statistically identical outcome generation. These results suggest human decision making is rational and model based and not consistent with model-free learning.

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Identification of differential and functionally active miRNAs in both anaplastic lymphoma kinase (ALK)+ and ALK- anaplastic large-cell lymphoma.

Publication Date: 2010 Aug 30 PMID: 20805506
Authors: Merkel, O. - Hamacher, F. - Laimer, D. - Sifft, E. - Trajanoski, Z. - Scheideler, M. - Egger, G. - Hassler, M. R. - Thallinger, C. - Schmatz, A. - Turner, S. D. - Greil, R. - Kenner, L.
Journal: Proc Natl Acad Sci U S A

Aberrant anaplastic lymphoma kinase (ALK) expression is a defining feature of many human cancers and was identified first in anaplastic large-cell lymphoma (ALCL), an aggressive non-Hodgkin T-cell lymphoma. Since that time, many studies have set out to identify the mechanisms used by aberrant ALK toward tumorigenesis. We have identified a distinct profile of micro-RNAs (miRNAs) that characterize ALCL; furthermore, this profile distinguishes ALK(+) from ALK(-) subtypes, and thus points toward potential mechanisms of tumorigenesis induced by aberrant ALK. Using a nucleophosmin-ALK transgenic mouse model as well as human primary ALCL tumor tissues and human ALCL-derived cell lines, we reveal a set of overlapping deregulated miRNAs that might be implicated in the development and progression of ALCL. Importantly, ALK(+) and ALK(-) ALCL could be distinguished by a distinct profile of "oncomirs": Five members of the miR-17-92 cluster were expressed more highly in ALK(+) ALCL, whereas miR-155 was expressed more than 10-fold higher in ALK(-) ALCL. Moreover, miR-101 was down-regulated in all ALCL model systems, but its forced expression attenuated cell proliferation only in ALK(+) and not in ALK(-) cell lines, perhaps suggesting different modes of ALK-dependent regulation of its target proteins. Furthermore, inhibition of mTOR, which is targeted by miR-101, led to reduced tumor growth in engrafted ALCL mouse models. In addition to future therapeutical and diagnostic applications, it will be of interest to study the physiological implications and prognostic value of the identified miRNA profiles.

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Proline-rich tyrosine kinase-2 is critical for CD8 T-cell short-lived effector fate.

Publication Date: 2010 Aug 30 PMID: 20805505
Authors: Beinke, S. - Phee, H. - Clingan, J. M. - Schlessinger, J. - Matloubian, M. - Weiss, A.
Journal: Proc Natl Acad Sci U S A

T-cell interactions with antigen-presenting cells are important for CD8 T-cell effector or memory fate determination. The integrin leukocyte function-associated antigen-1 (LFA-1) mediates T-cell adhesion but the contribution of LFA-1-induced signaling pathways to T-cell responses is poorly understood. Here we demonstrate that proline-rich tyrosine kinase-2 (PYK2) deficiency impairs CD8 T-cell activation by synergistic LFA-1 and T-cell receptor stimulation. Furthermore, PYK2 is essential for LFA-1-mediated CD8 T-cell adhesion and LFA-1 costimulation of CD8 T-cell migration. During lymphocytic choriomeningitis virus infection in vivo, PYK2 deficiency results in a specific loss of short-lived effector CD8 T cells but does not affect memory-precursor CD8 T-cell development. Similarly, lack of LFA-1 primarily impairs the generation of short-lived effector cells. Thus, PYK2 facilitates LFA-1-dependent CD8 T-cell responses and promotes CD8 T-cell short-lived effector fate, suggesting that PYK2 may be an interesting therapeutic target to suppress exacerbated CD8 T-cell responses.

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Wnt/{beta}-catenin signaling is involved in the induction and maintenance of primitive hematopoiesis in the vertebrate embryo.

Publication Date: 2010 Aug 30 PMID: 20805504
Authors: Tran, H. T. - Sekkali, B. - Van Imschoot, G. - Janssens, S. - Vleminckx, K.
Journal: Proc Natl Acad Sci U S A

The formation of primitive (embryonic) blood in vertebrates is mediated by spatio-temporally restricted signaling between different tissue layers. In Xenopus, in which primitive blood originates in the ventral blood island, this involves the secretion of bone morphogenetic protein (BMP) ligands by the ectoderm that signal to the underlying mesoderm during gastrulation. Using novel transgenic reporter lines, we report that the canonical Wnt/beta-catenin pathway is also activated in the blood islands in Xenopus. Furthermore, Wnt-reporter activity was also detected in the blood islands of the mouse yolk sac. By using morpholino-mediated depletion in Xenopus, we identified Wnt4 as the ligand that is expressed in the mesoderm of the ventral blood island and is essential for the expression of hematopoietic and erythroid marker genes. Injection of an inducible Wnt-interfering construct further showed that, during gastrulation, Wnt/beta-catenin signaling is required both in the mesoderm and in the overlying ectoderm for the formation of the ventral blood island. Using recombination assays with embryonic explants, we document that ectodermal BMP4 expression is dependent on Wnt4 signals from the mesoderm. Our results thus reveal a unique role for Wnt4-mediated canonical signaling in the formation and maintenance of the ventral blood island in Xenopus.

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Genome mining and genetic analysis of cypemycin biosynthesis reveal an unusual class of posttranslationally modified peptides.

Publication Date: 2010 Aug 30 PMID: 20805503
Authors: Claesen, J. - Bibb, M.
Journal: Proc Natl Acad Sci U S A

Posttranslational modification of amino acids confers a range of structural features and activities on ribosomally synthesized peptides, many of which have potent antimicrobial or other biological activities. Cypemycin is an extensively modified linear peptide produced by Streptomyces sp. OH-4156 with potent in vitro activity against mouse leukemia cells. Cypemycin does not contain lanthionine bridges but exhibits some of the structural features of lantibiotics, notably dehydrated threonines (dehydrobutyrines) and a C-terminal S-[(Z)-2-aminovinyl]-D-cysteine. Consequently it was classified as a member of the lantibiotic family of posttranslationally modified peptides. Cypemycin also possesses two l-allo-isoleucine residues and an N-terminal N,N-dimethylalanine, both unique amino acid modifications. We identified and heterologously expressed the cypemycin biosynthetic gene cluster and performed a mutational analysis of each individual gene. We show that even the previously described modifications are carried out by unusual enzymes or via a modification pathway unrelated to lantibiotic biosynthesis. Bioinformatic analysis revealed the widespread occurrence of cypemycin-like gene clusters within the bacterial kingdom and in the Archaea. Cypemycin is the founding member of an unusual class of posttranslationally modified ribosomally synthesized peptides, the linaridins.

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Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis.

Publication Date: 2010 Aug 30 PMID: 20805502
Authors: Liu, W. T. - Yang, Y. L. - Xu, Y. - Lamsa, A. - Haste, N. M. - Yang, J. Y. - Ng, J. - Gonzalez, D. - Ellermeier, C. D. - Straight, P. D. - Pevzner, P. A. - Pogliano, J. - Nizet, V. - Pogliano, K. - Dorrestein, P. C.
Journal: Proc Natl Acad Sci U S A

During bacterial cannibalism, a differentiated subpopulation harvests nutrients from their genetically identical siblings to allow continued growth in nutrient-limited conditions. Hypothesis-driven imaging mass spectrometry (IMS) was used to identify metabolites active in a Bacillus subtilis cannibalism system in which sporulating cells lyse nonsporulating siblings. Two candidate molecules with sequences matching the products of skfA and sdpC, genes for the proposed cannibalistic factors sporulation killing factor (SKF) and sporulation delaying protein (SDP), respectively, were identified and the structures of the final products elucidated. SKF is a cyclic 26-amino acid (aa) peptide that is posttranslationally modified with one disulfide and one cysteine thioether bridged to the alpha-position of a methionine, a posttranslational modification not previously described in biology. SDP is a 42-residue peptide with one disulfide bridge. In spot test assays on solid medium, overproduced SKF and SDP enact a cannibalistic killing effect with SDP having higher potency. However, only purified SDP affected B. subtilis cells in liquid media in fluorescence microscopy and growth assays. Specifically, SDP treatment delayed growth in a concentration-dependent manner, caused increases in cell permeability, and ultimately caused cell lysis accompanied by the production of membrane tubules and spheres. Similarly, SDP but not SKF was able to inhibit the growth of the pathogens Staphylococcus aureus and Staphylococcus epidermidis with comparable IC(50) to vancomycin. This investigation, with the identification of SKF and SDP structures, highlights the strength of IMS in investigations of metabolic exchange of microbial colonies and also demonstrates IMS as a promising approach to discover novel biologically active molecules.

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Focal Mullerian duct retention in male mice with constitutively activated {beta}-catenin expression in the Mullerian duct mesenchyme.

Publication Date: 2010 Aug 30 PMID: 20805501
Authors: Tanwar, P. S. - Zhang, L. - Tanaka, Y. - Taketo, M. M. - Donahoe, P. K. - Teixeira, J. M.
Journal: Proc Natl Acad Sci U S A

Mullerian-inhibiting substance (MIS), which is produced by fetal Sertoli cells shortly after commitment of the bipotential gonads to testicular differentiation, causes Mullerian duct (MD) regression. In the fetal female gonads, MIS is not expressed and the MDs will differentiate into the internal female reproductive tract. We have investigated whether dysregulated beta-catenin activity affects MD regression by expressing a constitutively activated nuclear form of beta-catenin in the MD mesenchyme. We show that constitutively activated (CA) beta-catenin causes focal retention of MD tissue in the epididymides and vasa deferentia. In adult mutant mice, the retained MD tissues express alpha-smooth muscle actin and desmin, which are markers for uterine differentiation. MD retention inhibited the folding complexity of the developing epididymides and usually led to obstructive azoospermia by spermatoceles. The MDs of urogenital ridges from mutant female embryos showed less regression with added MIS in organ culture compared with control MDs when analyzed by whole mount in situ hybridization for Wnt7a as a marker for the MD epithelium. CA beta-catenin did not appear to affect expression of either MIS in the embryonic testes or its type II receptor (AMHR2) in the MD mesenchyme nor did it inhibit pSmad1/5/8 nuclear accumulation, suggesting that dysregulated beta-catenin must inhibit MD regression independently of MIS signaling. These studies suggest that dysregulated Wnt/beta-catenin signaling in the MD mesenchyme might also be a contributing factor in persistent Mullerian duct syndrome, a form of male pseudohermaphroditism, and development of spermatoceles.

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Ubiquitination of lysine-331 by Kaposi's sarcoma-associated herpesvirus protein K5 targets HFE for lysosomal degradation.

Publication Date: 2010 Aug 30 PMID: 20805500
Authors: Rhodes, D. A. - Boyle, L. H. - Boname, J. M. - Lehner, P. J. - Trowsdale, J.
Journal: Proc Natl Acad Sci U S A

The nonclassical MHC class I-related (MHC-I) molecule HFE controls cellular iron homeostasis by a mechanism that has not been fully elucidated. We examined the regulation of HFE by K5, the E3 ubiquitin ligase encoded by Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8), that is known to down-regulate classical MHC-I. K5 down-regulated HFE efficiently, using polyubiquitination of the membrane proximal lysine in the HFE cytoplasmic tail (K331), to target the molecule for degradation via ESCRT1/TSG101-dependent sorting from endosomes to multivesicular bodies (MVBs)/lysosomes. In the primary effusion lymphoma cell line BC-3, which carries latent KSHV, HFE was degraded rapidly upon virus reactivation. HFE was ubiquitinated on lysine-331 in unactivated BC-3 cells, conditions where K5 was not detectable, consistent with an endogenous E3 ubiquitin ligase controlling HFE expression. The results show regulated expression of HFE by ubiquitination, consistent with a role in cellular iron homeostasis, a molecular mechanism targeted by KSHV to achieve a positive iron balance.

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Endosomal-sorting complexes required for transport (ESCRT) pathway-dependent endosomal traffic regulates the localization of active Src at focal adhesions.

Publication Date: 2010 Aug 30 PMID: 20805499
Authors: Tu, C. - Ortega-Cava, C. F. - Winograd, P. - Stanton, M. J. - Reddi, A. L. - Dodge, I. - Arya, R. - Dimri, M. - Clubb, R. J. - Naramura, M. - Wagner, K. U. - Band, V. - Band, H.
Journal: Proc Natl Acad Sci U S A

Active Src localization at focal adhesions (FAs) is essential for cell migration. How this pool is linked mechanistically to the large pool of Src at late endosomes (LEs)/lysosomes (LY) is not well understood. Here, we used inducible Tsg101 gene deletion, TSG101 knockdown, and dominant-negative VPS4 expression to demonstrate that the localization of activated cellular Src and viral Src at FAs requires the endosomal-sorting complexes required for transport (ESCRT) pathway. Tsg101 deletion also led to impaired Src-dependent activation of STAT3 and focal adhesion kinase and reduced cell migration. Impairment of the ESCRT pathway or Rab7 function led to the accumulation of active Src at aberrant LE/LY compartments followed by its loss. Analyses using fluorescence recovery after photo-bleaching show that dynamic mobility of Src in endosomes is ESCRT pathway-dependent. These results reveal a critical role for an ESCRT pathway-dependent LE/LY trafficking step in Src function by promoting localization of active Src to FAs.

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Cdc42 interacting protein 4 (CIP4) is essential for integrin-dependent T-cell trafficking.

Publication Date: 2010 Aug 30 PMID: 20805498
Authors: Koduru, S. - Kumar, L. - Massaad, M. J. - Ramesh, N. - Le Bras, S. - Ozcan, E. - Oyoshi, M. K. - Kaku, M. - Fujiwara, Y. - Kremer, L. - King, S. - Fuhlbrigge, R. - Rodig, S. - Sage, P. - Carman, C. - Alcaide, P. - Luscinskas, F. W. - Geha, R. S.
Journal: Proc Natl Acad Sci U S A

The F-BAR domain containing protein CIP4 (Cdc42 interacting protein 4) interacts with Cdc42 and WASP/N-WASP and is thought to participate in the assembly of filamentous actin. CIP4(-/-) mice had normal T- and B-lymphocyte development but impaired T cell-dependent antibody production, IgG antibody affinity maturation, and germinal center (GC) formation, despite an intact CD40L-CD40 axis. CIP4(-/-) mice also had impaired contact hypersensitivity (CHS) to haptens, and their T cells failed to adoptively transfer CHS. Ovalbumin-activated CD4(+) effector T cells from CIP4(-/-)/OT-II mice migrated poorly to antigen-challenged skin. Activated CIP4(-/-) T cells exhibited impaired adhesion and polarization on immobilized VCAM-1 and ICAM-1 and defective arrest and transmigration across murine endothelial cell monolayers under shear flow conditions. These results demonstrate an important role for CIP4 in integrin-dependent T cell-dependent antibody responses and GC formation and in integrin-mediated recruitment of effector T cells to cutaneous sites of antigen-driven immune reactions.

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Central serotonin neurons are required for arousal to CO2.

Publication Date: 2010 Aug 30 PMID: 20805497
Authors: Buchanan, G. F. - Richerson, G. B.
Journal: Proc Natl Acad Sci U S A

There is a long-standing controversy about the role of serotonin in sleep/wake control, with competing theories that it either promotes sleep or causes arousal. Here, we show that there is a marked increase in wakefulness when all serotonin neurons are genetically deleted in mice hemizygous for ePet1-Cre and homozygous for floxed Lmx1b (Lmx1b(f/f/p)). However, this only occurs at cool ambient temperatures and can be explained by a thermoregulatory defect that leads to an increase in motor activity to generate heat. Because some serotonin neurons are stimulated by CO(2), and serotonin activates thalamocortical networks, we hypothesized that serotonin neurons cause arousal in response to hypercapnia. We found that Lmx1b(f/f/p) mice completely lacked any arousal response to inhalation of 10% CO(2) (with 21% O(2) in balance N(2)) but had normal arousal responses to hypoxia, sound, and air puff. We propose that serotonin neurons mediate the potentially life-saving arousal response to hypercapnia. Impairment of this response may contribute to sudden unexpected death in epilepsy, sudden infant death syndrome, and sleep apnea.

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Rapidly fatal myeloproliferative disorders in mice with deletion of Casitas B-cell lymphoma (Cbl) and Cbl-b in hematopoietic stem cells.

Publication Date: 2010 Aug 30 PMID: 20805496
Authors: Naramura, M. - Nandwani, N. - Gu, H. - Band, V. - Band, H.
Journal: Proc Natl Acad Sci U S A

Casitas B-cell lymphoma (Cbl)-family E3 ubiquitin ligases are negative regulators of tyrosine kinase signaling. Recent work has revealed a critical role of Cbl in the maintenance of hematopoietic stem cell (HSC) homeostasis, and mutations in CBL have been identified in myeloid malignancies. Here we show that, in contrast to Cbl or Cbl-b single-deficient mice, concurrent loss of Cbl and Cbl-b in the HSC compartment leads to an early-onset lethal myeloproliferative disease in mice. Cbl, Cbl-b double-deficient bone marrow cells are hypersensitive to cytokines, and show altered biochemical response to thrombopoietin. Thus, Cbl and Cbl-b play redundant but essential roles in HSC regulation, whose breakdown leads to hematological abnormalities that phenocopy crucial aspects of mutant Cbl-driven human myeloid malignancies.

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Embryonic gonadotropin-releasing hormone signaling is necessary for maturation of the male reproductive axis.

Publication Date: 2010 Aug 30 PMID: 20805495
Authors: Wen, S. - Ai, W. - Alim, Z. - Boehm, U.
Journal: Proc Natl Acad Sci U S A

Gonadotropin-releasing hormone (GnRH) signaling regulates reproductive physiology in mammals. GnRH is released by a subset of hypothalamic neurons and binds to GnRH receptor (GnRHR) on gonadotropes in the anterior pituitary gland to control production and secretion of gonadotropins that in turn regulate the activity of the gonads. Central control of reproduction is well understood in adult animals, but GnRH signaling has also been implicated in the development of the reproductive axis. To investigate the role of GnRH signaling during development, we selectively ablated GnRHR-expressing cells in mice. This genetic strategy permitted us to identify an essential stage in male reproductive axis development, which depends on embryonic GnRH signaling. Our experiments revealed a striking dichotomy in the gonadotrope population of the fetal anterior pituitary gland. We show that luteinizing hormone-expressing gonadotropes, but not follicle-stimulating hormone-expressing gonadotropes, express the GnRHR at embryonic day 16.75. Furthermore, we demonstrate that an embryonic increase in luteinizing hormone secretion is needed to promote development of follicle-stimulating hormone-expressing gonadotropes, which might be mediated by paracrine interactions within the pituitary. Moreover, migration of GnRH neurons into the hypothalamus appeared normal with appropriate axonal connections to the median eminence, providing genetic evidence against autocrine regulation of GnRH neurons. Surprisingly, genetic ablation of GnRHR expressing cells significantly increased the number of GnRH neurons in the anterior hypothalamus, suggesting an unexpected role of GnRH signaling in establishing the size of the GnRH neuronal population. Our experiments define a functional role of embryonic GnRH signaling.

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How to sample the carbon isotopes of tropical ecosystems without leaving your armchair.

Publication Date: 2010 Aug 30 PMID: 20805494
Authors: Ayliffe, L. K.
Journal: Proc Natl Acad Sci U S A

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Preference of RIG-I for short viral RNA molecules in infected cells revealed by next-generation sequencing.

Publication Date: 2010 Aug 30 PMID: 20805493
Authors: Baum, A. - Sachidanandam, R. - Garcia-Sastre, A.
Journal: Proc Natl Acad Sci U S A

Intracellular detection of virus infections is a critical component of innate immunity carried out by molecules known as pathogen recognition receptors (PRRs). Activation of PRRs by their respective pathogen-associated molecular patterns (PAMPs) leads to production of proinflamatory cytokines, including type I IFN, and the establishment of an antiviral state in the host. Out of all PRRs found to date, retinoic acid inducible gene I (RIG-I) has been shown to play a key role in recognition of RNA viruses. On the basis of in vitro and transfection studies, 5'ppp RNA produced during virus replication is thought to bind and activate this important sensor. However, the nature of RNA molecules that interact with endogenous RIG-I during the course of viral infection has not been determined. In this work we use next-generation RNA sequencing to show that RIG-I preferentially associates with shorter, 5'ppp containing viral RNA molecules in infected cells. We found that during Sendai infection RIG-I specifically bound the genome of the defective interfering (DI) particle and did not bind the full-length virus genome or any other viral RNAs. In influenza-infected cells RIG-I preferentially associated with shorter genomic segments as well as subgenomic DI particles. Our analysis for the first time identifies RIG-I PAMPs under natural infection conditions and implies that full-length genomes of single segmented RNA virus families are not bound by RIG-I during infection.

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Comprehensive phylogeny of apid bees reveals the evolutionary origins and antiquity of cleptoparasitism.

Publication Date: 2010 Aug 30 PMID: 20805492
Authors: Cardinal, S. - Straka, J. - Danforth, B. N.
Journal: Proc Natl Acad Sci U S A

Apidae is the most speciose and behaviorally diverse family of bees. It includes solitary, eusocial, socially parasitic, and an exceptionally high proportion of cleptoparasitic species. Cleptoparasitic bees, which are brood parasites in the nests of other bees, have long caused problems in resolving the phylogenetic relationships within Apidae based on morphological data because of the tendency for parasites to converge on a suite of traits, making it difficult to differentiate similarity caused by common ancestry from convergence. Here, we resolve the evolutionary history of apid cleptoparasitism by conducting a detailed, comprehensive molecular phylogenetic analysis of all 33 apid tribes (based on 190 species), including representatives from every hypothesized origin of cleptoparasitism. Based on Bayesian ancestral state reconstruction, we show that cleptoparasitism has arisen just four times in Apidae, which is fewer times than previously estimated. Our results indicate that 99% of cleptoparasitic apid bees form a monophyletic group. Divergence time estimates reveal that cleptoparasitism is an ancient behavior in bees that first evolved in the late Cretaceous 95 Mya [95% highest posterior density (HPD) = 87-103]. Our phylogenetic analysis of the Apidae sheds light on the macroevolution of a bee family that is of evolutionary, ecological, and economic importance.

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Ancient pinnate leaf mimesis among lacewings.

Publication Date: 2010 Aug 30 PMID: 20805491
Authors: Wang, Y. - Liu, Z. - Wang, X. - Shih, C. - Zhao, Y. - Engel, M. S. - Ren, D.
Journal: Proc Natl Acad Sci U S A

Insects have evolved diverse methods of predator avoidance, many of which implicate complex adaptations of their wings (e.g., Phylliidae, Nymphalidae, Notodontidae). Among these, angiosperm leaf mimicry is one of the most dramatic, although the historical origins of such modifications are unclear owing to a dearth of paleontological records. Here, we report evidence of pinnate leaf mimesis in two lacewings (Neuroptera): Bellinympha filicifolia Y. Wang, Ren, Liu & Engel gen. et sp. nov. and Bellinympha dancei Y. Wang, Ren, Shih & Engel, sp. nov., from the Middle Jurassic, representing a 165-million-year-old specialization between insects and contemporaneous gymnosperms of the Cycadales or Bennettitales. Furthermore, such lacewings demonstrate a preangiosperm origin for leaf mimesis, revealing a lost evolutionary scenario of interactions between insects and gymnosperms. The current fossil record suggests that this enigmatic lineage became extinct during the Early Cretaceous, apparently closely correlated with the decline of Cycadales and Bennettitales at that time, and perhaps owing to the changing floral environment resulted from the rise of flowering plants.

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Multipotential hematopoietic blast colony-forming cells exhibit delays in self-generation and lineage commitment.

Publication Date: 2010 Aug 30 PMID: 20805490
Authors: Metcalf, D. - Ng, A. - Mifsud, S. - Di Rago, L.
Journal: Proc Natl Acad Sci U S A

Murine hematopoietic blast colony-forming cells (BL-CFCs) are able to generate up to 30,000 progeny blast cells within 10 d in agar cultures. Contained in these populations are large numbers of lineage-committed progenitor cells in the granulocytic and macrophage lineages. Sequential analyses of blast colonies revealed that self-generation of BL-CFCs occurs but is surprisingly late in clonal expansion, as is the emergence of progenitor cells committed to megakaryocytic and eosinophil lineages. Self-generating BL-CFCs were highly enriched in lineage(-) Kit(+) Sca1(+) CD34(-) Flt3R(-) populations, and colonies generated by such cells contained colony-forming units-spleen and formed erythroid and lymphoid progeny in vivo. The data suggest the existence of a hierarchical structure in BL-CFC populations with at least a subset being cells assayable as colony-forming units-spleen. Because BL-CFCs can self-generate and are able to generate lymphoid and myeloid populations, BL-CFCs appear to be ideal cells in which to analyze the processes of self-generation and lineage commitment in clonal in vitro cultures.

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D1 and D2 dopamine receptors in separate circuits cooperate to drive associative long-term potentiation in the prefrontal cortex.

Publication Date: 2010 Aug 30 PMID: 20805489
Authors: Xu, T. X. - Yao, W. D.
Journal: Proc Natl Acad Sci U S A

Dopamine release associated with motivational arousal is thought to drive goal-directed learning and consolidation of acquired memories. This dopamine hypothesis of learning and motivation directly suggests that dopamine is necessary for modifications of excitatory synapses in dopamine terminal fields, including the prefrontal cortex (PFC), to "stamp in" posttrial memory traces. It is unknown how such enabling occurs in native circuits tightly controlled by GABAergic inhibitory tone. Here we report that dopamine, via both D1-class receptors (D1Rs) and D2-class receptors (D2Rs), enables the induction of spike timing-dependent long-term potentiation (t-LTP) in layer V PFC pyramidal neurons over a "window" of more than 30 ms that is otherwise closed under intact inhibitory constraint. Dopamine acts at D2Rs in local GABAergic interneurons to suppress inhibitory transmission, gating the induction of t-LTP. Moreover, dopamine activates postsynaptic D1Rs in excitatory synapses to allow t-LTP induction at a substantially extended, normally ineffective, timing interval (+30 ms), thus increasing the associability of prepost coincident stimuli. Although the D2R-mediated disinhibition alone is sufficient to gate t-LTP at a normal timing (+10 ms), t-LTP at +30 ms requires concurrent activation of both D1Rs and D2Rs. Our results illustrate a previously unrecognized circuit-level mechanism by which dopamine receptors in separate microcircuits cooperate to drive Hebbian synaptic plasticity across a significant temporal window under intact inhibition. This mechanism should be important in functioning of interconnected PFC microcircuits, in which D1Rs and D2Rs are not colocalized but their coactivation is necessary.

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Prior knowledge of illumination for 3D perception in the human brain.

Publication Date: 2010 Aug 30 PMID: 20805488
Authors: Gerardin, P. - Kourtzi, Z. - Mamassian, P.
Journal: Proc Natl Acad Sci U S A

In perceiving 3D shape from ambiguous shading patterns, humans use the prior knowledge that the light is located above their head and slightly to the left. Although this observation has fascinated scientists and artists for a long time, the neural basis of this "light from above left" preference for the interpretation of 3D shape remains largely unexplored. Combining behavioral and functional MRI measurements coupled with multivoxel pattern analysis, we show that activations in early visual areas predict best the light source direction irrespective of the perceived shape, but activations in higher occipitotemporal and parietal areas predict better the perceived 3D shape irrespective of the light direction. These findings demonstrate that illumination is processed earlier than the representation of 3D shape in the visual system. In contrast to previous suggestions, we propose that prior knowledge about illumination is processed in a bottom-up manner and influences the interpretation of 3D structure at higher stages of processing.

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The serine/arginine-rich protein SF2/ASF regulates protein sumoylation.

Publication Date: 2010 Aug 30 PMID: 20805487
Authors: Pelisch, F. - Gerez, J. - Druker, J. - Schor, I. E. - Munoz, M. J. - Risso, G. - Petrillo, E. - Westman, B. J. - Lamond, A. I. - Arzt, E. - Srebrow, A.
Journal: Proc Natl Acad Sci U S A

Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF.

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Oil sands development contributes elements toxic at low concentrations to the Athabasca River and its tributaries.

Publication Date: 2010 Aug 30 PMID: 20805486
Authors: Kelly, E. N. - Schindler, D. W. - Hodson, P. V. - Short, J. W. - Radmanovich, R. - Nielsen, C. C.
Journal: Proc Natl Acad Sci U S A

We show that the oil sands industry releases the 13 elements considered priority pollutants (PPE) under the US Environmental Protection Agency's Clean Water Act, via air and water, to the Athabasca River and its watershed. In the 2008 snowpack, all PPE except selenium were greater near oil sands developments than at more remote sites. Bitumen upgraders and local oil sands development were sources of airborne emissions. Concentrations of mercury, nickel, and thallium in winter and all 13 PPE in summer were greater in tributaries with watersheds more disturbed by development than in less disturbed watersheds. In the Athabasca River during summer, concentrations of all PPE were greater near developed areas than upstream of development. At sites downstream of development and within the Athabasca Delta, concentrations of all PPE except beryllium and selenium remained greater than upstream of development. Concentrations of some PPE at one location in Lake Athabasca near Fort Chipewyan were also greater than concentration in the Athabasca River upstream of development. Canada's or Alberta's guidelines for the protection of aquatic life were exceeded for seven PPE-cadmium, copper, lead, mercury, nickel, silver, and zinc-in melted snow and/or water collected near or downstream of development.

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Ancient DNA reveals extreme egg morphology and nesting behavior in New Zealand's extinct moa.

Publication Date: 2010 Aug 30 PMID: 20805485
Authors: Huynen, L. - Gill, B. J. - Millar, C. D. - Lambert, D. M.
Journal: Proc Natl Acad Sci U S A

New Zealand's extinct flightless moa radiated rapidly into a large number of morphologically diverse species, which produced an equally large range of egg morphologies. The exact number of moa species, as well as the characteristics of the eggs they laid, remains contentious. Moreover, like most extinct species, we understand little about their nesting and incubation habits. We used a modified ancient DNA extraction procedure to recover exogenous mitochondrial and nuclear DNA from the inside and outside surfaces of moa eggs. We used sequences from the inside of 69 eggshells to directly assign these remains to seven of the 10 currently recognized moa species. In addition we were able to assign, to the species level, six of the rare reconstructed "whole" eggs. These molecular results enabled us to identify two distinct lineages within the genus Euryapteryx. Members of these lineages differed in eggshell thickness, with one lineage being characterized by a relatively thin eggshell. Unexpectedly, several thin-shelled eggs were also shown to belong to the heaviest moa of the genera Dinornis, Euryapteryx and Emeus, making these, to our knowledge, the most fragile of all avian eggs measured to date. Moreover, sex-specific DNA recovered from the outer surfaces of eggshells belonging to species of Dinornis and Euryapteryx suggest that these very thin eggs were likely to have been incubated by the lighter males. The thin nature of the eggshells of these larger species of moa, even if incubated by the male, suggests that egg breakage in these species would have been common if the typical contact method of avian egg incubation was used.

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Social interaction modulates autonomic, inflammatory, and depressive-like responses to cardiac arrest and cardiopulmonary resuscitation.

Publication Date: 2010 Aug 30 PMID: 20805484
Authors: Norman, G. J. - Zhang, N. - Morris, J. S. - Karelina, K. - Berntson, G. G. - Devries, A. C.
Journal: Proc Natl Acad Sci U S A

Psychological factors, including depression and social isolation, are important determinants of cardiovascular health. The current study uses a well-validated mouse model of cardiac arrest/cardiopulmonary resuscitation (CA/CPR) to examine the effect of social environment on several pathophysiological and behavioral responses to cerebral ischemia. Male experimental mice were either housed in pairs with an ovariectomized female or socially isolated for the duration of the experiment. Cardiac arrest increased the mRNA expression of the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6, as well as the microglia marker MAC-1; expression of each of these factors, except IL-6, was further increased among socially isolated mice. Furthermore, socially isolated animals exposed to the CA/CPR procedure displayed significantly higher levels of neuronal cell death and microglia staining within the hippocampus at 7 d following surgery. Social isolation also exacerbated CA/CPR-induced depressive-like behavior and cardiac autonomic dysregulation. In the absence of ischemic damage, social environment had no significant effect on the expression of neuronal cell death, autonomic cardiac control, or behavior. Together, these data suggest that social factors influence the pathophysiological trajectory following cardiac arrest.

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Visual motion aftereffect from understanding motion language.

Publication Date: 2010 Aug 30 PMID: 20805483
Authors: Dils, A. T. - Boroditsky, L.
Journal: Proc Natl Acad Sci U S A

Do people spontaneously form visual mental images when understanding language, and if so, how truly visual are these representations? We test whether processing linguistic descriptions of motion produces sufficiently vivid mental images to cause direction-selective motion adaptation in the visual system (i.e., cause a motion aftereffect illusion). We tested for motion aftereffects (MAEs) following explicit motion imagery, and after processing literal or metaphorical motion language (without instructions to imagine). Intentionally imagining motion produced reliable MAEs. The aftereffect from processing motion language gained strength as people heard more and more of a story (participants heard motion stories in four installments, with a test after each). For the last two story installments, motion language produced reliable MAEs across participants. Individuals differed in how early in the story this effect appeared, and this difference was predicted by the strength of an individual's MAE from imagining motion. Strong imagers (participants who showed the largest MAEs from imagining motion) were more likely to show an MAE in the course of understanding motion language than were weak imagers. The results demonstrate that processing language can spontaneously create sufficiently vivid mental images to produce direction-selective adaptation in the visual system. The timecourse of adaptation suggests that individuals may differ in how efficiently they recruit visual mechanisms in the service of language understanding. Further, the results reveal an intriguing link between the vividness of mental imagery and the nature of the processes and representations involved in language understanding.

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Spontaneous EEG oscillations reveal periodic sampling of visual attention.

Publication Date: 2010 Aug 30 PMID: 20805482
Authors: Busch, N. A. - Vanrullen, R.
Journal: Proc Natl Acad Sci U S A

An important effect of sustained attention is the facilitation of perception. Although the term "sustained" suggests that this beneficial effect endures continuously as long as something is attended, we present electrophysiological evidence that perception at attended locations is actually modulated periodically. Subjects detected brief light flashes that were presented peripherally at locations that were either attended or unattended. We analyzed the correlation between detection performance for attended and unattended stimuli and the phase of ongoing EEG oscillations, which relate to subsecond fluctuations of neuronal excitability. Although on average, detection performance was improved by attention-indicated by reduced detection thresholds at attended locations-we found that detection performance for attended stimuli actually fluctuated over time along with the phase of spontaneous oscillations in the ( approximately 7 Hz) frequency band just before stimulus onset. This fluctuation was absent for unattended stimuli. This pattern of results suggests that "sustained" attention in fact exerts its facilitative effect on perception in a periodic fashion.

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Image-matching during ant navigation occurs through saccade-like body turns controlled by learned visual features.

Publication Date: 2010 Aug 30 PMID: 20805481
Authors: Lent, D. D. - Graham, P. - Collett, T. S.
Journal: Proc Natl Acad Sci U S A

Visual memories of landmarks play a major role in guiding the habitual foraging routes of ants and bees, but how these memories engage visuo-motor control systems during guidance is poorly understood. We approach this problem through a study of image matching, a navigational strategy in which insects reach a familiar place by moving so that their current retinal image transforms to match a memorized snapshot of the scene viewed from that place. Analysis of how navigating wood ants correct their course when close to a goal reveals a significant part of the mechanism underlying this transformation. Ants followed a short route to an inconspicuous feeder positioned at a fixed distance from a vertical luminance edge. They responded to an unexpected jump of the edge by turning to face the new feeder position specified by the edge. Importantly, the initial speed of the turn increased linearly with the turn's amplitude. This correlation implies that the ants' turns are driven initially by their prior calculation of the angular difference between the current retinal position of the edge and its desired position in their memorized view. Similar turns keep ants to their path during unperturbed routes. The neural circuitry mediating image-matching is thus concerned not only with the storage of views, but also with making exact comparisons between the retinal positions of a visual feature in a memorized view and of the same feature in the current retinal image.

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A transmembrane formin nucleates subapical actin assembly and controls tip-focused growth in pollen tubes.

Publication Date: 2010 Aug 30 PMID: 20805480
Authors: Cheung, A. Y. - Niroomand, S. - Zou, Y. - Wu, H. M.
Journal: Proc Natl Acad Sci U S A

Pollen tubes are highly polarized plant cells specialized in delivering sperm for fertilization. Pollen tube growth is rapid, occurs exclusively at the tip, and can reach distances thousands of times the diameter of the pollen grain without cell division, thus representing an excellent model system for studying asymmetric cell growth. In flowering plants, pollen tube growth is dependent on the actin cytoskeleton, which supports an efficient vesicle trafficking system to deliver membrane and cell-wall materials to the tube tip. A highly dynamic subapical actin structure and an apical vesicular zone are known to be critical for the tip-growth process. How this apical organization is maintained, how the subapical actin structure is assembled, and direct evidence for its functional coupling with tip growth remain to be established. Here, we show that a tip-located, cell membrane-anchored actin-nucleating protein, the Arabidopsis formin homology5 (FH5), stimulates actin assembly from the subapical membrane, provides actin filaments for vesicular trafficking to the apical dome, and mediates assembly of the subapical actin structure. Moreover, FH5-expressing pollen tubes provided a unique opportunity to demonstrate that assembly of the subapical actin structure is concomitant with the acquisition of rapid tip growth, providing further support for their functional coupling. Together, our results show that FH5 plays a pivotal role in establishing the subapical actin and apical vesicular organization critical for tip-focused growth in pollen tubes.

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An unusual dinosaur from the Late Cretaceous of Romania and the island rule.

Publication Date: 2010 Aug 31 PMID: 20805479
Authors: Sues, H. D.
Journal: Proc Natl Acad Sci U S A

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Enhancement of antigen-specific Treg vaccination in vivo.

Publication Date: 2010 Aug 30 PMID: 20805478
Authors: Daniel, C. - Wennhold, K. - Kim, H. J. - von Boehmer, H.
Journal: Proc Natl Acad Sci U S A

The conversion of naive T cells into Treg can be achieved in vivo by delivery of antigen under subimmunogenic conditions. Here we have examined several drugs for their ability to enhance the conversion process in vivo and have found that the rapamycin analog everolimus potently enhances Treg conversion by interfering with T-cell costimulation, reducing cell division and thereby activation of DNA methyltransferase 1 as well as by reducing T-cell activation through the ATP-gated P2x7 receptor controlling Ca2(+) influx. The resulting Tregs exhibit increased stability of Foxp3 expression even when generated in TGFbeta-containing media in vitro. Thus the mammalian target of rapamycin (mTOR) inhibitor everolimus in addition to inhibiting immune responses enhances Treg conversion by several distinct pathways. The converted Tregs can be further expanded by injection of IL-2/IL-2ab complexes. These complexes also increase the number of CD25(+)Foxp3(-) cells that, however, do not represent cytokine secreting effector cells but anergic cells, some of which can secrete IL-10 and can themselves be considered regulatory T cells as well. The combined use of everolimus and IL-2/IL-2ab complexes in vivo makes it feasible to achieve highly effective antigen-driven conversion of naive T cells into Treg and their expansion in vivo and thereby the described protocols constitute important tools to achieve immunological tolerance by Treg vaccination.

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Membrane-shaping host reticulon proteins play crucial roles in viral RNA replication compartment formation and function.

Publication Date: 2010 Aug 30 PMID: 20805477
Authors: Diaz, A. - Wang, X. - Ahlquist, P.
Journal: Proc Natl Acad Sci U S A

Positive-strand RNA viruses replicate their genomes on membranes with virus-induced rearrangements such as single- or double-membrane vesicles, but the mechanisms of such rearrangements, including the role of host proteins, are poorly understood. Brome mosaic virus (BMV) RNA synthesis occurs in approximately 70 nm, negatively curved endoplasmic reticulum (ER) membrane invaginations induced by multifunctional BMV protein 1a. We show that BMV RNA replication is inhibited 80-90% by deleting the reticulon homology proteins (RHPs), a family of membrane-shaping proteins that normally induce and stabilize positively curved peripheral ER membrane tubules. In RHP-depleted cells, 1a localized normally to perinuclear ER membranes and recruited the BMV 2a(pol) polymerase. However, 1a failed to induce ER replication compartments or to recruit viral RNA templates. Partial RHP depletion allowed formation of functional replication vesicles but reduced their diameter by 30-50%. RHPs coimmunoprecipitated with 1a and 1a expression redirected >50% of RHPs from peripheral ER tubules to the interior of BMV-induced RNA replication compartments on perinuclear ER. Moreover, RHP-GFP fusions retained 1a interaction but shifted 1a-induced membrane rearrangements from normal vesicles to double membrane layers, a phenotype also induced by excess 1a-interacting 2a(pol). Thus, RHPs interact with 1a, are incorporated into RNA replication compartments, and are required for multiple 1a functions in replication compartment formation and function. The results suggest possible RHP roles in the bodies and necks of replication vesicles.

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In utero exposure to dioxin causes neocortical dysgenesis through the actions of p27Kip1.

Publication Date: 2010 Aug 30 PMID: 20805476
Authors: Mitsuhashi, T. - Yonemoto, J. - Sone, H. - Kosuge, Y. - Kosaki, K. - Takahashi, T.
Journal: Proc Natl Acad Sci U S A

Dioxins have been reported to exert various adverse effects, including cell-cycle dysregulation in vitro and impairment of spatial learning and memory after in utero exposure in rodents. Furthermore, children born to mothers who are exposed to dioxin analogs polychlorinated dibenzofurans or polychlorinated biphenyls have developmental impairments in cognitive functions. Here, we show that in utero exposure to dioxins in mice alters differentiation patterns of neural progenitors and leads to decreased numbers of non-GABAergic neurons and thinner deep neocortical layers. This reduction in number of non-GABAergic neurons is assumed to be caused by accumulation of cyclin-dependent kinase inhibitor p27(Kip1) in nuclei of neural progenitors. Lending support to this presumption, mice lacking p27(Kip1) are not susceptible to in utero dioxin exposure. These results show that environmental pollutants may affect neocortical histogenesis through alterations of functions of specific gene(s)/protein(s) (in our case, dioxins), exerting adverse effects by altering functions of p27(Kip1).

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Elr-type proteins protect Xenopus Dead end mRNA from miR-18-mediated clearance in the soma.

Publication Date: 2010 Aug 30 PMID: 20805475
Authors: Koebernick, K. - Loeber, J. - Arthur, P. K. - Tarbashevich, K. - Pieler, T.
Journal: Proc Natl Acad Sci U S A

Segregation of the future germ line defines a crucial cell fate decision during animal development. In Xenopus, germ cells are specified by inheritance of vegetally localized maternal determinants, including a group of specific mRNAs. Here, we show that the vegetal localization elements (LE) of Xenopus Dead end (XDE) and of several other germ-line-specific, vegetally localized transcripts mediate germ cell-specific stabilization and somatic clearance of microinjected reporter mRNA in Xenopus embryos. The part of XDE-LE critical for somatic RNA clearance exhibits homology to zebrafish nanos1 and appears to be targeted by Xenopus miR-18 for somatic mRNA clearance. Xenopus Elr-type proteins of the vegetal localization complex can alleviate somatic RNA clearance of microinjected XDE-LE and endogenous XDE mRNA. ElrB1 synergizes with Xenopus Dead end protein in the stabilization of XDE-LE mRNA. Taken together, our findings unveil a functional link of vegetal mRNA localization and the protection of germ-line mRNAs from somatic clearance.

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Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein.

Publication Date: 2010 Aug 30 PMID: 20805474
Authors: Jaras, M. - Johnels, P. - Hansen, N. - Agerstam, H. - Tsapogas, P. - Rissler, M. - Lassen, C. - Olofsson, T. - Bjerrum, O. W. - Richter, J. - Fioretos, T.
Journal: Proc Natl Acad Sci U S A

Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome, formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test whether IL1RAP expression distinguishes normal (Ph(-)) and leukemic (Ph(+)) cells within the CML CD34(+)CD38(-) cell compartment, we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method, we sorted cells directly into drops on slides to investigate their Ph-chromosome status. Interestingly, we found that the CML CD34(+)CD38(-)IL1RAP(+) cells were Ph(+), whereas CML CD34(+)CD38(-)IL1RAP(-) cells were almost exclusively Ph(-). By performing long-term culture-initiating cell assays on the two cell populations, we found that Ph(+) and Ph(-) candidate CML stem cells could be prospectively separated. In addition, by generating an anti-IL1RAP antibody, we provide proof of concept that IL1RAP can be used as a target on CML CD34(+)CD38(-) cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell surface biomarker distinguishing Ph(+) from Ph(-) candidate CML stem cells and opens up a previously unexplored avenue for therapy of CML.

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Functional comparison of the effects of TARPs and cornichons on AMPA receptor trafficking and gating.

Publication Date: 2010 Aug 30 PMID: 20805473
Authors: Shi, Y. - Suh, Y. H. - Milstein, A. D. - Isozaki, K. - Schmid, S. M. - Roche, K. W. - Nicoll, R. A.
Journal: Proc Natl Acad Sci U S A

Glutamate receptors of the AMPA subtype (AMPARs) mediate fast synaptic transmission in the brain. These ionotropic receptors rely on auxiliary subunits known as transmembrane AMPAR regulatory proteins (TARPs) for both trafficking and gating. Recently, a second family of AMPAR binding proteins, referred to as cornichons, were identified and also proposed to function as auxiliary subunits. Cornichons are transmembrane proteins that modulate AMPAR function in expression systems much like TARPs. In the present study we compare the role of cornichons in controlling AMPA receptor function in neurons and HEK cells to that of TARPs. Cornichons mimic some, but not all, of the actions of TARPs in HEK cells; their role in neurons, however, is more limited. Although expressed cornichons can affect the trafficking of AMPARs, they were not detected on the surface of neurons and failed to alter the kinetics of endogenous AMPARs. This neuronal role is more consistent with that of an endoplasmic reticulum (ER) chaperone rather than a bona fide auxiliary subunit.

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Major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the Democratic Republic of Congo.

Publication Date: 2010 Aug 30 PMID: 20805472
Authors: Rimoin, A. W. - Mulembakani, P. M. - Johnston, S. C. - Smith, J. O. - Kisalu, N. K. - Kinkela, T. L. - Blumberg, S. - Thomassen, H. A. - Pike, B. L. - Fair, J. N. - Wolfe, N. D. - Shongo, R. L. - Graham, B. S. - Formenty, P. - Okitolonda, E. - Hensley, L. E. - Meyer, H. - Wright, L. L. - Muyembe, J. J.
Journal: Proc Natl Acad Sci U S A

Studies on the burden of human monkeypox in the Democratic Republic of the Congo (DRC) were last conducted from 1981 to 1986. Since then, the population that is immunologically naive to orthopoxviruses has increased significantly due to cessation of mass smallpox vaccination campaigns. To assess the current risk of infection, we analyzed human monkeypox incidence trends in a monkeypox-enzootic region. Active, population-based surveillance was conducted in nine health zones in central DRC. Epidemiologic data and biological samples were obtained from suspected cases. Cumulative incidence (per 10,000 population) and major determinants of infection were compared with data from active surveillance in similar regions from 1981 to 1986. Between November 2005 and November 2007, 760 laboratory-confirmed human monkeypox cases were identified in participating health zones. The average annual cumulative incidence across zones was 5.53 per 10,000 (2.18-14.42). Factors associated with increased risk of infection included: living in forested areas, male gender, age < 15, and no prior smallpox vaccination. Vaccinated persons had a 5.2-fold lower risk of monkeypox than unvaccinated persons (0.78 vs. 4.05 per 10,000). Comparison of active surveillance data in the same health zone from the 1980s (0.72 per 10,000) and 2006-07 (14.42 per 10,000) suggests a 20-fold increase in human monkeypox incidence. Thirty years after mass smallpox vaccination campaigns ceased, human monkeypox incidence has dramatically increased in rural DRC. Improved surveillance and epidemiological analysis is needed to better assess the public health burden and develop strategies for reducing the risk of wider spread of infection.

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Repression of Wnt signaling by a Fer-type nonreceptor tyrosine kinase.

Publication Date: 2010 Aug 30 PMID: 20805471
Authors: Putzke, A. P. - Rothman, J. H.
Journal: Proc Natl Acad Sci U S A

The Wnt signaling pathway must be properly modulated to ensure an appropriate output: pathological conditions result from either insufficient or excessive levels of Wnt signal. For example, hyperactivation of the Wnt pathway is associated with various cancers and subnormal Wnt signaling can lead to increased invasiveness of tumor cells. We found that the Caenorhabditis elegans ortholog of the Fer nonreceptor tyrosine kinase, FRK-1, limits Wnt signaling by preventing the adhesion complex-associated beta-catenin, HMP-2, from participating in Wnt-dependent specification of the endoderm during embryogenesis. Removal of FRK-1 function results in relocalization of HMP-2 to the nucleus of epidermal cells, and allows it to substitute for WRM-1, the nuclear beta-catenin that normally transduces the Wnt signal during endoderm development. APR-1, the C. elegans APC ortholog, is similarly required to prevent HMP-2 relocalization and keeps it from participating in Wnt signal transduction; this finding partially explains the paradoxical observation that APR-1 acts either negatively or positively in Wnt signaling, depending on context. The apparent hyperactivation of the Wnt response in the absence of FRK-1 leads to hyperproliferation in the endoderm, as is also seen when WRM-1 is overexpressed in wild-type embryos. The specification and proliferation activities of Wnt signaling are separable: although the Tcf/Lef factor POP-1 acts in Wnt-dependent endoderm specification, it is not apparently required for hyperproliferation resulting from excessive Wnt signaling. These findings highlight a role for a Fer-type kinase in setting the proper levels of Wnt signaling and demonstrate the importance of this modulation in ensuring appropriate cell division.

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Correction for Bowman et al., Protein folded states are kinetic hubs.

Publication Date: 2010 Aug 30 PMID: 20805470
Authors:
Journal: Proc Natl Acad Sci U S A

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Insulin-like growth factor 1 treatment extends longevity in a mouse model of human premature aging by restoring somatotroph axis function.

Publication Date: 2010 Aug 30 PMID: 20805469
Authors: Marino, G. - Ugalde, A. P. - Fernandez, A. F. - Osorio, F. G. - Fueyo, A. - Freije, J. M. - Lopez-Otin, C.
Journal: Proc Natl Acad Sci U S A

Zmpste24 (also called FACE-1) is a metalloproteinase involved in the maturation of lamin A, an essential component of the nuclear envelope. Zmpste24-deficient mice exhibit multiple defects that phenocopy human accelerated aging processes such as Hutchinson-Gilford progeria syndrome. In this work, we report that progeroid Zmpste24(-/)(-) mice present profound transcriptional alterations in genes that regulate the somatotroph axis, together with extremely high circulating levels of growth hormone (GH) and a drastic reduction in plasma insulin-like growth factor 1 (IGF-1). We also show that recombinant IGF-1 treatment restores the proper balance between IGF-1 and GH in Zmpste24(-/)(-) mice, delays the onset of many progeroid features, and significantly extends the lifespan of these progeroid animals. Our findings highlight the importance of IGF/GH balance in longevity and may be of therapeutic interest for devastating human progeroid syndromes associated with nuclear envelope abnormalities.

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Refined LexA transactivators and their use in combination with the Drosophila Gal4 system.

Publication Date: 2010 Aug 30 PMID: 20805468
Authors: Yagi, R. - Mayer, F. - Basler, K.
Journal: Proc Natl Acad Sci U S A

The use of binary transcriptional systems offers many advantages for experimentally manipulating gene activity, as exemplified by the success of the Gal4/UAS system in Drosophila. To expand the number of applications, a second independent transactivator (TA) is desirable. Here, we present the optimization of an additional system based on LexA and show how it can be applied. We developed a series of LexA TAs, selectively suppressible via Gal80, that exhibit high transcriptional activity and low detrimental effects when expressed in vivo. In combination with Gal4, an appropriately selected LexA TA permits to program cells with a distinct balance and independent outputs of the two TAs. We demonstrate how the two systems can be combined for manipulating communicating cell populations, converting transient tissue-specific expression patterns into heritable, constitutive activities, and defining cell territories by intersecting TA expression domains. Finally, we describe a versatile enhancer trap system that allows swapping TA and generating mosaics composed of Gal4 and LexA TA-expressing cells. The optimized LexA system facilitates precise analyses of complex biological phenomena and signaling pathways in Drosophila.

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Reply to Reams et al.: Quantifying forest cover change at local and global scales.

Publication Date: 2010 Aug 30 PMID: 20805467
Authors: Hansen, M. C. - Stehman, S. V. - Potapov, P. V.
Journal: Proc Natl Acad Sci U S A

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Remote sensing alone is insufficient for quantifying changes in forest cover.

Publication Date: 2010 Aug 30 PMID: 20805466
Authors: Reams, G. A. - Brewer, C. K. - Guldin, R. W.
Journal: Proc Natl Acad Sci U S A

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Chk1 promotes replication fork progression by controlling replication initiation.

Publication Date: 2010 Aug 30 PMID: 20805465
Authors: Petermann, E. - Woodcock, M. - Helleday, T.
Journal: Proc Natl Acad Sci U S A

DNA replication starts at initiation sites termed replication origins. Metazoan cells contain many more potential origins than are activated (fired) during each S phase. Origin activation is controlled by the ATR checkpoint kinase and its downstream effector kinase Chk1, which suppresses origin firing in response to replication blocks and during normal S phase by inhibiting the cyclin-dependent kinase Cdk2. In addition to increased origin activation, cells deficient in Chk1 activity display reduced rates of replication fork progression. Here we investigate the causal relationship between increased origin firing and reduced replication fork progression. We use the Cdk inhibitor roscovitine or RNAi depletion of Cdc7 to inhibit origin firing in Chk1-inhibited or RNAi-depleted cells. We report that Cdk inhibition and depletion of Cdc7 can alleviate the slow replication fork speeds in Chk1-deficient cells. Our data suggest that increased replication initiation leads to slow replication fork progression and that Chk1 promotes replication fork progression during normal S phase by controlling replication origin activity.

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Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain.

Publication Date: 2010 Aug 30 PMID: 20805464
Authors: Nadal, M. - Mas, P. J. - Blanco, A. G. - Arnan, C. - Sola, M. - Hart, D. J. - Coll, M.
Journal: Proc Natl Acad Sci U S A

During viral replication, herpesviruses package their DNA into the procapsid by means of the terminase protein complex. In human cytomegalovirus (herpesvirus 5), the terminase is composed of subunits UL89 and UL56. UL89 cleaves the long DNA concatemers into unit-length genomes of appropriate length for encapsidation. We used ESPRIT, a high-throughput screening method, to identify a soluble purifiable fragment of UL89 from a library of 18,432 randomly truncated ul89 DNA constructs. The purified protein was crystallized and its three-dimensional structure was solved. This protein corresponds to the key nuclease domain of the terminase and shows an RNase H/integrase-like fold. We demonstrate that UL89-C has the capacity to process the DNA and that this function is dependent on Mn(2+) ions, two of which are located at the active site pocket. We also show that the nuclease function can be inactivated by raltegravir, a recently approved anti-AIDS drug that targets the HIV integrase.

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Shedding light on photoperiodism.

Publication Date: 2010 Aug 27 PMID: 20802157
Authors: Provencio, I.
Journal: Proc Natl Acad Sci U S A

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RNA helicase A is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.

Publication Date: 2010 Aug 27 PMID: 20802156
Authors: Huo, L. - Wang, Y. N. - Xia, W. - Hsu, S. C. - Lai, C. C. - Li, L. Y. - Chang, W. C. - Wang, Y. - Hsu, M. C. - Yu, Y. L. - Huang, T. H. - Ding, Q. - Chen, C. H. - Tsai, C. H. - Hung, M. C.
Journal: Proc Natl Acad Sci U S A

EGF induces the translocation of EGF receptor (EGFR) from the cell surface to the nucleus where EGFR activates gene transcription through its binding to an AT-rich sequence (ATRS) of the target gene promoter. However, how EGFR, without a DNA-binding domain, can bind to the gene promoter is unclear. In the present study, we show that RNA helicase A (RHA) is an important mediator for EGFR-induced gene transactivation. EGF stimulates the interaction of EGFR with RHA in the nucleus of cancer cells. The EGFR/RHA complex then associates with the target gene promoter through binding of RHA to the ATRS of the target gene promoter to activate its transcription. Knockdown of RHA expression in cancer cells abrogates the binding of EGFR to the target gene promoter, thereby reducing EGF/EGFR-induced gene expression. In addition, interruption of EGFR-RHA interaction decreases the EGFR-induced promoter activity. Consistently, we observed a positive correlation of the nuclear expression of EGFR, RHA, and cyclin D1 in human breast cancer samples. These results indicate that RHA is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.

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Mammalian endoplasmic reticulum stress sensor IRE1 signals by dynamic clustering.

Publication Date: 2010 Aug 26 PMID: 20798350
Authors: Li, H. - Korennykh, A. V. - Behrman, S. L. - Walter, P.
Journal: Proc Natl Acad Sci U S A

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), an intracellular signaling pathway that adjusts the protein folding capacity of the ER according to need. If homeostasis in the ER protein folding environment cannot be reestablished, cells commit to apoptosis. The ER-resident transmembrane kinase-endoribonuclease inositol-requiring enzyme 1 (IRE1) is the best characterized UPR signal transduction molecule. In yeast, Ire1 oligomerizes upon activation in response to an accumulation of misfolded proteins in the ER. Here we show that the salient mechanistic features of IRE1 activation are conserved: mammalian IRE1 oligomerizes in the ER membrane and oligomerization correlates with the onset of IRE1 phosphorylation and RNase activity. Moreover, the kinase/RNase module of human IRE1 activates cooperatively in vitro, indicating that formation of oligomers larger than four IRE1 molecules takes place upon activation. High-order IRE1 oligomerization thus emerges as a conserved mechanism of IRE1 signaling. IRE1 signaling attenuates after prolonged ER stress. IRE1 then enters a refractive state even if ER stress remains unmitigated. Attenuation includes dissolution of IRE1 clusters, IRE1 dephosphorylation, and decline in endoribonuclease activity. Thus IRE1 activity is governed by a timer that may be important in switching the UPR from the initially cytoprotective phase to the apoptotic mode.

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Signatures of founder effects, admixture, and selection in the Ashkenazi Jewish population.

Publication Date: 2010 Aug 26 PMID: 20798349
Authors: Bray, S. M. - Mulle, J. G. - Dodd, A. F. - Pulver, A. E. - Wooding, S. - Warren, S. T.
Journal: Proc Natl Acad Sci U S A

The Ashkenazi Jewish (AJ) population has long been viewed as a genetic isolate, yet it is still unclear how population bottlenecks, admixture, or positive selection contribute to its genetic structure. Here we analyzed a large AJ cohort and found higher linkage disequilibrium (LD) and identity-by-descent relative to Europeans, as expected for an isolate. However, paradoxically we also found higher genetic diversity, a sign of an older or more admixed population but not of a long-term isolate. Recent reports have reaffirmed that the AJ population has a common Middle Eastern origin with other Jewish Diaspora populations, but also suggest that the AJ population, compared with other Jews, has had the most European admixture. Our analysis indeed revealed higher European admixture than predicted from previous Y-chromosome analyses. Moreover, we also show that admixture directly correlates with high LD, suggesting that admixture has increased both genetic diversity and LD in the AJ population. Additionally, we applied extended haplotype tests to determine whether positive selection can account for the level of AJ-prevalent diseases. We identified genomic regions under selection that account for lactose and alcohol tolerance, and although we found evidence for positive selection at some AJ-prevalent disease loci, the higher incidence of the majority of these diseases is likely the result of genetic drift following a bottleneck. Thus, the AJ population shows evidence of past founding events; however, admixture and selection have also strongly influenced its current genetic makeup.

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Resolving postglacial phylogeography using high-throughput sequencing.

Publication Date: 2010 Aug 26 PMID: 20798348
Authors: Emerson, K. J. - Merz, C. R. - Catchen, J. M. - Hohenlohe, P. A. - Cresko, W. A. - Bradshaw, W. E. - Holzapfel, C. M.
Journal: Proc Natl Acad Sci U S A

The distinction between model and nonmodel organisms is becoming increasingly blurred. High-throughput, second-generation sequencing approaches are being applied to organisms based on their interesting ecological, physiological, developmental, or evolutionary properties and not on the depth of genetic information available for them. Here, we illustrate this point using a low-cost, efficient technique to determine the fine-scale phylogenetic relationships among recently diverged populations in a species. This application of restriction site-associated DNA tags (RAD tags) reveals previously unresolved genetic structure and direction of evolution in the pitcher plant mosquito, Wyeomyia smithii, from a southern Appalachian Mountain refugium following recession of the Laurentide Ice Sheet at 22,000-19,000 B.P. The RAD tag method can be used to identify detailed patterns of phylogeography in any organism regardless of existing genomic data, and, more broadly, to identify incipient speciation and genome-wide variation in natural populations in general.

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BK channels play a counter-adaptive role in drug tolerance and dependence.

Publication Date: 2010 Aug 26 PMID: 20798347
Authors: Ghezzi, A. - Pohl, J. B. - Wang, Y. - Atkinson, N. S.
Journal: Proc Natl Acad Sci U S A

Disturbance of neural activity by sedative drugs has been proposed to trigger a homeostatic response that resists unfavorable changes in net cellular excitability, leading to tolerance and dependence. The Drosophila slo gene encodes a BK-type Ca(2+)-activated K(+) channel implicated in functional tolerance to alcohol and volatile anesthetics. We hypothesized that increased expression of BK channels induced by these drugs constitutes the homeostatic adaptation conferring resistance to sedative drugs. In contrast to the dogmatic view that BK channels act as neural depressants, we show that drug-induced slo expression enhances excitability by reducing the neuronal refractory period. Although this neuroadaptation directly counters some effects of anesthetics, it also causes long-lasting enhancement of seizure susceptibility, a common symptom of drug withdrawal. These data provide a possible mechanism for the long-standing counter-adaptive theory for drug tolerance in which homeostatic adaptations triggered by drug exposure to produce drug tolerance become counter-adaptive after drug clearance and result in symptoms of dependence.

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Paracrinology of islets and the paracrinopathy of diabetes.

Publication Date: 2010 Aug 26 PMID: 20798346
Authors: Unger, R. H. - Orci, L.
Journal: Proc Natl Acad Sci U S A

New results have brought to light the importance of the regulation of glucagon by beta-cells in the development of diabetes. In this perspective, we examine the normal paracrinology of alpha- and beta-cells in nondiabetic pancreatic islets. We propose a Sherringtonian model of coordinated reciprocal secretory responses of these juxtaposed cells that secrete glucagon and insulin, hormones with opposing actions on the liver. As insulin is a powerful inhibitor of glucagon, we propose that within-islet inhibition of alpha-cells by beta-cells creates an insulin-to-glucagon ratio that maintains glycemic stability even in extremes of glucose influx or efflux. By contrast, in type 1 diabetes mellitus, alpha-cells lack constant action of high insulin levels from juxtaposed beta-cells. Replacement with exogenous insulin does not approach paracrine levels of secreted insulin except with high doses that "overinsulinize" the peripheral insulin targets, thereby promoting glycemic volatility. Based on the stable normoglycemia of mice with type 1 diabetes during suppression of glucagon with leptin, we conclude that, in the absence of paracrine regulation of alpha-cells, tonic inhibition of alpha-cells improves the dysregulated glucose homeostasis. These results have considerable medical implications, as they suggest new approaches to normalize the extreme volatility of glycemia in diabetic patients.

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G-quadruplex structures in RNA stimulate mitochondrial transcription termination and primer formation.

Publication Date: 2010 Aug 26 PMID: 20798345
Authors: Wanrooij, P. H. - Uhler, J. P. - Simonsson, T. - Falkenberg, M. - Gustafsson, C. M.
Journal: Proc Natl Acad Sci U S A

The human mitochondrial transcription machinery generates the primers required for initiation of leading-strand DNA replication. According to one model, the 3' end of the primer is defined by transcription termination at conserved sequence block II (CSB II) in the mitochondrial DNA control region. We here demonstrate that this site-specific termination event is caused by G-quadruplex structures formed in nascent RNA upon transcription of CSB II. We also demonstrate that a poly-dT stretch downstream of CSB II has a modest stimulatory effect on the termination efficiency. The mechanism is reminiscent of Rho-independent transcription termination in prokaryotes, with the exception that a G-quadruplex structure replaces the hairpin loop formed in bacterial mRNA during transcription of terminator sequences.

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Crystal engineering of lattice metrics of perhalometallate salts and MOFs.

Publication Date: 2010 Aug 26 PMID: 20798344
Authors: Adams, C. J. - Haddow, M. F. - Lusi, M. - Orpen, A. G.
Journal: Proc Natl Acad Sci U S A

The synthesis of the salt 3 and metallo-organic framework (MOF) [{(4,4(')-bipy)CoBr(2)}(n)] 4 by a range of solid state (mechanochemical and thermochemical) and solution methods is reported; they are isostructural with their respective chloride analogues 1 and 2. 3 and 4 can be interconverted by means of HBr elimination and absorption. Single phases of controlled composition and general formula [4,4(')-H(2)bipy][CoBr(4-x)Cl(x)] 5(x) may be prepared from 2 and 4 by solid-gas reactions involving HBr or HCl respectively. Crystalline single phase samples of 5(x) and [{(4,4(')-bipy)CoBr(2-x)Cl(x)}(n)] 6(x) were prepared by solid-state mechanochemical routes, allowing fine control over the composition and unit cell volume of the product. Collectively these methods enable continuous variation of the unit cell dimensions of the salts [4,4(')-H(2)bipy][CoBr(4-x)Cl(x)] (5(x)) and the MOFs [{(4,4(')-bipy)CoBr(2-x)Cl(x)}(n)] (6(x)) by varying the bromide to chloride ratio and establish a means of controlling MOF composition and the lattice metrics, and so the physical and chemical properties that derive from it.

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Nanopore DNA sequencing with MspA.

Publication Date: 2010 Aug 26 PMID: 20798343
Authors: Derrington, I. M. - Butler, T. Z. - Collins, M. D. - Manrao, E. - Pavlenok, M. - Niederweis, M. - Gundlach, J. H.
Journal: Proc Natl Acad Sci U S A

Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing.

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Biomimetic cilia arrays generate simultaneous pumping and mixing regimes.

Publication Date: 2010 Aug 26 PMID: 20798342
Authors: Shields, A. R. - Fiser, B. L. - Evans, B. A. - Falvo, M. R. - Washburn, S. - Superfine, R.
Journal: Proc Natl Acad Sci U S A

Living systems employ cilia to control and to sense the flow of fluids for many purposes, such as pumping, locomotion, feeding, and tissue morphogenesis. Beyond their use in biology, functional arrays of artificial cilia have been envisaged as a potential biomimetic strategy for inducing fluid flow and mixing in lab-on-a-chip devices. Here we report on fluid transport produced by magnetically actuated arrays of biomimetic cilia whose size approaches that of their biological counterparts, a scale at which advection and diffusion compete to determine mass transport. Our biomimetic cilia recreate the beat shape of embryonic nodal cilia, simultaneously generating two sharply segregated regimes of fluid flow: Above the cilia tips their motion causes directed, long-range fluid transport, whereas below the tips we show that the cilia beat generates an enhanced diffusivity capable of producing increased mixing rates. These two distinct types of flow occur simultaneously and are separated in space by less than 5 mum, approximately 20% of the biomimetic cilium length. While this suggests that our system may have applications as a versatile microfluidics device, we also focus on the biological implications of our findings. Our statistical analysis of particle transport identifying an enhanced diffusion regime provides novel evidence for the existence of mixing in ciliated systems, and we demonstrate that the directed transport regime is Poiseuille-Couette flow, the first analytical model consistent with biological measurements of fluid flow in the embryonic node.

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Charges in the hydrophobic interior of proteins.

Publication Date: 2010 Aug 26 PMID: 20798341
Authors: Isom, D. G. - Castaneda, C. A. - Cannon, B. R. - Velu, P. D. - Garcia-Moreno, E. B
Journal: Proc Natl Acad Sci U S A

Charges are inherently incompatible with hydrophobic environments. Presumably for this reason, ionizable residues are usually excluded from the hydrophobic interior of proteins and are found instead at the surface, where they can interact with bulk water. Paradoxically, ionizable groups buried in the hydrophobic interior of proteins play essential roles, especially in biological energy transduction. To examine the unusual properties of internal ionizable groups we measured the pK(a) of glutamic acid residues at 25 internal positions in a stable form of staphylococcal nuclease. Two of 25 Glu residues titrated with normal pK(a) near 4.5; the other 23 titrated with elevated pK(a) values ranging from 5.2-9.4, with an average value of 7.7. Trp fluorescence and far-UV circular dichroism were used to monitor the effects of internal charges on conformation. These data demonstrate that although charges buried in proteins are indeed destabilizing, charged side chains can be buried readily in the hydrophobic core of stable proteins without the need for specialized structural adaptations to stabilize them, and without inducing any major conformational reorganization. The apparent dielectric effect experienced by the internal charges is considerably higher than the low dielectric constants of hydrophobic matter used to represent the protein interior in electrostatic continuum models of proteins. The high thermodynamic stability required for proteins to withstand the presence of buried charges suggests a pathway for the evolution of enzymes, and it underscores the need to mind thermodynamic stability in any strategy for engineering novel or altered enzymatic active sites in proteins.

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Label-free imaging, detection, and mass measurement of single viruses by surface plasmon resonance.

Publication Date: 2010 Aug 26 PMID: 20798340
Authors: Wang, S. - Shan, X. - Patel, U. - Huang, X. - Lu, J. - Li, J. - Tao, N.
Journal: Proc Natl Acad Sci U S A

We report on label-free imaging, detection, and mass/size measurement of single viral particles in solution by high-resolution surface plasmon resonance microscopy. Diffraction of propagating plasmon waves along a metal surface by the viral particles creates images of the individual particles, which allow us to detect the binding of the viral particles to surfaces functionalized with and without antibodies. We show that the intensity of the particle image is related to the mass of the particle, from which we determine the mass and mass distribution of influenza viral particles with a mass detection limit of approximately 1 ag (or 0.2 fg/mm(2)). This work demonstrates a multiplexed method to measure the masses of individual viral particles and to study the binding activity of the viral particles.

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Functional interactions between membrane-bound transporters and membranes.

Publication Date: 2010 Aug 23 PMID: 20798065
Authors: Ojemyr, L. N. - Lee, H. J. - Gennis, R. B. - Brzezinski, P.
Journal: Proc Natl Acad Sci U S A

One key role of many cellular membranes is to hold a transmembrane electrochemical ion gradient that stores free energy, which is used, for example, to generate ATP or to drive transmembrane transport processes. In mitochondria and many bacteria, the gradient is maintained by proton-transport proteins that are part of the respiratory (electron-transport) chain. Even though our understanding of the structure and function of these proteins has increased significantly, very little is known about the specific role of functional protein-membrane and membrane-mediated protein-protein interactions. Here, we have investigated the effect of membrane incorporation on proton-transfer reactions within the membrane-bound proton pump cytochrome c oxidase. The results show that the membrane acts to accelerate proton transfer into the enzyme's catalytic site and indicate that the intramolecular proton pathway is wired via specific amino acid residues to the two-dimensional space defined by the membrane surface. We conclude that the membrane not only acts as a passive barrier insulating the interior of the cell from the exterior solution, but also as a component of the energy-conversion machinery.

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Highly sensitive and selective odorant sensor using living cells expressing insect olfactory receptors.

Publication Date: 2010 Aug 31 PMID: 20798064
Authors: Misawa, N. - Mitsuno, H. - Kanzaki, R. - Takeuchi, S.
Journal: Proc Natl Acad Sci U S A

This paper describes a highly sensitive and selective chemical sensor using living cells (Xenopus laevis oocytes) within a portable fluidic device. We constructed an odorant sensor whose sensitivity is a few parts per billion in solution and can simultaneously distinguish different types of chemicals that have only a slight difference in double bond isomerism or functional group such as horizontal line OH, horizontal line CHO and horizontal line C( horizontal lineO) horizontal line . We developed a semiautomatic method to install cells to the fluidic device and achieved stable and reproducible odorant sensing. In addition, we found that the sensor worked for multiple-target chemicals and can be integrated with a robotic system without any noise reduction systems. Our developed sensor is compact and easy to replace in the system. We believe that the sensor can potentially be incorporated into a portable system for monitoring environmental and physical conditions.

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Discriminating early stage A{beta}42 monomer structures using chirality-induced 2DIR spectroscopy in a simulation study.

Publication Date: 2010 Aug 23 PMID: 20798063
Authors: Zhuang, W. - Sgourakis, N. G. - Li, Z. - Garcia, A. E. - Mukamel, S.
Journal: Proc Natl Acad Sci U S A

Elucidating the structural features of the Abeta monomer, the peptide constituent of amyloid fibrils found in Alzheimer's disease, can enable a direct characterization of aggregation pathways. Recent studies support the view that the ensemble of Abeta42 monomers is a mixture of diverse ordered and disordered conformational species, which can be classified according to the formation of a characteristic beta-hairpin conformation in a certain region. Despite the disparity in the structural features of these species, commonly used spectroscopic techniques such as NMR may not directly trace the conformational dynamics in the ensemble due to the limited time resolution and the lack of well-resolved spectral features for different comformers. Here we use molecular dynamics simulations combined with simulations of two-dimensional IR (2DIR) spectra to investigate the structure of these species, their interchange kinetics, and their spectral features. We show that while the discrimination efficiency of the ordinary, nonchiral 2DIR signal is limited due to its intrinsic dependence on common order parameters that are dominated by the generally unstructured part of the sequence, signals with carefully designed chirality-sensitive pulse configurations have the high resolution required for differentiating the various monomer structures. Our combined simulation studies indicate the power of the chirality-induced (CI) 2DIR technique in investigating early events in Abeta42 aggregation and open the possibility for their use as a novel experimental tool.

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Bioinspired optofluidic FRET lasers via DNA scaffolds.

Publication Date: 2010 Aug 23 PMID: 20798062
Authors: Sun, Y. - Shopova, S. I. - Wu, C. S. - Arnold, S. - Fan, X.
Journal: Proc Natl Acad Sci U S A

Optofluidic dye lasers hold great promise for adaptive photonic devices, compact and wavelength-tunable light sources, and micro total analysis systems. To date, however, nearly all those lasers are directly excited by tuning the pump laser into the gain medium absorption band. Here we demonstrate bioinspired optofluidic dye lasers excited by FRET, in which the donor-acceptor distance, ratio, and spatial configuration can be precisely controlled by DNA scaffolds. The characteristics of the FRET lasers such as spectrum, threshold, and energy conversion efficiency are reported. Through DNA scaffolds, nearly 100% energy transfer can be maintained regardless of the donor and acceptor concentration. As a result, efficient FRET lasing is achieved at an unusually low acceptor concentration of micromolar, over 1,000 times lower than that in conventional optofluidic dye lasers. The lasing threshold is on the order of muJ/mm(2). Various DNA scaffold FRET lasers are demonstrated to illustrate vast possibilities in optofluidic laser designs. Our work opens a door to many researches and applications such as intracavity bio/chemical sensing, biocontrolled photonic devices, and biophysics.

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Multidomain assembled states of Hck tyrosine kinase in solution.

Publication Date: 2010 Aug 23 PMID: 20798061
Authors: Yang, S. - Blachowicz, L. - Makowski, L. - Roux, B.
Journal: Proc Natl Acad Sci U S A

An approach combining small-angle X-ray solution scattering (SAXS) data with coarse-grained (CG) simulations is developed to characterize the assembly states of Hck, a member of the Src-family kinases, under various conditions in solution. First, a basis set comprising a small number of assembly states is generated from extensive CG simulations. Second, a theoretical SAXS profile for each state in the basis set is computed by using the Fast-SAXS method. Finally, the relative population of the different assembly states is determined via a Bayesian-based Monte Carlo procedure seeking to optimize the theoretical scattering profiles against experimental SAXS data. The study establishes the concept of basis-set supported SAXS (BSS-SAXS) reconstruction combining computational and experimental techniques. Here, BSS-SAXS reconstruction is used to reveal the structural organization of Hck in solution and the different shifts in the equilibrium population of assembly states upon the binding of different signaling peptides.

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Assembly of Q{beta} viral RNA polymerase with host translational elongation factors EF-Tu and -Ts.

Publication Date: 2010 Aug 23 PMID: 20798060
Authors: Takeshita, D. - Tomita, K.
Journal: Proc Natl Acad Sci U S A

Replication and transcription of viral RNA genomes rely on host-donated proteins. Qbeta virus infects Escherichia coli and replicates and transcribes its own genomic RNA by Qbeta replicase. Qbeta replicase requires the virus-encoded RNA-dependent RNA polymerase (beta-subunit), and the host-donated translational elongation factors EF-Tu and -Ts, as active core subunits for its RNA polymerization activity. Here, we present the crystal structure of the core Qbeta replicase, comprising the beta-subunit, EF-Tu and -Ts. The beta-subunit has a right-handed structure, and the EF-Tu:Ts binary complex maintains the structure of the catalytic core crevasse of the beta-subunit through hydrophobic interactions, between the finger and thumb domains of the beta-subunit and domain-2 of EF-Tu and the coiled-coil motif of EF-Ts, respectively. These hydrophobic interactions are required for the expression and assembly of the Qbeta replicase complex. Thus, EF-Tu and -Ts have chaperone-like functions in the maintenance of the structure of the active Qbeta replicase. Modeling of the template RNA and the growing RNA in the catalytic site of the Qbeta replicase structure also suggests that structural changes of the RNAs and EF-Tu:Ts should accompany processive RNA polymerization and that EF-Tu:Ts in the Qbeta replicase could function to modulate the RNA folding and structure.

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Superconductivity at ~100 K in dense SiH4(H2)2 predicted by first principles.

Publication Date: 2010 Aug 23 PMID: 20798059
Authors: Li, Y. - Gao, G. - Xie, Y. - Ma, Y. - Cui, T. - Zou, G.
Journal: Proc Natl Acad Sci U S A

Motivated by the potential high-temperature superconductivity in hydrogen-rich materials, the high-pressure structures of SiH(4)(H(2))(2) in the pressure range 50-300 GPa were extensively explored by using a genetic algorithm. An intriguing layered orthorhombic (Ccca) structure was revealed to be energetically stable above 248 GPa with the inclusion of zero-point energy. The Ccca structure is metallic and composed of hydrogen shared SiH(8) dodecahedra layers intercalated by orientationally ordered molecular H(2). Application of the Allen-Dynes modified McMillan equation yields remarkably high superconducting temperatures of 98-107 K at 250 GPa, among the highest values reported so far for phonon-mediated superconductors. Analysis reveals a unique superconducting mechanism that the direct interactions between H(2) and SiH(4) molecules at high pressure play the major role in the high superconductivity, while the contribution from H(2) vibrons is minor.

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Surface Chemistry Special Feature: Imprinting self-assembled patterns of lines at a semiconductor surface, using heat, light, or electrons.

Publication Date: 2010 Aug 23 PMID: 20798058
Authors: Harikumar, K. R. - McNab, I. R. - Polanyi, J. C. - Zabet-Khosousi, A. - Hofer, W. A.
Journal: Proc Natl Acad Sci U S A

The fabrication of nano devices at surfaces makes conflicting demands of mobility for self-assembly (SA) and immobility for permanence. The solution proposed in earlier work from this laboratory involved pattern formation in physisorbed molecules by SA, followed by localized reaction to chemically imprint the pattern substantially unchanged, a procedure we termed molecular-scale imprinting (MSI). Here, as proof of generality we extended this procedure, previously applied to imprinting circles on Si(111)-7 x 7, to SA lines of 1-chloropentane (CP) on Si(100)-2 x 1. The physisorbed lines consisted of pairs of CP that grew perpendicular to the Si dimer rows, as shown by scanning tunneling microscopy and ab initio theory. Chemical reaction of these lines with the surface was triggered in separate experiments by three different modes of energization: heat, electrons, or light. In all cases the CP molecules underwent MSI with a Si atom beneath so that the physisorbed lines of CP pairs were imprinted as chemisorbed lines of Cl pairs.

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RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription.

Publication Date: 2010 Aug 23 PMID: 20798057
Authors: Huang, X. - Wang, D. - Weiss, D. R. - Bushnell, D. A. - Kornberg, R. D. - Levitt, M.
Journal: Proc Natl Acad Sci U S A

A structurally conserved element, the trigger loop, has been suggested to play a key role in substrate selection and catalysis of RNA polymerase II (pol II) transcription elongation. Recently resolved X-ray structures showed that the trigger loop forms direct interactions with the beta-phosphate and base of the matched nucleotide triphosphate (NTP) through residues His1085 and Leu1081, respectively. In order to understand the role of these two critical residues in stabilizing active site conformation in the dynamic complex, we performed all-atom molecular dynamics simulations of the wild-type pol II elongation complex and its mutants in explicit solvent. In the wild-type complex, we found that the trigger loop is stabilized in the "closed" conformation, and His1085 forms a stable interaction with the NTP. Simulations of point mutations of His1085 are shown to affect this interaction; simulations of alternative protonation states, which are inaccessible through experiment, indicate that only the protonated form is able to stabilize the His1085-NTP interaction. Another trigger loop residue, Leu1081, stabilizes the incoming nucleotide position through interaction with the nucleotide base. Our simulations of this Leu mutant suggest a three-component mechanism for correctly positioning the incoming NTP in which (i) hydrophobic contact through Leu1081, (ii) base stacking, and (iii) base pairing work together to minimize the motion of the incoming NTP base. These results complement experimental observations and provide insight into the role of the trigger loop on transcription fidelity.

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H-NS forms a superhelical protein scaffold for DNA condensation.

Publication Date: 2010 Aug 23 PMID: 20798056
Authors: Arold, S. T. - Leonard, P. G. - Parkinson, G. N. - Ladbury, J. E.
Journal: Proc Natl Acad Sci U S A

The histone-like nucleoid structuring (H-NS) protein plays a fundamental role in DNA condensation and is a key regulator of enterobacterial gene expression in response to changes in osmolarity, pH, and temperature. The protein is capable of high-order self-association via interactions of its oligomerization domain. Using crystallography, we have solved the structure of this complete domain in an oligomerized state. The observed superhelical structure establishes a mechanism for the self-association of H-NS via both an N-terminal antiparallel coiled-coil and a second, hitherto unidentified, helix-turn-helix dimerization interface at the C-terminal end of the oligomerization domain. The helical scaffold suggests the formation of a H-NS:plectonemic DNA nucleoprotein complex that is capable of explaining published biophysical and functional data, and establishes a unifying structural basis for coordinating the DNA packaging and transcription repression functions of H-NS.

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Efficacy of geoengineering to limit 21st century sea-level rise.

Publication Date: 2010 Aug 30 PMID: 20798055
Authors: Moore, J. C. - Jevrejeva, S. - Grinsted, A.
Journal: Proc Natl Acad Sci U S A

Geoengineering has been proposed as a feasible way of mitigating anthropogenic climate change, especially increasing global temperatures in the 21st century. The two main geoengineering options are limiting incoming solar radiation, or modifying the carbon cycle. Here we examine the impact of five geoengineering approaches on sea level; SO(2) aerosol injection into the stratosphere, mirrors in space, afforestation, biochar, and bioenergy with carbon sequestration. Sea level responds mainly at centennial time scales to temperature change, and has been largely driven by anthropogenic forcing since 1850. Making use a model of sea-level rise as a function of time-varying climate forcing factors (solar radiation, volcanism, and greenhouse gas emissions) we find that sea-level rise by 2100 will likely be 30 cm higher than 2000 levels despite all but the most aggressive geoengineering under all except the most stringent greenhouse gas emissions scenarios. The least risky and most desirable way of limiting sea-level rise is bioenergy with carbon sequestration. However aerosol injection or a space mirror system reducing insolation at an accelerating rate of 1 W m(-2) per decade from now to 2100 could limit or reduce sea levels. Aerosol injection delivering a constant 4 W m(-2) reduction in radiative forcing (similar to a 1991 Pinatubo eruption every 18 months) could delay sea-level rise by 40-80 years. Aerosol injection appears to fail cost-benefit analysis unless it can be maintained continuously, and damage caused by the climate response to the aerosols is less than about 0.6% Global World Product.

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Four-electron oxidation of p-hydroxylaminobenzoate to p-nitrobenzoate by a peroxodiferric complex in AurF from Streptomyces thioluteus.

Publication Date: 2010 Aug 23 PMID: 20798054
Authors: Li, N. - Korboukh, V. K. - Krebs, C. - Bollinger, J. M. Jr
Journal: Proc Natl Acad Sci U S A

The nonheme di-iron oxygenase, AurF, converts p-aminobenzoate (Ar-NH(2), where Ar = 4-carboxyphenyl) to p-nitrobenzoate (Ar-NO(2)) in the biosynthesis of the antibiotic, aureothin, by Streptomyces thioluteus. It has been reported that this net six-electron oxidation proceeds in three consecutive, two-electron steps, through p-hydroxylaminobenzoate (Ar-NHOH) and p-nitrosobenzoate (Ar-NO) intermediates, with each step requiring one equivalent of O(2) and two exogenous reducing equivalents. We recently demonstrated that a peroxodiiron(III/III) complex (peroxo- -AurF) formed by addition of O(2) to the diiron(II/II) enzyme ( -AurF) effects the initial oxidation of Ar-NH(2), generating a mu-(oxo)diiron(III/III) form of the enzyme (mu-oxo- -AurF) and (presumably) Ar-NHOH. Here we show that peroxo- -AurF also oxidizes Ar-NHOH. Unexpectedly, this reaction proceeds through to the Ar-NO(2) final product, a four-electron oxidation, and produces -AurF, with which O(2) can combine to regenerate peroxo- -AurF. Thus, conversion of Ar-NHOH to Ar-NO(2) requires only a single equivalent of O(2) and (starting from -AurF or peroxo- -AurF) is fully catalytic in the absence of exogenous reducing equivalents, by contrast to the published stoichiometry. This novel type of four-electron N-oxidation is likely also to occur in the reaction sequences of nitro-installing di-iron amine oxygenases in the biosyntheses of other natural products.

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Metallic ferromagnetism in the Kondo lattice.

Publication Date: 2010 Aug 23 PMID: 20798053
Authors: Yamamoto, S. J. - Si, Q.
Journal: Proc Natl Acad Sci U S A

Metallic magnetism is both ancient and modern, occurring in such familiar settings as the lodestone in compass needles and the hard drive in computers. Surprisingly, a rigorous theoretical basis for metallic ferromagnetism is still largely missing. The Stoner approach perturbatively treats Coulomb interactions when the latter need to be large, whereas the Nagaoka approach incorporates thermodynamically negligible holes into a half-filled band. Here, we show that the ferromagnetic order of the Kondo lattice is amenable to an asymptotically exact analysis over a range of interaction parameters. In this ferromagnetic phase, the conduction electrons and local moments are strongly coupled but the Fermi surface does not enclose the latter (i.e., it is "small"). Moreover, non-Fermi-liquid behavior appears over a range of frequencies and temperatures. Our results provide the basis to understand some long-standing puzzles in the ferromagnetic heavy fermion metals, and raise the prospect for a new class of ferromagnetic quantum phase transitions.

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Adaptor protein is essential for insect cytokine signaling in hemocytes.

Publication Date: 2010 Aug 23 PMID: 20798052
Authors: Oda, Y. - Matsumoto, H. - Kurakake, M. - Ochiai, M. - Ohnishi, A. - Hayakawa, Y.
Journal: Proc Natl Acad Sci U S A

Growth-blocking peptide (GBP) is an insect cytokine that stimulates a class of immune cells called plasmatocytes to adhere to one another and to foreign surfaces. Although extensive structure-activity studies have been performed on the GBP and its mutants in Lepidoptera Pseudaletia separata, the signaling pathway of GBP-dependent activation of plasmatocytes remains unknown. We identified an adaptor protein (P77) with a molecular mass of 77 kDa containing SH2/SH3 domain binding motifs and an immunoreceptor tyrosine-based activation motif (ITAM)-like domain in the cytoplasmic region of the C terminus. Although P77 showed no capacity for direct binding with GBP, its cytoplasmic tyrosine residues were specifically phosphorylated within seconds after GBP was added to a plasmatocyte suspension. Tyrosine phosphorylation of P77 also was observed when hemocytes were incubated with Enterobactor cloacae or Micrococcus luteus, but this phosphorylation was found to be induced by GBP released from hemocytes stimulated by the pathogens. Tyrosine phosphorylation of the integrin beta subunit also was detected in plasmatocytes stimulated by GBP. Double-stranded RNAs targeting P77 not only decreased GBP-dependent tyrosine phosphorylation of the integrin beta subunit, but also abolished GBP-induced spreading of plasmatocytes on foreign surfaces. P77 RNAi larvae also showed significantly higher mortality than control larvae after infection with Serratia marcescens, indicating that P77 is essential for GBP to mediate a normal innate cellular immunity in insects. These results demonstrate that GBP signaling in plasmatocytes requires the adaptor protein P77, and that active P77-assisted tyrosine phosphorylation of integrins is critical for the activation of plasmatocytes.

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FGF receptor-4 (FGFR4) polymorphism acts as an activity switch of a membrane type 1 matrix metalloproteinase-FGFR4 complex.

Publication Date: 2010 Aug 23 PMID: 20798051
Authors: Sugiyama, N. - Varjosalo, M. - Meller, P. - Lohi, J. - Chan, K. M. - Zhou, Z. - Alitalo, K. - Taipale, J. - Keski-Oja, J. - Lehti, K.
Journal: Proc Natl Acad Sci U S A

Tumor cells use membrane type 1 matrix metalloproteinase (MT1-MMP) for invasion and metastasis. However, the signaling mechanisms that underlie MT1-MMP regulation in cancer have remained unclear. Using a systematic gain-of-function kinome screen for MT1-MMP activity, we have here identified kinases that significantly enhance MT1-MMP activity in tumor cells. In particular, we discovered an MT1-MMP/FGF receptor-4 (FGFR4) membrane complex that either stimulates or suppresses MT1-MMP and FGFR4 activities, depending on a tumor progression-associated polymorphism in FGFR4. The FGFR4-R388 allele, linked to poor cancer prognosis, increased collagen invasion by decreasing lysosomal MT1-MMP degradation. FGFR4-R388 induced MT1-MMP phosphorylation and endosomal stabilization, and surprisingly, the increased MT1-MMP in return enhanced FGFR4-R388 autophosphorylation. A phosphorylation-defective MT1-MMP was stabilized on the cell surface, where it induced simultaneous FGFR4-R388 internalization and dissociation of cell-cell junctions. In contrast, the alternative FGFR4-G388 variant down-regulated MT1-MMP, and the overexpression of MT1-MMP and particularly its phosphorylation-defective mutant vice versa induced FGFR4-G388 degradation. These results provide a mechanistic basis for FGFR4-R388 function in cancer invasion.

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Neuron densities vary across and within cortical areas in primates.

Publication Date: 2010 Aug 23 PMID: 20798050
Authors: Collins, C. E. - Airey, D. C. - Young, N. A. - Leitch, D. B. - Kaas, J. H.
Journal: Proc Natl Acad Sci U S A

The numbers and proportion of neurons in areas and regions of cortex were determined for a single cortical hemisphere from two prosimian galagos, one New World owl monkey, one Old World macaque monkey, and one baboon. The results suggest that there is a common plan of cortical organization across the species examined here and also differences that suggest greater specializations in the Old World monkeys. In all primates examined, primary visual cortex (V1) was the most neuron-dense cortical area and the secondary visual areas had higher-than-average densities. Primary auditory and somatosensory areas tended to have high densities in the Old World macaque and baboon. Neuronal density varies less across cortical areas in prosimian galagos than in the Old World monkeys. Thus, cortical architecture varies greatly within and across primate species, but cell density is greater in cortex devoted to the early stages of sensory processing.

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The apical-basal cell polarity determinant Crumbs regulates Hippo signaling in Drosophila.

Publication Date: 2010 Aug 23 PMID: 20798049
Authors: Chen, C. L. - Gajewski, K. M. - Hamaratoglu, F. - Bossuyt, W. - Sansores-Garcia, L. - Tao, C. - Halder, G.
Journal: Proc Natl Acad Sci U S A

Defects in apical-basal cell polarity and abnormal expression of cell polarity determinants are often associated with cancer in vertebrates. In Drosophila, abnormal expression of apical-basal determinants can cause neoplastic phenotypes, including loss of cell polarity and overproliferation. However, the pathways through which apical-basal polarity determinants affect growth are poorly understood. Here, we investigated the mechanism by which the apical determinant Crumbs (Crb) affects growth in Drosophila imaginal discs. Overexpression of Crb causes severe overproliferation, and we found that loss of Crb similarly results in overgrowth of imaginal discs. Crb gain and loss of function caused defects in Hippo signaling, a key signaling pathway that controls tissue growth in Drosophila and mammals. Manipulation of Crb levels caused the up-regulation of Hippo target genes, genetically interacted with known Hippo pathway components, and required Yorkie, a transcriptional coactivator that acts downstream in the Hippo pathway, for target gene induction and overgrowth. Interestingly, Crb regulates growth and cell polarity through different motifs in its intracellular domain. A juxtamembrane FERM domain-binding motif is responsible for growth regulation and induction of Hippo target gene expression, whereas Crb uses a PDZ-binding motif to form a complex with other polarity factors. The Hippo pathway component Expanded, an apically localized adaptor protein, is mislocalized in both crb mutant cells and Crb overexpressing tissues, whereas the other Hippo pathway components, Fat and Merlin, are unaffected. Taken together, our data show that Crb regulates growth through Hippo signaling, and thus identify Crb as a previously undescribed upstream input into the Hippo pathway.

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Perceptual learning beyond retinotopic reference frame.

Publication Date: 2010 Aug 23 PMID: 20798048
Authors: Zhang, E. - Li, W.
Journal: Proc Natl Acad Sci U S A

Repetitive experience with the same visual stimulus and task can remarkably improve behavioral performance on the task. This well-known perceptual-learning phenomenon is usually specific to the trained retinal- or visual-field location, which is taken as an indication of plastic changes in retinotopic visual areas. In previous studies of perceptual learning, however, a change in stimulus location on the retina is accompanied by positional changes of the stimulus in nonretinotopic frames of reference, such as relative to the head and other objects. It is unclear, therefore, whether the putative location specificity is exclusively retinotopic or if it could also depend on nonretinotopic representation of the stimulus, which is particularly important for multisensory and sensorimotor integration as well as for maintenance of stable visual percepts. Here, by manipulating subjects' gaze direction to control spatial and retinal locations of stimuli independently, we found that, when the stimulated retinal regions were held constant, the improvement with training in motion-direction discrimination of two successively displayed stimuli was restricted to the relative spatial position of the stimuli but independent of their absolute locations in head- and world-centered frame. These findings indicate location specificity of perceptual learning beyond retinotopic frame of reference, suggesting a pliable spatiotopic mechanism that can be specifically shaped by experience for better spatiotemporal integration of the learned stimuli.

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Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors.

Publication Date: 2010 Aug 23 PMID: 20798047
Authors: Lo, S. C. - Pripuzova, N. - Li, B. - Komaroff, A. L. - Hung, G. C. - Wang, R. - Alter, H. J.
Journal: Proc Natl Acad Sci U S A

Chronic fatigue syndrome (CFS) is a serious systemic illness of unknown cause. A recent study identified DNA from a xenotropic murine leukemia virus-related virus (XMRV) in peripheral blood mononuclear cells (PBMCs) from 68 of 101 patients (67%) by nested PCR, as compared with 8 of 218 (3.7%) healthy controls. However, four subsequent reports failed to detect any murine leukemia virus (MLV)-related virus gene sequences in blood of CFS patients. We examined 41 PBMC-derived DNA samples from 37 patients meeting accepted diagnostic criteria for CFS and found MLV-like virus gag gene sequences in 32 of 37 (86.5%) compared with only 3 of 44 (6.8%) healthy volunteer blood donors. No evidence of mouse DNA contamination was detected in the PCR assay system or the clinical samples. Seven of 8 gag-positive patients tested again positive in a sample obtained nearly 15 y later. In contrast to the reported findings of near-genetic identity of all XMRVs, we identified a genetically diverse group of MLV-related viruses. The gag and env sequences from CFS patients were more closely related to those of polytropic mouse endogenous retroviruses than to those of XMRVs and were even less closely related to those of ecotropic MLVs. Further studies are needed to determine whether the same strong association with MLV-related viruses is found in other groups of patients with CFS, whether these viruses play a causative role in the development of CFS, and whether they represent a threat to the blood supply.

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Notch signaling specifies prosensory domains via lateral induction in the developing mammalian inner ear.

Publication Date: 2010 Aug 23 PMID: 20798046
Authors: Hartman, B. H. - Reh, T. A. - Bermingham-McDonogh, O.
Journal: Proc Natl Acad Sci U S A

During inner ear morphogenesis, the process of prosensory specification defines the specific regions of the otic epithelium that will give rise to the six separate inner ear organs essential for hearing and balance. The mechanism of prosensory specification is not fully understood, but there is evidence that the Notch intercellular signaling pathway plays a critical role. The Notch ligand Jagged1 (Jag1) is expressed in the prosensory domains, and mutation of Jag1 impairs sensory formation. Furthermore, pharmacological inhibition of Notch in vitro during prosensory specification disrupts the prosensory process. Additionally, activation of Notch by cDNA electroporation in chick otocysts results in formation of ectopic sensory patches. Here we test whether Notch activity is sufficient for prosensory specification in the mouse, using a Cre-/loxP approach to conditionally activate the Notch pathway in nonsensory regions of the inner ear epithelia during different stages of otic vesicle morphogenesis. We find that broad ectopic activation of Notch at very early developmental stages causes induction of prosensory markers throughout the entire otic epithelium. At later stages of development, activation of Notch in nonsensory regions leads to induction of sensory patches that later differentiate to form complete ectopic sensory structures. Activation of Notch in isolated nonsensory cells results in lateral induction of Jag1 expression in neighboring cells and spreading of prosensory specification to the adjacent cells through an intercellular mechanism. These results support a model where activation of Notch and propagation through lateral induction promote prosensory character in specific regions of the developing otocyst.

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Ezh2, the histone methyltransferase of PRC2, regulates the balance between self-renewal and differentiation in the cerebral cortex.

Publication Date: 2010 Aug 23 PMID: 20798045
Authors: Pereira, J. D. - Sansom, S. N. - Smith, J. - Dobenecker, M. W. - Tarakhovsky, A. - Livesey, F. J.
Journal: Proc Natl Acad Sci U S A

Multipotent progenitor cells of the cerebral cortex balance self-renewal and differentiation to produce complex neural lineages in a fixed temporal order in a cell-autonomous manner. We studied the role of the polycomb epigenetic system, a chromatin-based repressive mechanism, in controlling cortical progenitor cell self-renewal and differentiation. We found that the histone methyltransferase of polycomb repressive complex 2 (PCR2), enhancer of Zeste homolog 2 (Ezh2), is essential for controlling the rate at which development progresses within cortical progenitor cell lineages. Loss of function of Ezh2 removes the repressive mark of trimethylated histone H3 at lysine 27 (H3K27me3) in cortical progenitor cells and also prevents its establishment in postmitotic neurons. Removal of this repressive chromatin modification results in marked up-regulation in gene expression, the consequence of which is a shift in the balance between self-renewal and differentiation toward differentiation, both directly to neurons and indirectly via basal progenitor cell genesis. Although the temporal order of neurogenesis and gliogenesis are broadly conserved under these conditions, the timing of neurogenesis, the relative numbers of different cell types, and the switch to gliogenesis are all altered, narrowing the neurogenic period for progenitor cells and reducing their neuronal output. As a consequence, the timing of cortical development is altered significantly after loss of PRC2 function.

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Angiotensin-converting enzyme 2 attenuates atherosclerotic lesions by targeting vascular cells.

Publication Date: 2010 Aug 23 PMID: 20798044
Authors: Zhang, C. - Zhao, Y. X. - Zhang, Y. H. - Zhu, L. - Deng, B. P. - Zhou, Z. L. - Li, S. Y. - Lu, X. T. - Song, L. L. - Lei, X. M. - Tang, W. B. - Wang, N. - Pan, C. M. - Song, H. D. - Liu, C. X. - Dong, B. - Zhang, Y. - Cao, Y.
Journal: Proc Natl Acad Sci U S A

Angiotensin-converting enzyme 2 (ACE2) is a newly discovered homolog of ACE whose actions oppose those of angiotensin II (AngII). However, the underlying mechanisms by which ACE2 effectively suppresses early atherosclerotic lesions remain poorly understood. Here, we show, both in vitro and in vivo, that ACE2 inhibited the development of early atherosclerotic lesions by suppressing the growth of vascular smooth muscle cells (VSMCs) and improving endothelial function. In a relatively large cohort animal study (66 rabbits), aortic segments transfected by Ad-ACE2 showed significantly attenuated fatty streak formation, neointimal macrophage infiltration, and alleviation of impaired endothelial function. Segments also showed decreased expression of monocyte chemoattractant protein 1, lectin-like oxidized low-density lipoprotein receptor 1, and proliferating cell nuclear antigen, which led to the delayed onset of atherosclerotic lesions. At the cellular level, ACE2 significantly modulated AngII-induced growth and migration in human umbilical vein endothelial cells and VSMCs. The antiatherosclerotic effect of ACE2 involved down-regulation of the ERK-p38, JAK-STAT, and AngII-ROS-NF-kappaB signaling pathways and up-regulation of the PI3K-Akt pathway. These findings revealed the molecular mechanisms of the antiatherosclerotic activity of ACE2 and suggested that modulation of ACE2 could offer a therapeutic option for treating atherosclerosis.

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Extracellular DNA traps promote thrombosis.

Publication Date: 2010 Aug 23 PMID: 20798043
Authors: Fuchs, T. A. - Brill, A. - Duerschmied, D. - Schatzberg, D. - Monestier, M. - Myers, D. D. Jr - Wrobleski, S. K. - Wakefield, T. W. - Hartwig, J. H. - Wagner, D. D.
Journal: Proc Natl Acad Sci U S A

Neutrophil extracellular traps (NETs) are part of the innate immune response to infections. NETs are a meshwork of DNA fibers comprising histones and antimicrobial proteins. Microbes are immobilized in NETs and encounter a locally high and lethal concentration of effector proteins. Recent studies show that NETs are formed inside the vasculature in infections and noninfectious diseases. Here we report that NETs provide a heretofore unrecognized scaffold and stimulus for thrombus formation. NETs perfused with blood caused platelet adhesion, activation, and aggregation. DNase or the anticoagulant heparin dismantled the NET scaffold and prevented thrombus formation. Stimulation of platelets with purified histones was sufficient for aggregation. NETs recruited red blood cells, promoted fibrin deposition, and induced a red thrombus, such as that found in veins. Markers of extracellular DNA traps were detected in a thrombus and plasma of baboons subjected to deep vein thrombosis, an example of inflammation-enhanced thrombosis. Our observations indicate that NETs are a previously unrecognized link between inflammation and thrombosis and may further explain the epidemiological association of infection with thrombosis.

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Patients, patience, and the publication process.

Publication Date: 2010 Aug 23 PMID: 20798042
Authors: Schekman, R.
Journal: Proc Natl Acad Sci U S A

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Conserved RNaseII domain protein functions in cytoplasmic mRNA decay and suppresses Arabidopsis decapping mutant phenotypes.

Publication Date: 2010 Aug 23 PMID: 20798041
Authors: Zhang, W. - Murphy, C. - Sieburth, L. E.
Journal: Proc Natl Acad Sci U S A

Both transcription and RNA decay are critical for normal gene regulation. Arabidopsis mutants with defects in VARICOSE (VCS), a decapping complex scaffold protein, lack mRNA decapping and 5'-to-3' decay. These mutants show either severe or suppressed phenotypes, depending on the Arabidopsis accession. Here, we show that the molecular basis for this variation is the SUPPRESSOR OF VARICOSE (SOV), a locus that encodes a conserved, cytoplasmically localized RRP44-like RNaseII-domain protein. In vivo RNA decay assays suggest that active forms of this protein carry out decay on mRNA substrates that overlap with those of the decapping complex. Members of this conserved gene family encode proteins lacking the PIN domain, suggesting that SOV is not a functional component of the RNA exosome.

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Hyper telomere recombination accelerates replicative senescence and may promote premature aging.

Publication Date: 2010 Aug 23 PMID: 20798040
Authors: Hagelstrom, R. T. - Blagoev, K. B. - Niedernhofer, L. J. - Goodwin, E. H. - Bailey, S. M.
Journal: Proc Natl Acad Sci U S A

Werner syndrome and Bloom syndrome result from defects in the RecQ helicases Werner (WRN) and Bloom (BLM), respectively, and display premature aging phenotypes. Similarly, XFE progeroid syndrome results from defects in the ERCC1-XPF DNA repair endonuclease. To gain insight into the origin of cellular senescence and human aging, we analyzed the dependence of sister chromatid exchange (SCE) frequencies on location [i.e., genomic (G-SCE) vs. telomeric (T-SCE) DNA] in primary human fibroblasts deficient in WRN, BLM, or ERCC1-XPF. Consistent with our other studies, we found evidence of elevated T-SCE in telomerase-negative but not telomerase-positive backgrounds. In telomerase-negative WRN-deficient cells, T-SCE-but not G-SCE-frequencies were significantly increased compared with controls. In contrast, SCE frequencies were significantly elevated in BLM-deficient cells irrespective of genome location. In ERCC1-XPF-deficient cells, neither T- nor G-SCE frequencies differed from controls. A theoretical model was developed that allowed an in silico investigation into the cellular consequences of increased T-SCE frequency. The model predicts that in cells with increased T-SCE, the onset of replicative senescence is dramatically accelerated even though the average rate of telomere loss has not changed. Premature cellular senescence may act as a powerful tumor-suppressor mechanism in telomerase-deficient cells with mutations that cause T-SCE levels to rise. Furthermore, T-SCE-driven premature cellular senescence may be a factor contributing to accelerated aging in Werner and Bloom syndromes, but not XFE progeroid syndrome.

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Noninvasive method for assessing the human circadian clock using hair follicle cells.

Publication Date: 2010 Aug 31 PMID: 20798039
Authors: Akashi, M. - Soma, H. - Yamamoto, T. - Tsugitomi, A. - Yamashita, S. - Yamamoto, T. - Nishida, E. - Yasuda, A. - Liao, J. K. - Node, K.
Journal: Proc Natl Acad Sci U S A

A thorough understanding of the circadian clock requires qualitative evaluation of circadian clock gene expression. Thus far, no simple and effective method for detecting human clock gene expression has become available. This limitation has greatly hampered our understanding of human circadian rhythm. Here we report a convenient, reliable, and less invasive method for detecting human clock gene expression using biopsy samples of hair follicle cells from the head or chin. We show that the circadian phase of clock gene expression in hair follicle cells accurately reflects that of individual behavioral rhythms, demonstrating that this strategy is appropriate for evaluating the human peripheral circadian clock. Furthermore, using this method, we indicate that rotating shift workers suffer from a serious time lag between circadian gene expression rhythms and lifestyle. Qualitative evaluation of clock gene expression in hair follicle cells, therefore, may be an effective approach for studying the human circadian clock in the clinical setting.

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Disruption of HDAC/CoREST/REST repressor by dnREST reduces genome silencing and increases virulence of herpes simplex virus.

Publication Date: 2010 Aug 23 PMID: 20798038
Authors: Du, T. - Zhou, G. - Khan, S. - Gu, H. - Roizman, B.
Journal: Proc Natl Acad Sci U S A

In nonneuronal cells, herpes simplex virus 1 overcomes host defenses, replicates, and ultimately kills the infected cell. Among the host defenses suppressed by the virus is a repressor complex whose key components are histone deacetylase (HDAC) 1 or 2, RE-1 silencing transcription factor (REST), corepressor of REST (CoREST), and lysine-specific demethylase (LSD) 1. In neurons innervating cells at the portal of entry into the body, the virus establishes a "latent" infection in which viral DNA is silenced with the exception of a family of genes. The question posed here is whether the virus hijacks this repressor complex to silence itself in neurons during the latent state. To test this hypothesis, we inserted into the wild-type virus genome a wild-type REST [recombinant (R) 111], a dominant-negative REST (dnREST) lacking the N- and C-terminal repressor domains (R112), or an insertion control consisting of tandem repeats of stop codons (R113). The recombinant virus R112 carrying the dnREST replicated better and was more virulent than the wild-type parent or the other recombinant viruses when administered by the corneal or i.p. routes. Moreover, in contrast to other recombinants, corneal route inoculation by R112 recombinant virus resulted in higher DNA copy numbers, higher levels of infectious virus in eye, trigeminal ganglion, or brain, and virtually complete destruction of trigeminal ganglia in mice that may ultimately succumb to infection. These results support an earlier conclusion that the HDAC/CoREST/REST/LSD1 repressor complex is a significant component of the host innate immunity and are consistent with the hypothesis that HSV-1 hijacks the repressor to silence itself during latent infection.

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Epigenetic effects of polymorphic Y chromosomes modulate chromatin components, immune response, and sexual conflict.

Publication Date: 2010 Aug 23 PMID: 20798037
Authors: Lemos, B. - Branco, A. T. - Hartl, D. L.
Journal: Proc Natl Acad Sci U S A

Genetic conflicts between sexes and generations provide a foundation for understanding the functional evolution of sex chromosomes and sexually dimorphic phenotypes. Y chromosomes of Drosophila contain multi-megabase stretches of satellite DNA repeats and a handful of protein-coding genes that are monomorphic within species. Nevertheless, polymorphic variation in heterochromatic Y chromosomes of Drosophila result in genome-wide gene expression variation. Here we show that such naturally occurring Y-linked regulatory variation (YRV) can be detected in somatic tissues and contributes to the epigenetic balance of heterochromatin/euchromatin at three distinct loci showing position-effect variegation (PEV). Moreover, polymorphic Y chromosomes differentially affect the expression of thousands of genes in XXY female genotypes in which Y-linked protein-coding genes are not transcribed. The data show a disproportionate influence of YRV on the variable expression of genes whose protein products localize to the nucleus, have nucleic-acid binding activity, and are involved in transcription, chromosome organization, and chromatin assembly. These include key components such as HP1, Trithorax-like (GAGA factor), Su(var)3-9, Brahma, MCM2, ORC2, and inner centromere protein. Furthermore, mitochondria-related genes, immune response genes, and transposable elements are also disproportionally affected by Y chromosome polymorphism. These functional clusterings may arise as a consequence of the involvement of Y-linked heterochromatin in the origin and resolution of genetic conflicts between males and females. Taken together, our results indicate that Y chromosome heterochromatin serves as a major source of epigenetic variation in natural populations that interacts with chromatin components to modulate the expression of biologically relevant phenotypic variation.

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Mouse retroviruses and chronic fatigue syndrome: Does X (or P) mark the spot?

Publication Date: 2010 Aug 23 PMID: 20798036
Authors: Courgnaud, V. - Battini, J. L. - Sitbon, M. - Mason, A. L.
Journal: Proc Natl Acad Sci U S A

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Defective membrane expression of the Na+-HCO3- cotransporter NBCe1 is associated with familial migraine.

Publication Date: 2010 Aug 23 PMID: 20798035
Authors: Suzuki, M. - Van Paesschen, W. - Stalmans, I. - Horita, S. - Yamada, H. - Bergmans, B. A. - Legius, E. - Riant, F. - De Jonghe, P. - Li, Y. - Sekine, T. - Igarashi, T. - Fujimoto, I. - Mikoshiba, K. - Shimadzu, M. - Shiohara, M. - Braverman, N. - Al-Gazali, L. - Fujita, T. - Seki, G.
Journal: Proc Natl Acad Sci U S A

Homozygous mutations in SLC4A4, encoding the electrogenic Na(+)-HCO(3)(-) cotransporter NBCe1, have been known to cause proximal renal tubular acidosis (pRTA) and ocular abnormalities. In this study, we report two sisters with pRTA, ocular abnormalities, and hemiplegic migraine. Genetic analysis ruled out pathological mutations in the known genes for familial hemiplegic migraine, but identified a homozygous 65-bp deletion (Delta65bp) in the C terminus of NBCe1, corresponding to the codon change S982NfsX4. Several heterozygous members of this family also presented glaucoma and migraine with or without aura. Despite the normal electrogenic activity in Xenopus oocytes, the Delta65bp mutant showed almost no transport activity due to a predominant cytosolic retention in mammalian cells. Furthermore, coexpression experiments uncovered a dominant negative effect of the mutant through hetero-oligomer formation with wild-type NBCe1. Among other pRTA pedigrees with different NBCe1 mutations, we identified four additional homozygous patients with migraine. The immunohistological and functional analyses of these mutants demonstrate that the near total loss of NBCe1 activity in astrocytes can cause migraine potentially through dysregulation of synaptic pH.

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Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats.

Publication Date: 2010 Aug 23 PMID: 20798034
Authors: Hargus, G. - Cooper, O. - Deleidi, M. - Levy, A. - Lee, K. - Marlow, E. - Yow, A. - Soldner, F. - Hockemeyer, D. - Hallett, P. J. - Osborn, T. - Jaenisch, R. - Isacson, O.
Journal: Proc Natl Acad Sci U S A

Recent advances in deriving induced pluripotent stem (iPS) cells from patients offer new possibilities for biomedical research and clinical applications, as these cells could be used for autologous transplantation. We differentiated iPS cells from patients with Parkinson's disease (PD) into dopaminergic (DA) neurons and show that these DA neurons can be transplanted without signs of neurodegeneration into the adult rodent striatum. The PD patient iPS (PDiPS) cell-derived DA neurons survived at high numbers, showed arborization, and mediated functional effects in an animal model of PD as determined by reduction of amphetamine- and apomorphine-induced rotational asymmetry, but only a few DA neurons projected into the host striatum at 16 wk after transplantation. We next applied FACS for the neural cell adhesion molecule NCAM on differentiated PDiPS cells before transplantation, which resulted in surviving DA neurons with functional effects on amphetamine-induced rotational asymmetry in a 6-OHDA animal model of PD. Morphologically, we found that PDiPS cell-derived non-DA neurons send axons along white matter tracts into specific close and remote gray matter target areas in the adult brain. Such findings establish the transplantation of human PDiPS cell-derived neurons as a long-term in vivo method to analyze potential disease-related changes in a physiological context. Our data also demonstrate proof of principle of survival and functional effects of PDiPS cell-derived DA neurons in an animal model of PD and encourage further development of differentiation protocols to enhance growth and function of implanted PDiPS cell-derived DA neurons in regard to potential therapeutic applications.

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12/15-lipoxygenase-derived lipid peroxides control receptor tyrosine kinase signaling through oxidation of protein tyrosine phosphatases.

Publication Date: 2010 Aug 23 PMID: 20798033
Authors: Conrad, M. - Sandin, A. - Forster, H. - Seiler, A. - Frijhoff, J. - Dagnell, M. - Bornkamm, G. W. - Radmark, O. - Hooft van Huijsduijnen, R. - Aspenstrom, P. - Bohmer, F. - Ostman, A.
Journal: Proc Natl Acad Sci U S A

Protein tyrosine phosphatases (PTPs) are regulated through reversible oxidation of the active-site cysteine. Previous studies have implied soluble reactive oxygen species (ROS), like H(2)O(2), as the mediators of PTP oxidation. The potential role(s) of peroxidized lipids in PTP oxidation have not been described. This study demonstrates that increases in cellular lipid peroxides, induced by disruption of glutathione peroxidase 4, induce cellular PTP oxidation and reduce the activity of PDGF receptor targeting PTPs. These effects were accompanied by site-selective increased PDGF beta-receptor phosphorylation, sensitive to 12/15-lipoxygenase (12/15-LOX) inhibitors, and increased PDGF-induced cytoskeletal rearrangements. Importantly, the 12/15-LOX-derived 15-OOH-eicosatetraenoic acid lipid peroxide was much more effective than H(2)O(2) in induction of in vitro PTP oxidation. Our study thus establishes that lipid peroxides are previously unrecognized inducers of oxidation of PTPs. This identifies a pathway for control of receptor tyrosine kinase signaling, which might also be involved in the etiology of diseases associated with increased lipid peroxidation.

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Fine-tuning of neuronal architecture requires two profilin isoforms.

Publication Date: 2010 Aug 23 PMID: 20798032
Authors: Michaelsen, K. - Murk, K. - Zagrebelsky, M. - Dreznjak, A. - Jockusch, B. M. - Rothkegel, M. - Korte, M.
Journal: Proc Natl Acad Sci U S A

Two profilin isoforms (PFN1 and PFN2a) are expressed in the mammalian brain. Although profilins are essential for regulating actin dynamics in general, the specific role of these isoforms in neurons has remained elusive. We show that knockdown of the neuron-specific PFN2a results in a significant reduction in dendrite complexity and spine numbers of hippocampal neurons. Overexpression of PFN1 in PFN2a-deficient neurons prevents the loss of spines but does not restore dendritic complexity. Furthermore, we show that profilins are involved in differentially regulating actin dynamics downstream of the pan-neurotrophin receptor (p75(NTR)), a receptor engaged in modulating neuronal morphology. Overexpression of PFN2a restores the morphological changes in dendrites caused by p75(NTR) overexpression, whereas PFN1 restores the normal spine density. Our data assign specific functions to the two PFN isoforms, possibly attributable to different affinities for potent effectors also involved in actin dynamics, and suggest that they are important for the signal-dependent fine-tuning of neuronal architecture.

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AMPA receptors gate spine Ca2+ transients and spike-timing-dependent potentiation.

Publication Date: 2010 Aug 23 PMID: 20798031
Authors: Holbro, N. - Grunditz, A. - Wiegert, J. S. - Oertner, T. G.
Journal: Proc Natl Acad Sci U S A

Spike timing-dependent long-term potentiation (t-LTP) is the embodiment of Donald Hebb's postulated rule for associative memory formation. Pre- and postsynaptic action potentials need to be precisely correlated in time to induce this form of synaptic plasticity. NMDA receptors have been proposed to detect correlated activity and to trigger synaptic plasticity. However, the slow kinetic of NMDA receptor currents is at odds with the millisecond precision of coincidence detection. Here we show that AMPA receptors are responsible for the extremely narrow time window for t-LTP induction. Furthermore, we visualized synergistic interactions between AMPA and NMDA receptors and back-propagating action potentials on the level of individual spines. Supralinear calcium signals were observed for spike timings that induced t-LTP and were most pronounced in spines well isolated from the dendrite. We conclude that AMPA receptors gate the induction of associative synaptic plasticity by regulating the temporal precision of coincidence detection.

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Dynamics of heat shock protein 90 C-terminal dimerization is an important part of its conformational cycle.

Publication Date: 2010 Aug 24 PMID: 20736353
Authors: Ratzke, C. - Mickler, M. - Hellenkamp, B. - Buchner, J. - Hugel, T.
Journal: Proc Natl Acad Sci U S A

The molecular chaperone heat shock protein 90 (Hsp90) is an important and abundant protein in eukaryotic cells, essential for the activation of a large set of signal transduction and regulatory proteins. During the functional cycle, the Hsp90 dimer performs large conformational rearrangements. The transient N-terminal dimerization of Hsp90 has been extensively investigated, under the assumption that the C-terminal interface is stably dimerized. Using a fluorescence-based single molecule assay and Hsp90 dimers caged in lipid vesicles, we were able to separately observe and kinetically analyze N- and C-terminal dimerizations. Surprisingly, the C-terminal dimer opens and closes with fast kinetics. The occupancy of the unexpected C-terminal open conformation can be modulated by nucleotides bound to the N-terminal domain and by N-terminal deletion mutations, clearly showing a communication between the two terminal domains. Moreover our findings suggest that the C- and N-terminal dimerizations are anticorrelated. This changes our view on the conformational cycle of Hsp90 and shows the interaction of two dimerization domains.

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The brain's fight against aging.

Publication Date: 2010 Aug 31 PMID: 20736352
Authors: Mandairon, N. - Didier, A.
Journal: Proc Natl Acad Sci U S A

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Translationally controlled tumor protein is a conserved mitotic growth integrator in animals and plants.

Publication Date: 2010 Aug 24 PMID: 20736351
Authors: Brioudes, F. - Thierry, A. M. - Chambrier, P. - Mollereau, B. - Bendahmane, M.
Journal: Proc Natl Acad Sci U S A

The growth of an organism and its size determination require the tight regulation of cell proliferation and cell growth. However, the mechanisms and regulatory networks that control and integrate these processes remain poorly understood. Here, we address the biological role of Arabidopsis translationally controlled tumor protein (AtTCTP) and test its shared functions in animals and plants. The data support a role of plant AtTCTP as a positive regulator of mitotic growth by specifically controlling the duration of the cell cycle. We show that, in contrast to animal TCTP, plant AtTCTP is not implicated in regulating postmitotic growth. Consistent with this finding, plant AtTCTP can fully rescue cell proliferation defects in Drosophila loss of function for dTCTP. Furthermore, Drosophila dTCTP is able to fully rescue cell proliferation defects in Arabidopsis tctp knockouts. Our data provide evidence that TCTP function in regulating cell division is part of a conserved growth regulatory pathway shared between plants and animals. The study also suggests that, although the cell division machinery is shared in all multicellular organisms to control growth, cell expansion can be uncoupled from cell division in plants but not in animals.

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Altered distributions of Gemini of coiled bodies and mitochondria in motor neurons of TDP-43 transgenic mice.

Publication Date: 2010 Aug 24 PMID: 20736350
Authors: Shan, X. - Chiang, P. M. - Price, D. L. - Wong, P. C.
Journal: Proc Natl Acad Sci U S A

TAR DNA-binding protein-43 (TDP-43), a DNA/RNA-binding protein involved in RNA transcription and splicing, has been associated with the pathophysiology of neurodegenerative diseases, including ALS. However, the function of TDP-43 in motor neurons remains undefined. Here we use both gain- and loss-of-function approaches to determine roles of TDP-43 in motor neurons. Mice expressing human TDP-43 in neurons exhibited growth retardation and premature death that are characterized by abnormal intranuclear inclusions composed of TDP-43 and fused in sarcoma/translocated in liposarcoma (FUS/TLS), and massive accumulation of mitochondria in TDP-43-negative cytoplasmic inclusions in motor neurons, lack of mitochondria in motor axon terminals, and immature neuromuscular junctions. Whereas an elevated level of TDP-43 disrupts the normal nuclear distribution of survival motor neuron (SMN)-associated Gemini of coiled bodies (GEMs) in motor neurons, its absence prevents the formation of GEMs in the nuclei of these cells. Moreover, transcriptome-wide deep sequencing analysis revealed that a decrease in abundance of neurofilament transcripts contributed to the reduction of caliber of motor axons in TDP-43 mice. In concert, our findings indicate that TDP-43 participates in pathways critical for motor neuron physiology, including those that regulate the normal distributions of SMN-associated GEMs in the nucleus and mitochondria in the cytoplasm.

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En masse in vitro functional profiling of the axonal mechanosensitivity of sensory neurons.

Publication Date: 2010 Aug 24 PMID: 20736349
Authors: Usoskin, D. - Zilberter, M. - Linnarsson, S. - Hjerling-Leffler, J. - Uhlen, P. - Harkany, T. - Ernfors, P.
Journal: Proc Natl Acad Sci U S A

Perception of the environment relies on somatosensory neurons. Mechanosensory, proprioceptor and many nociceptor subtypes of these neurons have specific mechanosensitivity profiles to adequately differentiate stimulus patterns. Nevertheless, the cellular basis of differential mechanosensation remains largely elusive. Successful transduction of sensory information relies on the recruitment of sensory neurons and mechanosensation occurring at their peripheral axonal endings in vivo. Conspicuously, existing in vitro models aimed to decipher molecular mechanisms of mechanosensation test single sensory neuron somata at any one time. Here, we introduce a compartmental in vitro chamber design to deliver precisely controlled mechanical stimulation of sensory axons with synchronous real-time imaging of Ca(2+) transients in neuronal somata that reliably reflect action potential firing patterns. We report of three previously not characterized types of mechanosensitive neuron subpopulations with distinct intrinsic axonal properties tuned specifically to static indentation or vibration stimuli, showing that different classes of sensory neurons are tuned to specific types of mechanical stimuli. Primary receptor currents of vibration neurons display rapidly adapting conductance reliably detected for every single stimulus during vibration and are consistently converted into action potentials. This result allows for the characterization of two critical steps of mechanosensation in vivo: primary signal detection and signal conversion into specific action potential firing patterns in axons.

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Nephromyces, a beneficial apicomplexan symbiont in marine animals.

Publication Date: 2010 Aug 24 PMID: 20736348
Authors: Saffo, M. B. - McCoy, A. M. - Rieken, C. - Slamovits, C. H.
Journal: Proc Natl Acad Sci U S A

With malaria parasites (Plasmodium spp.), Toxoplasma, and many other species of medical and veterinary importance its iconic representatives, the protistan phylum Apicomplexa has long been defined as a group composed entirely of parasites and pathogens. We present here a report of a beneficial apicomplexan: the mutualistic marine endosymbiont Nephromyces. For more than a century, the peculiar structural and developmental features of Nephromyces, and its unusual habitat, have thwarted characterization of the phylogenetic affinities of this eukaryotic microbe. Using short-subunit ribosomal DNA (SSU rDNA) sequences as key evidence, with sequence identity confirmed by fluorescence in situ hybridization (FISH), we show that Nephromyces, originally classified as a chytrid fungus, is actually an apicomplexan. Inferences from rDNA data are further supported by the several apicomplexan-like structural features in Nephromyces, including especially the strong resemblance of Nephromyces infective stages to apicomplexan sporozoites. The striking emergence of the mutualistic Nephromyces from a quintessentially parasitic clade accentuates the promise of this organism, and the three-partner symbiosis of which it is a part, as a model for probing the factors underlying the evolution of mutualism, pathogenicity, and infectious disease.

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Cortico-striatal connections predict control over speed and accuracy in perceptual decision making.

Publication Date: 2010 Aug 23 PMID: 20733082
Authors: Forstmann, B. U. - Anwander, A. - Schafer, A. - Neumann, J. - Brown, S. - Wagenmakers, E. J. - Bogacz, R. - Turner, R.
Journal: Proc Natl Acad Sci U S A

When people make decisions they often face opposing demands for response speed and response accuracy, a process likely mediated by response thresholds. According to the striatal hypothesis, people decrease response thresholds by increasing activation from cortex to striatum, releasing the brain from inhibition. According to the STN hypothesis, people decrease response thresholds by decreasing activation from cortex to subthalamic nucleus (STN); a decrease in STN activity is likewise thought to release the brain from inhibition and result in responses that are fast but error-prone. To test these hypotheses-both of which may be true-we conducted two experiments on perceptual decision making in which we used cues to vary the demands for speed vs. accuracy. In both experiments, behavioral data and mathematical model analyses confirmed that instruction from the cue selectively affected the setting of response thresholds. In the first experiment we used ultra-high-resolution 7T structural MRI to locate the STN precisely. We then used 3T structural MRI and probabilistic tractography to quantify the connectivity between the relevant brain areas. The results showed that participants who flexibly change response thresholds (as quantified by the mathematical model) have strong structural connections between presupplementary motor area and striatum. This result was confirmed in an independent second experiment. In general, these findings show that individual differences in elementary cognitive tasks are partly driven by structural differences in brain connectivity. Specifically, these findings support a cortico-striatal control account of how the brain implements adaptive switches between cautious and risky behavior.

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Notch signaling is required for the generation of hair cells and supporting cells in the mammalian inner ear.

Publication Date: 2010 Aug 23 PMID: 20733081
Authors: Pan, W. - Jin, Y. - Stanger, B. - Kiernan, A. E.
Journal: Proc Natl Acad Sci U S A

Sensorineural deafness and balance dysfunction are common impairments in humans frequently caused by defects in the sensory epithelium of the inner ear, composed of hair cells and supporting cells. Lineage studies have shown that hair cells and supporting cells arise from a common progenitor, but how these progenitors are generated remains unknown. Although various molecules have been implicated in the development of the sensory progenitors, none has been shown to be required for the specification of these progenitors in the mammalian inner ear. Here, using both loss-of-function and gain-of-function approaches, we show that Jagged1 (JAG1)-mediated Notch signaling is both required and sufficient for the generation of the sensory progenitors. Specifically, we find that loss of JAG1 signaling leads to smaller sensory progenitor regions without initial effects on proliferation or cell death, indicating that JAG1 is involved in initial specification events. To further test whether Notch signaling is involved in specification of the sensory progenitors, we transiently expressed an activated form of the Notch1 receptor (NICD) using a combined Tet-On/Cre induction system in the mouse. NICD expression resulted in ectopic hair cells and supporting cells in the nonsensory regions of the cochlea and vestibule. These data indicate that Notch specifies sensory progenitors in the inner ear, and that induction of Notch may be important for regenerating or replacing hair cells and supporting cells in the mammalian inner ear.

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AMPA receptors are exocytosed in stimulated spines and adjacent dendrites in a Ras-ERK-dependent manner during long-term potentiation.

Publication Date: 2010 Aug 23 PMID: 20733080
Authors: Patterson, M. A. - Szatmari, E. M. - Yasuda, R.
Journal: Proc Natl Acad Sci U S A

The exocytosis of AMPA receptors is a key step in long-term potentiation (LTP), yet the timing and location of exocytosis and the signaling pathways involved in exocytosis during synaptic plasticity are not fully understood. Here we combine two-photon uncaging with two-photon imaging of a fluorescent label of surface AMPA receptors to monitor individual AMPA receptor exocytosis events near spines undergoing LTP. AMPA receptors that reached the stimulated spine came from a combination of preexisting surface receptors (70-90%) and newly exocytosed receptors (10-30%). We observed exocytosis in both the dendrite and spine under basal conditions. The rate of AMPA receptor exocytosis increased approximately 5-fold during LTP induction and decayed to the basal level within approximately 1 min, both in the stimulated spine and in the dendrite within approximately 3 mum of the stimulated spine. AMPA receptors inserted in the spine were trapped in the spine in an activity-dependent manner. The activity-dependent exocytosis required the Ras-ERK pathway, but not CaMKII. Thus, diffusive Ras-ERK signaling presumably serves as an important means for signaling from synapses to dendritic shafts to recruit AMPA receptors into synapses during LTP.

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Evolutionary origin of a functional gonadotropin in the pituitary of the most primitive vertebrate, hagfish.

Publication Date: 2010 Aug 23 PMID: 20733079
Authors: Uchida, K. - Moriyama, S. - Chiba, H. - Shimotani, T. - Honda, K. - Miki, M. - Takahashi, A. - Sower, S. A. - Nozaki, M.
Journal: Proc Natl Acad Sci U S A

Hagfish, which lack both jaws and vertebrae, are considered the most primitive vertebrate known, living or extinct. Hagfish have long been the enigma of vertebrate evolution not only because of their evolutionary position, but also because of our lack of knowledge on fundamental processes. Key elements of the reproductive endocrine system in hagfish have yet to be elucidated. Here, the presence and identity of a functional glycoprotein hormone (GPH) have been elucidated from the brown hagfish Paramyxine atami. The hagfish GPH consists of two subunits, alpha and beta, which are synthesized and colocalized in the same cells of the adenohypophysis. The cellular and transcriptional activities of hagfish GPHalpha and -beta were significantly correlated with the developmental stages of the gonad. The purified native GPH induced the release of gonadal sex steroids in vitro. From our phylogenetic analysis, we propose that ancestral glycoprotein alpha-subunit 2 (GPA2) and beta-subunit 5 (GPB5) gave rise to GPHalpha and GPHbeta of the vertebrate glycoprotein hormone family, respectively. The identified hagfish GPHalpha and -beta subunits appear to be the typical gnathostome GPHalpha and -beta subunits based on the sequence and phylogenetic analyses. We hypothesize that the identity of a single functional GPH of the hagfish, hagfish GTH, provides critical evidence for the existence of a pituitary-gonadal system in the earliest divergent vertebrate that likely evolved from an ancestral, prevertebrate exclusively neuroendocrine mechanism by gradual emergence of a previously undescribed control level, the pituitary, which is not found in the Protochordates.

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Structure-based modeling of the functional HIV-1 intasome and its inhibition.

Publication Date: 2010 Aug 23 PMID: 20733078
Authors: Krishnan, L. - Li, X. - Naraharisetty, H. L. - Hare, S. - Cherepanov, P. - Engelman, A.
Journal: Proc Natl Acad Sci U S A

The intasome is the basic recombination unit of retroviral integration, comprising the integrase protein and the ends of the viral DNA made by reverse transcription. Clinical inhibitors preferentially target the DNA-bound form of integrase as compared with the free protein, highlighting the critical requirement for detailed understanding of HIV-1 intasome structure and function. Although previous biochemical studies identified integrase residues that contact the DNA, structural details of protein-protein and protein-DNA interactions within the functional intasome were lacking. The recent crystal structure of the prototype foamy virus (PFV) integrase-viral DNA complex revealed numerous details of this related integration machine. Structures of drug-bound PFV intasomes moreover elucidated the mechanism of inhibitor action. Herein we present a model for the HIV-1 intasome assembled using the PFV structure as template. Our results pinpoint previously identified protein-DNA contacts within the quaternary structure and reveal hitherto unknown roles for Arg20 and Lys266 in DNA binding and integrase function. Models for clinical inhibitors bound at the HIV-1 integrase active site were also constructed and compared with previous studies. Our findings highlight the structural basis for HIV-1 integration and define the mechanism of its inhibition, which should help in formulating new drugs to inhibit viruses resistant to first-in-class compounds.

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Characterization of Prochlorococcus clades from iron-depleted oceanic regions.

Publication Date: 2010 Aug 23 PMID: 20733077
Authors: Rusch, D. B. - Martiny, A. C. - Dupont, C. L. - Halpern, A. L. - Venter, J. C.
Journal: Proc Natl Acad Sci U S A

Prochlorococcus describes a diverse and abundant genus of marine photosynthetic microbes. It is primarily found in oligotrophic waters across the globe and plays a crucial role in energy and nutrient cycling in the ocean ecosystem. The abundance, global distribution, and availability of isolates make Prochlorococcus a model system for understanding marine microbial diversity and biogeochemical cycling. Analysis of 73 metagenomic samples from the Global Ocean Sampling expedition acquired in the Atlantic, Pacific, and Indian Oceans revealed the presence of two uncharacterized Prochlorococcus clades. A phylogenetic analysis using six different genetic markers places the clades close to known lineages adapted to high-light environments. The two uncharacterized clades consistently cooccur and dominate the surface waters of high-temperature, macronutrient-replete, and low-iron regions of the Eastern Equatorial Pacific upwelling and the tropical Indian Ocean. They are genetically distinct from each other and other high-light Prochlorococcus isolates and likely define a previously unrecognized ecotype. Our detailed genomic analysis indicates that these clades comprise organisms that are adapted to iron-depleted environments by reducing their iron quota through the loss of several iron-containing proteins that likely function as electron sinks in the photosynthetic pathway in other Prochlorococcus clades from high-light environments. The presence and inferred physiology of these clades may explain why Prochlorococcus populations from iron-depleted regions do not respond to iron fertilization experiments and further expand our understanding of how phytoplankton adapt to variations in nutrient availability in the ocean.

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Identification of dendritic antigen-presenting cells in the zebrafish.

Publication Date: 2010 Aug 23 PMID: 20733076
Authors: Lugo-Villarino, G. - Balla, K. M. - Stachura, D. L. - Banuelos, K. - Werneck, M. B. - Traver, D.
Journal: Proc Natl Acad Sci U S A

In mammals, dendritic cells (DCs) form the key link between the innate and adaptive immune systems. DCs act as immune sentries in various tissues and, upon encountering pathogen, engulf and traffic foreign antigen to secondary lymphoid tissues, stimulating antigen-specific T lymphocytes. Although DCs are of fundamental importance in orchestrating the mammalian immune response, their presence and function in nonmammalian vertebrates is largely unknown. Because teleosts possess one of the earliest recognizable adaptive immune systems, we sought to identify antigen-presenting cells (APCs) in the zebrafish to better understand the potential origins of DCs and their evolutionary relationship to lymphocytes. Here we present the identification and characterization of a zebrafish APC subset strongly resembling mammalian DCs. Rare DCs are present in various adult tissues, and can be enriched by their affinity for the lectin peanut agglutinin (PNA). We show that PNA(hi) myeloid cells possess the classical morphological features of mammalian DCs as revealed by histochemical and ultrastructural analyses, phagocytose-labeled bacterial preparations in vivo, and exhibit expression of genes associated with DC function and antigen presentation, including il12, MHC class II invariant chain iclp1, and csf1r. Importantly, we show that PNA(hi) cells can activate T lymphocytes in an antigen-dependent manner. Together, these studies suggest that the cellular constituents responsible for antigen presentation are remarkably conserved from teleosts to mammals, and indicate that the zebrafish may serve as a unique model to study the origin of APC subsets and their evolutionary role as the link between the innate and adaptive immune systems.

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{alpha}-Synuclein gene duplication impairs reward learning.

Publication Date: 2010 Aug 23 PMID: 20733075
Authors: Keri, S. - Moustafa, A. A. - Myers, C. E. - Benedek, G. - Gluck, M. A.
Journal: Proc Natl Acad Sci U S A

alpha-Synuclein (SNCA) plays an important role in the regulation of dopaminergic neurotransmission and neurodegeneration in Parkinson disease. We investigated reward and punishment learning in asymptomatic carriers of a rare SNCA gene duplication who were healthy siblings of patients with Parkinson disease. Results revealed that healthy SNCA duplication carriers displayed impaired reward and intact punishment learning compared with noncarriers. These results demonstrate that a copy number variation of the SNCA gene is associated with selective impairments on reinforcement learning in asymptomatic carriers without the motor symptoms of Parkinson disease.

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Sex-specific association of X-linked Toll-like receptor 7 (TLR7) with male systemic lupus erythematosus.

Publication Date: 2010 Aug 23 PMID: 20733074
Authors: Shen, N. - Fu, Q. - Deng, Y. - Qian, X. - Zhao, J. - Kaufman, K. M. - Wu, Y. L. - Yu, C. Y. - Tang, Y. - Chen, J. Y. - Yang, W. - Wong, M. - Kawasaki, A. - Tsuchiya, N. - Sumida, T. - Kawaguchi, Y. - Howe, H. S. - Mok, M. Y. - Bang, S. Y. - Liu, F. L. - Chang, D. M. - Takasaki, Y. - Hashimoto, H. - Harley, J. B. - Guthridge, J. M. - Grossman, J. M. - Cantor, R. M. - Song, Y. W. - Bae, S. C. - Chen, S. - Hahn, B. H. - Lau, Y. L. - Tsao, B. P.
Journal: Proc Natl Acad Sci U S A

Systemic lupus erythematosus (SLE) is a multisystem, autoimmune disease that predominantly affects women. Previous findings that duplicated Toll-like receptor 7 (Tlr7) promotes lupus-like disease in male BXSB mice prompted us to evaluate TLR7 in human SLE. By using a candidate gene approach, we identified and replicated association of a TLR7 3'UTR SNP, rs3853839 (G/C), with SLE in 9,274 Eastern Asians (P(combined) = 6.5 x 10(-10)), with a stronger effect in male than female subjects [odds ratio, male vs. female = 2.33 (95% CI = 1.64-3.30) vs. 1.24 (95% CI = 1.14-1.34); P = 4.1 x 10(-4)]. G-allele carriers had increased TLR7 transcripts and more pronounced IFN signature than C-allele carriers; heterozygotes had 2.7-fold higher transcripts of G-allele than C-allele. These data established a functional polymorphism in type I IFN pathway gene TLR7 predisposing to SLE, especially in Chinese and Japanese male subjects.

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Niche and neutral models predict asymptotically equivalent species abundance distributions in high-diversity ecological communities.

Publication Date: 2010 Aug 23 PMID: 20733073
Authors: Chisholm, R. A. - Pacala, S. W.
Journal: Proc Natl Acad Sci U S A

A fundamental challenge in ecology is to understand the mechanisms that govern patterns of relative species abundance. Previous numerical simulations have suggested that complex niche-structured models produce species abundance distributions (SADs) that are qualitatively similar to those of very simple neutral models that ignore differences between species. However, in the absence of an analytical treatment of niche models, one cannot tell whether the two classes of model produce the same patterns via similar or different mechanisms. We present an analytical proof that, in the limit as diversity becomes large, a strong niche model give rises to exactly the same asymptotic form of SAD as the neutral model, and we verify the analytical predictions for a Panamanian tropical forest data set. Our results strongly suggest that neutral processes drive patterns of relative species abundance in high-diversity ecological communities, even when strong niche structure exists. However, neutral theory cannot explain what generates high diversity in the first place, and it may not be valid in low-diversity communities. Our results also confirm that neutral theory cannot be used to infer an absence of niche structure or to explain ecosystem function.

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Distinct 3'UTRs differentially regulate activity-dependent translation of brain-derived neurotrophic factor (BDNF).

Publication Date: 2010 Aug 23 PMID: 20733072
Authors: Lau, A. G. - Irier, H. A. - Gu, J. - Tian, D. - Ku, L. - Liu, G. - Xia, M. - Fritsch, B. - Zheng, J. Q. - Dingledine, R. - Xu, B. - Lu, B. - Feng, Y.
Journal: Proc Natl Acad Sci U S A

Expression of the brain-derived neurotrophic factor (BDNF) is under tight regulation to accommodate its intricate roles in controlling brain function. Transcription of BDNF initiates from multiple promoters in response to distinct stimulation cues. However, regardless which promoter is used, all BDNF transcripts are processed at two alternative polyadenylation sites, generating two pools of mRNAs that carry either a long or a short 3'UTR, both encoding the same BDNF protein. Whether and how the two distinct 3'UTRs may differentially regulate BDNF translation in response to neuronal activity changes is an intriguing and challenging question. We report here that the long BDNF 3'UTR is a bona fide cis-acting translation suppressor at rest whereas the short 3'UTR mediates active translation to maintain basal levels of BDNF protein production. Upon neuronal activation, the long BDNF 3'UTR, but not the short 3'UTR, imparts rapid and robust activation of translation from a reporter. Importantly, the endogenous long 3'UTR BDNF mRNA specifically undergoes markedly enhanced polyribosome association in the hippocampus in response to pilocarpine induced-seizure before transcriptional up-regulation of BDNF. Furthermore, BDNF protein level is quickly increased in the hippocampus upon seizure-induced neuronal activation, accompanied by a robust activation of the tropomyosin-related receptor tyrosine kinase B. These observations reveal a mechanism for activity-dependent control of BDNF translation and tropomyosin-related receptor tyrosine kinase B signaling in brain neurons.

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Natural killer cells in NOD.NK1.1 mice acquire cytolytic function during viral infection and provide protection against cytomegalovirus.

Publication Date: 2010 Aug 23 PMID: 20733071
Authors: Orr, M. T. - Beilke, J. N. - Proekt, I. - Lanier, L. L.
Journal: Proc Natl Acad Sci U S A

Resting natural killer (NK) cells in nonobese diabetic (NOD) mice have impaired immune functions compared with NK cells from other mouse strains. Here we investigated how NOD NK cells respond after mouse cytomegalovirus (MCMV) infection, using NOD mice congenic for the protective NK gene complex from C57BL/6 mice. Compared with C57BL/6 mice congenic for the H2 gene complex from NOD mice (B6.g7), NOD.NK1.1 mice fail to control early infection with MCMV. After MCMV infection, however, NOD.NK1.1 NK cells demonstrate increased cytolytic function, associated with higher expression of granzyme B, and undergo robust expansion. One week after infection, NOD.NK1.1 NK cells control MCMV replication as effectively as B6.g7 NK cells, even in the absence of T cells and B cells. Thus, the impaired cytotoxic function of NK cells in NOD mice is alleviated by viral infection, which enables NOD NK cells to efficiently control MCMV infection.

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Physical interaction between VIVID and white collar complex regulates photoadaptation in Neurospora.

Publication Date: 2010 Aug 23 PMID: 20733070
Authors: Chen, C. H. - Demay, B. S. - Gladfelter, A. S. - Dunlap, J. C. - Loros, J. J.
Journal: Proc Natl Acad Sci U S A

Photoadaptation, the ability to attenuate a light response on prolonged light exposure while remaining sensitive to escalating changes in light intensity, is essential for organisms to decipher time information appropriately, yet the underlying molecular mechanisms are poorly understood. In Neurospora crassa, VIVID (VVD), a small LOV domain containing blue-light photoreceptor protein, affects photoadaptation for most if not all light-responsive genes. We report that there is a physical interaction between VVD and the white collar complex (WCC), the primary blue-light photoreceptor and the transcription factor complex that initiates light-regulated transcriptional responses in Neurospora. Using two previously characterized VVD mutants, we show that the level of interaction is correlated with the level of WCC repression in constant light and that even light-insensitive VVD is sufficient partly to regulate photoadaptation in vivo. We provide evidence that a functional GFP-VVD fusion protein accumulates in the nucleus on light induction but that nuclear localization of VVD does not require light. Constitutively expressed VVD alone is sufficient to change the dynamics of photoadaptation. Thus, our results demonstrate a direct molecular connection between two of the most essential light signaling components in Neurospora, VVD and WCC, illuminating a previously uncharacterized process for light-sensitive eukaryotic cells.

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Linking transcription with DNA repair, damage tolerance, and genome duplication.

Publication Date: 2010 Aug 31 PMID: 20733069
Authors: McGlynn, P.
Journal: Proc Natl Acad Sci U S A

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A Trojan horse mechanism of bacterial pathogenesis against nematodes.

Publication Date: 2010 Aug 23 PMID: 20733068
Authors: Niu, Q. - Huang, X. - Zhang, L. - Xu, J. - Yang, D. - Wei, K. - Niu, X. - An, Z. - Bennett, J. W. - Zou, C. - Yang, J. - Zhang, K. Q.
Journal: Proc Natl Acad Sci U S A

Understanding the mechanisms of host-pathogen interaction can provide crucial information for successfully manipulating their relationships. Because of its genetic background and practical advantages over vertebrate model systems, the nematode Caenorhabditis elegans model has become an attractive host for studying microbial pathogenesis. Here we report a "Trojan horse" mechanism of bacterial pathogenesis against nematodes. We show that the bacterium Bacillus nematocida B16 lures nematodes by emitting potent volatile organic compounds that are much more attractive to worms than those from ordinary dietary bacteria. Seventeen B. nematocida-attractant volatile organic compounds are identified, and seven are individually confirmed to lure nematodes. Once the bacteria enter the intestine of nematodes, they secrete two proteases with broad substrate ranges but preferentially target essential intestinal proteins, leading to nematode death. This Trojan horse pattern of bacterium-nematode interaction enriches our understanding of microbial pathogenesis.

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Economic contract theory tests models of mutualism.

Publication Date: 2010 Aug 23 PMID: 20733067
Authors: Weyl, E. G. - Frederickson, M. E. - Yu, D. W. - Pierce, N. E.
Journal: Proc Natl Acad Sci U S A

Although mutualisms are common in all ecological communities and have played key roles in the diversification of life, our current understanding of the evolution of cooperation applies mostly to social behavior within a species. A central question is whether mutualisms persist because hosts have evolved costly punishment of cheaters. Here, we use the economic theory of employment contracts to formulate and distinguish between two mechanisms that have been proposed to prevent cheating in host-symbiont mutualisms, partner fidelity feedback (PFF) and host sanctions (HS). Under PFF, positive feedback between host fitness and symbiont fitness is sufficient to prevent cheating; in contrast, HS posits the necessity of costly punishment to maintain mutualism. A coevolutionary model of mutualism finds that HS are unlikely to evolve de novo, and published data on legume-rhizobia and yucca-moth mutualisms are consistent with PFF and not with HS. Thus, in systems considered to be textbook cases of HS, we find poor support for the theory that hosts have evolved to punish cheating symbionts; instead, we show that even horizontally transmitted mutualisms can be stabilized via PFF. PFF theory may place previously underappreciated constraints on the evolution of mutualism and explain why punishment is far from ubiquitous in nature.

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In planta changes in protein phosphorylation induced by the plant hormone abscisic acid.

Publication Date: 2010 Aug 23 PMID: 20733066
Authors: Kline, K. G. - Barrett-Wilt, G. A. - Sussman, M. R.
Journal: Proc Natl Acad Sci U S A

Abscisic acid (ABA) is a hormone that controls seed dormancy and germination as well as the overall plant response to important environmental stresses such as drought. Recent studies have demonstrated that the ABA-bound receptor binds to and inhibits a class of protein phosphatases. To identify more broadly the phosphoproteins affected by this hormone in vivo, we used (14)N/(15)N metabolic labeling to perform a quantitative untargeted mass spectrometric analysis of the Arabidopsis thaliana phosphoproteome following ABA treatment. We found that 50 different phosphopeptides had their phosphorylation state significantly altered by ABA over a treatment period lasting 5-30 min. Among these changes were increases in phosphorylation of subfamily 2 SNF1-related kinases and ABA-responsive basic leucine zipper transcription factors implicated in ABA signaling by previous in vitro studies. Furthermore, four members of the aquaporin family showed decreased phosphorylation at a carboxy-terminal serine which is predicted to cause closure of the water-transporting aquaporin gate, consistent with ABA's role in ameliorating the effect of drought. Finally, more than 20 proteins not previously known to be involved with ABA were found to have significantly altered phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that an expanded model of ABA signaling, beyond simple phosphatase inhibition, may be necessary. This quantitative proteomics dataset provides a more comprehensive, albeit incomplete, view both of the protein targets whose biochemical activities are likely to be controlled by ABA and of the nature of the emerging phosphorylation and dephosphorylation cascades triggered by this hormone.

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Fold-change detection and scalar symmetry of sensory input fields.

Publication Date: 2010 Aug 20 PMID: 20729472
Authors: Shoval, O. - Goentoro, L. - Hart, Y. - Mayo, A. - Sontag, E. - Alon, U.
Journal: Proc Natl Acad Sci U S A

Recent studies suggest that certain cellular sensory systems display fold-change detection (FCD): a response whose entire shape, including amplitude and duration, depends only on fold changes in input and not on absolute levels. Thus, a step change in input from, for example, level 1 to 2 gives precisely the same dynamical output as a step from level 2 to 4, because the steps have the same fold change. We ask what the benefit of FCD is and show that FCD is necessary and sufficient for sensory search to be independent of multiplying the input field by a scalar. Thus, the FCD search pattern depends only on the spatial profile of the input and not on its amplitude. Such scalar symmetry occurs in a wide range of sensory inputs, such as source strength multiplying diffusing/convecting chemical fields sensed in chemotaxis, ambient light multiplying the contrast field in vision, and protein concentrations multiplying the output in cellular signaling systems. Furthermore, we show that FCD entails two features found across sensory systems, exact adaptation and Weber's law, but that these two features are not sufficient for FCD. Finally, we present a wide class of mechanisms that have FCD, including certain nonlinear feedback and feed-forward loops. We find that bacterial chemotaxis displays feedback within the present class and hence, is expected to show FCD. This can explain experiments in which chemotaxis searches are insensitive to attractant source levels. This study, thus, suggests a connection between properties of biological sensory systems and scalar symmetry stemming from physical properties of their input fields.

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Mechanistic constraints from the substrate concentration dependence of enzymatic fluctuations.

Publication Date: 2010 Aug 20 PMID: 20729471
Authors: Moffitt, J. R. - Chemla, Y. R. - Bustamante, C.
Journal: Proc Natl Acad Sci U S A

The time it takes an enzyme to complete its reaction is a stochastic quantity governed by thermal fluctuations. With the advent of high-resolution methods of single-molecule manipulation and detection, it is now possible to observe directly this natural variation in the enzymatic cycle completion time and extract kinetic information from the statistics of its fluctuations. To this end, the inverse of the squared coefficient of variation, which we term n(min), is a useful measure of fluctuations because it places a strict lower limit on the number of kinetic states in the enzymatic mechanism. Here we show that there is a single general expression for the substrate dependence of n(min) for a wide range of kinetic models. This expression is governed by three kinetic parameters, which we term N(L), N(S), and alpha. These parameters have simple geometric interpretations and provide clear constraints on possible kinetic mechanisms. As a demonstration of this analysis, we fit the fluctuations in the dwell times of the packaging motor of the bacteriophage varphi29, revealing additional features of the nucleotide loading process in this motor. Because a diverse set of kinetic models display the same substrate dependence for their fluctuations, the expression for this general dependence may prove of use in the characterization and study of the dynamics of a wide range of enzymes.

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Conserved microRNA targeting in Drosophila is as widespread in coding regions as in 3'UTRs.

Publication Date: 2010 Aug 20 PMID: 20729470
Authors: Schnall-Levin, M. - Zhao, Y. - Perrimon, N. - Berger, B.
Journal: Proc Natl Acad Sci U S A

MicroRNAs (miRNAs) are a class of short noncoding RNAs that regulate protein-coding genes posttranscriptionally. In animals, most known miRNA targeting occurs within the 3'UTR of mRNAs, but the extent of biologically relevant targeting in the ORF or 5'UTR of mRNAs remains unknown. Here, we develop an algorithm (MinoTar-miRNA ORF Targets) to identify conserved regulatory motifs within protein-coding regions and use it to estimate the number of preferentially conserved miRNA-target sites in ORFs. We show that, in Drosophila, preferentially conserved miRNA targeting in ORFs is as widespread as it is in 3'UTRs and that, while far less abundant, conserved targets in Drosophila 5'UTRs number in the hundreds. Using our algorithm, we predicted a set of high-confidence ORF targets and selected seven miRNA-target pairs from among these for experimental validation. We observed down-regulation by the miRNA in five out of seven cases, indicating our approach can recover functional sites with high confidence. Additionally, we observed additive targeting by multiple sites within a single ORF. Altogether, our results demonstrate that the scale of biologically important miRNA targeting in ORFs is extensive and that computational tools such as ours can aid in the identification of such targets. Further evidence suggests that our results extend to mammals, but that the extent of ORF and 5'UTR targeting relative to 3'UTR targeting may be greater in Drosophila.

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Mas-related G-protein-coupled receptors inhibit pathological pain in mice.

Publication Date: 2010 Aug 19 PMID: 20724664
Authors: Guan, Y. - Liu, Q. - Tang, Z. - Raja, S. N. - Anderson, D. J. - Dong, X.
Journal: Proc Natl Acad Sci U S A

An important objective of pain research is to identify novel drug targets for the treatment of pathological persistent pain states, such as inflammatory and neuropathic pain. Mas-related G-protein-coupled receptors (Mrgprs) represent a large family of orphan receptors specifically expressed in small-diameter nociceptive primary sensory neurons. To determine the roles of Mrgprs in persistent pathological pain states, we exploited a mouse line in which a chromosomal locus spanning 12 Mrgpr genes was deleted (KO). Initial studies indicated that these KO mice show prolonged mechanical- and thermal-pain hypersensitivity after hind-paw inflammation compared with wild-type littermates. Here, we show that this mutation also enhances the windup response of dorsal-horn wide dynamic-range neurons, an electrophysiological model for the triggering of central pain sensitization. Deletion of the Mrgpr cluster also blocked the analgesic effect of intrathecally applied bovine adrenal medulla peptide 8-22 (BAM 8-22), an MrgprC11 agonist, on both inflammatory heat hyperalgesia and neuropathic mechanical allodynia. Spinal application of bovine adrenal medulla peptide 8-22 also significantly attenuated windup in wild-type mice, an effect eliminated in KO mice. These data suggest that members of the Mrgpr family, in particular MrgprC11, may constitute an endogenous inhibitory mechanism for regulating persistent pain in mice. Agonists for these receptors may, therefore, represent a class of antihyperalgesics for treating persistent pain with minimal side effects because of the highly specific expression of their targets.

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A general basis for quarter-power scaling in animals.

Publication Date: 2010 Aug 19 PMID: 20724663
Authors: Banavar, J. R. - Moses, M. E. - Brown, J. H. - Damuth, J. - Rinaldo, A. - Sibly, R. M. - Maritan, A.
Journal: Proc Natl Acad Sci U S A

It has been known for decades that the metabolic rate of animals scales with body mass with an exponent that is almost always <1, >2/3, and often very close to 3/4. The 3/4 exponent emerges naturally from two models of resource distribution networks, radial explosion and hierarchically branched, which incorporate a minimum of specific details. Both models show that the exponent is 2/3 if velocity of flow remains constant, but can attain a maximum value of 3/4 if velocity scales with its maximum exponent, 1/12. Quarter-power scaling can arise even when there is no underlying fractality. The canonical "fourth dimension" in biological scaling relations can result from matching the velocity of flow through the network to the linear dimension of the terminal "service volume" where resources are consumed. These models have broad applicability for the optimal design of biological and engineered systems where energy, materials, or information are distributed from a single source.

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Culture, distress, and oxytocin receptor polymorphism (OXTR) interact to influence emotional support seeking.

Publication Date: 2010 Aug 19 PMID: 20724662
Authors: Kim, H. S. - Sherman, D. K. - Sasaki, J. Y. - Xu, J. - Chu, T. Q. - Ryu, C. - Suh, E. M. - Graham, K. - Taylor, S. E.
Journal: Proc Natl Acad Sci U S A

Research has demonstrated that certain genotypes are expressed in different forms, depending on input from the social environment. To examine sensitivity to cultural norms regarding emotional support seeking as a type of social environment, we explored the behavioral expression of oxytocin receptor polymorphism (OXTR) rs53576, a gene previously related to socio-emotional sensitivity. Seeking emotional support in times of distress is normative in American culture but not in Korean culture. Consequently, we predicted a three-way interaction of culture, distress, and OXTR genotype on emotional support seeking. Korean and American participants (n = 274) completed assessments of psychological distress and emotional support seeking and were genotyped for OXTR. We found the predicted three-way interaction: among distressed American participants, those with the GG/AG genotypes reported seeking more emotional social support, compared with those with the AA genotype, whereas Korean participants did not differ significantly by genotype; under conditions of low distress, OXTR groups did not differ significantly in either cultural group. These findings suggest that OXTR rs53576 is sensitive to input from the social environment, specifically cultural norms regarding emotional social support seeking. These findings also indicate that psychological distress and culture are important moderators that shape behavioral outcomes associated with OXTR genotypes.

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Insights into the mechanism of polysaccharide dephosphorylation by a glucan phosphatase.

Publication Date: 2010 Aug 31 PMID: 20724661
Authors: Tagliabracci, V. S. - Roach, P. J.
Journal: Proc Natl Acad Sci U S A

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Polyubiquitin conjugation to NEMO by triparite motif protein 23 (TRIM23) is critical in antiviral defense.

Publication Date: 2010 Aug 19 PMID: 20724660
Authors: Arimoto, K. I. - Funami, K. - Saeki, Y. - Tanaka, K. - Okawa, K. - Takeuchi, O. - Akira, S. - Murakami, Y. - Shimotohno, K.
Journal: Proc Natl Acad Sci U S A

The rapid induction of type I IFN is a central event of the innate defense against viral infections and is tightly regulated by a number of cellular molecules. Viral components induce strong type I IFN responses through the activation of toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as an RNA helicase RIG-I and/or MDA5. According to recent studies, the NF-kappaB essential modulator (NEMO, also called IKKgamma) is crucial for this virus-induced antiviral response. However, the precise roles of signal activation by NEMO adaptor have not been elucidated. Here, we show that virus-induced IRF3 and NF-kappaB activation depends on the K(lys)-27-linked polyubiquitination to NEMO by the novel ubiquitin E3 ligase triparite motif protein 23 (TRIM23). Virus-induced IRF3 and NF-kappaB activation, as well as K27-linked NEMO polyubiquitination, were abrogated in TRIM23 knockdown cells, whereas TRIM23 knockdown had no effect on TNFalpha-mediated NF-kappaB activation. Furthermore, in NEMO-deficient mouse embryo fibroblast cells, IFN-stimulated response element-driven reporter activity was restored by ectopic expression of WT NEMO, as expected, but only partial recovery by NEMO K165/309/325/326/344R multipoints mutant on which TRIM23-mediated ubiquitin conjugation was substantially reduced. Thus, we conclude that TRIM23-mediated ubiquitin conjugation to NEMO is essential for TLR3- and RIG-I/MDA5-mediated antiviral innate and inflammatory responses.

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Synthesis and biological evaluation of fluorinated deoxynucleotide analogs based on bis-(difluoromethylene)triphosphoric acid.

Publication Date: 2010 Aug 19 PMID: 20724659
Authors: Surya Prakash, G. K. - Zibinsky, M. - Upton, T. G. - Kashemirov, B. A. - McKenna, C. E. - Oertell, K. - Goodman, M. F. - Batra, V. K. - Pedersen, L. C. - Beard, W. A. - Shock, D. D. - Wilson, S. H. - Olah, G. A.
Journal: Proc Natl Acad Sci U S A

It is difficult to overestimate the importance of nucleoside triphosphates in cellular chemistry: They are the building blocks for DNA and RNA and important sources of energy. Modifications of biologically important organic molecules with fluorine are of great interest to chemists and biologists because the size and electronegativity of the fluorine atom can be used to make defined structural alterations to biologically important molecules. Although the concept of nonhydrolyzable nucleotides has been around for some time, the progress in the area of modified triphosphates was limited by the lack of synthetic methods allowing to access bisCF(2)-substituted nucleotide analogs-one of the most interesting classes of nonhydrolyzable nucleotides. These compounds have "correct" polarity and the smallest possible steric perturbation compared to natural nucleotides. No other known nucleotides have these advantages, making bisCF(2)-substituted analogs unique. Herein, we report a concise route for the preparation of hitherto unknown highly acidic and polybasic bis(difluoromethylene)triphosphoric acid 1 using a phosphorous(III)/phosphorous(V) interconversion approach. The analog 1 compared to triphosphoric acid is enzymatically nonhydrolyzable due to substitution of two bridging oxygen atoms with CF(2) groups, maintaining minimal perturbations in steric bulkiness and overall polarity of the triphosphate polyanion. The fluorinated triphosphoric acid 1 was used for the preparation of the corresponding fluorinated deoxynucleotides (dNTPs). One of these dNTP analogs (dT) was demonstrated to fit into DNA polymerase beta (DNA pol beta) binding pocket by obtaining a 2.5 A resolution crystal structure of a ternary complex with the enzyme. Unexpected dominating effect of triphosphate/Mg(2+) interaction over Watson-Crick hydrogen bonding was found and discussed.

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Aquaporin-3 mediates hydrogen peroxide uptake to regulate downstream intracellular signaling.

Publication Date: 2010 Aug 19 PMID: 20724658
Authors: Miller, E. W. - Dickinson, B. C. - Chang, C. J.
Journal: Proc Natl Acad Sci U S A

Hydrogen peroxide (H(2)O(2)) produced by cell-surface NADPH Oxidase (Nox) enzymes is emerging as an important signaling molecule for growth, differentiation, and migration processes. However, how cells spatially regulate H(2)O(2) to achieve physiological redox signaling over nonspecific oxidative stress pathways is insufficiently understood. Here we report that the water channel Aquaporin-3 (AQP3) can facilitate the uptake of H(2)O(2) into mammalian cells and mediate downstream intracellular signaling. Molecular imaging with Peroxy Yellow 1 Methyl-Ester (PY1-ME), a new chemoselective fluorescent indicator for H(2)O(2), directly demonstrates that aquaporin isoforms AQP3 and AQP8, but not AQP1, can promote uptake of H(2)O(2) specifically through membranes in mammalian cells. Moreover, we show that intracellular H(2)O(2) accumulation can be modulated up or down based on endogenous AQP3 expression, which in turn can influence downstream cell signaling cascades. Finally, we establish that AQP3 is required for Nox-derived H(2)O(2) signaling upon growth factor stimulation. Taken together, our findings demonstrate that the downstream intracellular effects of H(2)O(2) can be regulated across biological barriers, a discovery that has broad implications for the controlled use of this potentially toxic small molecule for beneficial physiological functions.

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Quantitative 3D elemental microtomography of Cyclotella meneghiniana at 400-nm resolution.

Publication Date: 2010 Aug 18 PMID: 20720164
Authors: de Jonge, M. D. - Holzner, C. - Baines, S. B. - Twining, B. S. - Ignatyev, K. - Diaz, J. - Howard, D. L. - Legnini, D. - Miceli, A. - McNulty, I. - Jacobsen, C. J. - Vogt, S.
Journal: Proc Natl Acad Sci U S A

X-ray fluorescence tomography promises to map elemental distributions in unstained and unfixed biological specimens in three dimensions at high resolution and sensitivity, offering unparalleled insight in medical, biological, and environmental sciences. X-ray fluorescence tomography of biological specimens has been viewed as impractical-and perhaps even impossible for routine application-due to the large time required for scanning tomography and significant radiation dose delivered to the specimen during the imaging process. Here, we demonstrate submicron resolution X-ray fluorescence tomography of a whole unstained biological specimen, quantifying three-dimensional distributions of the elements Si, P, S, Cl, K, Ca, Mn, Fe, Cu, and Zn in the freshwater diatom Cyclotella meneghiniana with 400-nm resolution, improving the spatial resolution by over an order of magnitude. The resulting maps faithfully reproduce cellular structure revealing unexpected patterns that may elucidate the role of metals in diatom biology and of diatoms in global element cycles. With anticipated improvements in data acquisition and detector sensitivity, such measurements could become routine in the near future.

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miRNA-based mechanism for the commitment of multipotent progenitors to a single cellular fate.

Publication Date: 2010 Aug 18 PMID: 20720163
Authors: Mann, M. - Barad, O. - Agami, R. - Geiger, B. - Hornstein, E.
Journal: Proc Natl Acad Sci U S A

When stem cells and multipotent progenitors differentiate, they undergo fate restriction, enabling a single fate and blocking differentiation along alternative routes. We herein present a mechanism whereby such unequivocal commitment is achieved, based on microRNA (miRNA)-dependent repression of an alternative cell fate. We show that the commitment of monocyte RAW264.7 progenitors to active macrophage differentiation involves rapid up-regulation of miR-155 expression, which leads to the suppression of the alternative pathway, namely RANK ligand-induced osteoclastogenesis, by repressing the expression of MITF, a transcription factor essential for osteoclast differentiation. A temporal asymmetry, whereby miR-155 expression precedes and overrides the activation of the osteoclast transcriptional program, provides the means for coherent macrophage differentiation, even in the presence of osteoclastogenic signals. Based on these findings, we propose that miRNA may provide a general mechanism for the unequivocal commitment underlying stem cell differentiation.

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Single-molecule derivation of salt dependent base-pair free energies in DNA.

Publication Date: 2010 Aug 31 PMID: 20716688
Authors: Huguet, J. M. - Bizarro, C. V. - Forns, N. - Smith, S. B. - Bustamante, C. - Ritort, F.
Journal: Proc Natl Acad Sci U S A

Accurate knowledge of the thermodynamic properties of nucleic acids is crucial to predicting their structure and stability. To date most measurements of base-pair free energies in DNA are obtained in thermal denaturation experiments, which depend on several assumptions. Here we report measurements of the DNA base-pair free energies based on a simplified system, the mechanical unzipping of single DNA molecules. By combining experimental data with a physical model and an optimization algorithm for analysis, we measure the 10 unique nearest-neighbor base-pair free energies with 0.1 kcal mol(-1) precision over two orders of magnitude of monovalent salt concentration. We find an improved set of standard energy values compared with Unified Oligonucleotide energies and a unique set of 10 base-pair-specific salt-correction values. The latter are found to be strongest for AA/TT and weakest for CC/GG. Our unique energy values and salt corrections improve predictions of DNA unzipping forces and are fully compatible with melting temperatures for oligos. The method should make it possible to obtain free energies, enthalpies, and entropies in conditions not accessible by bulk methodologies.

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Structural basis for the regulation of NtcA-dependent transcription by proteins PipX and PII.

Publication Date: 2010 Aug 31 PMID: 20716687
Authors: Llacer, J. L. - Espinosa, J. - Castells, M. A. - Contreras, A. - Forchhammer, K. - Rubio, V.
Journal: Proc Natl Acad Sci U S A

PII, an ancient and widespread signaling protein, transduces nitrogen/carbon/energy abundance signals through interactions with target proteins. We clarify structurally how PII regulates gene expression mediated by the transcription factor NtcA, the global nitrogen regulator of cyanobacteria, shedding light on NtcA structure and function and on how NtcA is activated by 2-oxoglutarate (2OG) and coactivated by the nonenzymatic PII target, protein PipX. We determine for the cyanobacteria Synechococcus elongatus the crystal structures of the PII-PipX and PipX-NtcA complexes and of NtcA in active and inactive conformations (respective resolutions, 3.2, 2.25, 2.3, and 3.05 A). The structures and the conclusions derived from them are consistent with the results of present and prior site-directed mutagenesis and functional studies. A tudor-like domain (TLD) makes up most of the PipX structure and mediates virtually all the contacts of PipX with PII and NtcA. In the PII-PipX complex, one PII trimer sequesters the TLDs of three PipX molecules between its body and its extended T loops, preventing PipX activation of NtcA. Changes in T loop conformation triggered by 2OG explain PII-PipX dissociation when 2OG is bound. The structure of active dimeric NtcA closely resembles that of the active cAMP receptor protein (CRP). This strongly suggests that with these proteins DNA binding, transcription activation, and allosteric regulation occur by common mechanisms, although the effectors are different. The PipX-NtcA complex consists of one active NtcA dimer and two PipX monomers. PipX coactivates NtcA by stabilizing its active conformation and by possibly helping recruit RNA polymerase but not by providing extra DNA contacts.

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PKA phosphorylates histone deacetylase 5 and prevents its nuclear export, leading to the inhibition of gene transcription and cardiomyocyte hypertrophy.

Publication Date: 2010 Aug 31 PMID: 20716686
Authors: Ha, C. H. - Kim, J. Y. - Zhao, J. - Wang, W. - Jhun, B. S. - Wong, C. - Jin, Z. G.
Journal: Proc Natl Acad Sci U S A

Dynamic nucleocytoplasmic shuttling of class IIa histone deacetylases (HDACs) is a fundamental mechanism regulating gene transcription. Recent studies have identified several protein kinases that phosphorylate HDAC5, leading to its exportation from the nucleus. However, the negative regulatory mechanisms for HDAC5 nuclear exclusion remain largely unknown. Here we show that cAMP-activated protein kinase A (PKA) specifically phosphorylates HDAC5 and prevents its export from the nucleus, leading to suppression of gene transcription. PKA interacts directly with HDAC5 and phosphorylates HDAC5 at serine 280, an evolutionarily conserved site. Phosphorylation of HDAC5 by PKA interrupts the association of HDAC5 with protein chaperone 14-3-3 and hence inhibits stress signal-induced nuclear export of HDAC5. An HDAC5 mutant that mimics PKA-dependent phosphorylation localizes in the nucleus and acts as a dominant inhibitor for myocyte enhancer factor 2 transcriptional activity. Molecular manipulations of HDAC5 show that PKA-phosphorylated HDAC5 inhibits cardiac fetal gene expression and cardiomyocyte hypertrophy. Our findings identify HDAC5 as a substrate of PKA and reveal a cAMP/PKA-dependent pathway that controls HDAC5 nucleocytoplasmic shuttling and represses gene transcription. This pathway may represent a mechanism by which cAMP/PKA signaling modulates a wide range of biological functions and human diseases such as cardiomyopathy.

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CpG island clusters and pro-epigenetic selection for CpGs in protein-coding exons of HOX and other transcription factors.

Publication Date: 2010 Aug 31 PMID: 20716685
Authors: Branciamore, S. - Chen, Z. X. - Riggs, A. D. - Rodin, S. N.
Journal: Proc Natl Acad Sci U S A

CpG dinucleotides contribute to epigenetic mechanisms by being the only site for DNA methylation in mammalian somatic cells. They are also mutation hotspots and approximately 5-fold depleted genome-wide. We report here a study focused on CpG sites in the coding regions of Hox and other transcription factor genes, comparing methylated genomes of Homo sapiens, Mus musculus, and Danio rerio with nonmethylated genomes of Drosophila melanogaster and Caenorhabditis elegans. We analyzed 4-fold degenerate, synonymous codons with the potential for CpG. That is, we studied "silent" changes that do not affect protein products but could damage epigenetic marking. We find that DNA-binding transcription factors and other developmentally relevant genes show, only in methylated genomes, a bimodal distribution of CpG usage. Several genetic code-based tests indicate, again for methylated genomes only, that the frequency of silent CpGs in Hox genes is much greater than expectation. Also informative are NCG-GNN and NCC-GNN codon doublets, for which an unusually high rate of G to C and C to G transversions was observed at the third (silent) position of the first codon. Together these results are interpreted as evidence for strong "pro-epigenetic" selection acting to preserve CpG sites in coding regions of many genes controlling development. We also report that DNA-binding transcription factors and developmentally important genes are dramatically overrepresented in or near clusters of three or more CpG islands, suggesting a possible relationship between evolutionary preservation of CpG dinucleotides in both coding regions and CpG islands.

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Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti-trp RNA-binding attenuation protein.

Publication Date: 2010 Aug 31 PMID: 20713740
Authors: Sachleben, J. R. - McElroy, C. A. - Gollnick, P. - Foster, M. P.
Journal: Proc Natl Acad Sci U S A

Anti-TRAP (AT) is a small zinc-binding protein that regulates tryptophan biosynthesis in Bacillus subtilis by binding to tryptophan-bound trp RNA-binding attenuation protein (TRAP), thereby preventing it from binding RNA, and allowing transcription and translation of the trpEDCFBA operon. Crystallographic and sedimentation studies have shown that AT can homooligomerize to form a dodecamer, AT(12), composed of a tetramer of trimers, AT(3). Structural and biochemical studies suggest that only trimeric AT is active for binding to TRAP. Our chromatographic and spectroscopic data revealed that a large fraction of recombinantly overexpressed AT retains the N-formyl group (fAT), presumably due to incomplete N-formyl-methionine processing by peptide deformylase. Hydrodynamic parameters from NMR relaxation and diffusion measurements showed that fAT is exclusively trimeric (AT(3)), while (deformylated) AT exhibits slow exchange between both trimeric and dodecameric forms. We examined this equilibrium using NMR spectroscopy and found that oligomerization of active AT(3) to form inactive AT(12) is linked to protonation of the amino terminus. Global analysis of the pH dependence of the trimer-dodecamer equilibrium revealed a near physiological pK(a) for the N-terminal amine of AT and yielded a pH-dependent oligomerization equilibrium constant. Estimates of excluded volume effects due to molecular crowding suggest the oligomerization equilibrium may be physiologically important. Because deprotonation favors "active" trimeric AT and protonation favors "inactive" dodecameric AT, our findings illuminate a possible mechanism for sensing and responding to changes in cellular pH.

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Proton and cation transport activity of the M2 proton channel from influenza A virus.

Publication Date: 2010 Aug 31 PMID: 20713739
Authors: Leiding, T. - Wang, J. - Martinsson, J. - Degrado, W. F. - Arskold, S. P.
Journal: Proc Natl Acad Sci U S A

The M2 protein is a small, single-span transmembrane (TM) protein from the influenza A virus. This virus enters cells via endosomes; as the endosomes mature and become more acidic M2 facilitates proton transport into the viral interior, thereby disrupting matrix protein/RNA interactions required for infectivity. A mystery has been how protons can accumulate in the viral interior without developing a large electrical potential that impedes further inward proton translocation. Progress in addressing this question has been limited by the availability of robust methods of unidirectional insertion of the protein into virus-like vesicles. Using an optimized procedure for reconstitution, we show that M2 has antiporter-like activity, facilitating K(+) or Na(+) efflux when protons flow down a concentration gradient into the vesicles. Cation efflux is very small except under conditions mimicking those encountered by the endosomally entrapped virus, in which protons are flowing through the channel. This proton/cation exchange function is consistent with the known high proton selectivity of the channel. Thus, M2 acts as a proton uniporter that occasionally allows K(+) to flow to maintain electrical neutrality. Remarkably, as the pH inside M2-containing vesicles (pH(in)) decreases, the proton channel activity of M2 is inhibited, but its cation transport activity is activated. This reciprocal inhibition of proton flux and activation of cation flux with decreasing pH(in) first allows accumulation of protons in the early stages of acidification, then trapping of protons within the virus when low pH(in) is achieved.

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Amphiphile regulation of ion channel function by changes in the bilayer spring constant.

Publication Date: 2010 Aug 31 PMID: 20713738
Authors: Lundbaek, J. A. - Koeppe, R. E. 2nd - Andersen, O. S.
Journal: Proc Natl Acad Sci U S A

Many drugs are amphiphiles that, in addition to binding to a particular target protein, adsorb to cell membrane lipid bilayers and alter intrinsic bilayer physical properties (e.g., bilayer thickness, monolayer curvature, and elastic moduli). Such changes can modulate membrane protein function by altering the energetic cost (DeltaG(bilayer)) of bilayer deformations associated with protein conformational changes that involve the protein-bilayer interface. But amphiphiles have complex effects on the physical properties of lipid bilayers, meaning that the net change in DeltaG(bilayer) cannot be predicted from measurements of isolated changes in such properties. Thus, the bilayer contribution to the promiscuous regulation of membrane proteins by drugs and other amphiphiles remains unknown. To overcome this problem, we use gramicidin A (gA) channels as molecular force probes to measure the net effect of amphiphiles, at concentrations often used in biological research, on the bilayer elastic response to a change in the hydrophobic length of an embedded protein. The effects of structurally diverse amphiphiles can be described by changes in a phenomenological bilayer spring constant (H(B)) that summarizes the bilayer elastic properties, as sensed by a bilayer-spanning protein. Amphiphile-induced changes in H(B), measured using gA channels of a particular length, quantitatively predict changes in lifetime for channels of a different length-as well as changes in the inactivation of voltage-dependent sodium channels in living cells. The use of gA channels as molecular force probes provides a tool for quantitative, predictive studies of bilayer-mediated regulation of membrane protein function by amphiphiles.

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An explanation for conflicting records of Triassic-Jurassic plant diversity.

Publication Date: 2010 Aug 31 PMID: 20713737
Authors: Mander, L. - Kurschner, W. M. - McElwain, J. C.
Journal: Proc Natl Acad Sci U S A

Macrofossils (mostly leaves) and sporomorphs (pollen and spores) preserve conflicting records of plant biodiversity during the end-Permian (P-Tr), Triassic-Jurassic (Tr-J), and end-Cretaceous (K-T) mass extinctions. Estimates of diversity loss based on macrofossils are typically much higher than estimates of diversity loss based on sporomorphs. Macrofossils from the Tr-J of East Greenland indicate that standing species richness declined by as much as 85% in the Late Triassic, whereas sporomorph records from the same region, and from elsewhere in Europe, reveal little evidence of such catastrophic diversity loss. To understand this major discrepancy, we have used a new high-resolution dataset of sporomorph assemblages from Astartekloft, East Greenland, to directly compare the macrofossil and sporomorph records of Tr-J plant biodiversity. Our results show that sporomorph assemblages from the Tr-J boundary interval are 10-12% less taxonomically diverse than sporomorph assemblages from the Late Triassic, and that vegetation composition changed rapidly in the boundary interval as a result of emigration and/or extirpation of taxa rather than immigration and/or origination of taxa. An analysis of the representation of different plant groups in the macrofossil and sporomorph records at Astartekloft reveals that reproductively specialized plants, including cycads, bennettites and the seed-fern Lepidopteris are almost absent from the sporomorph record. These results provide a means of reconciling the macrofossil and sporomorph records of Tr-J vegetation change, and may help to understand vegetation change during the P-Tr and K-T mass extinctions and around the Paleocene-Eocene Thermal Maximum.

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Accelerated warming of the Southern Ocean and its impacts on the hydrological cycle and sea ice.

Publication Date: 2010 Aug 24 PMID: 20713736
Authors: Liu, J. - Curry, J. A.
Journal: Proc Natl Acad Sci U S A

The observed sea surface temperature in the Southern Ocean shows a substantial warming trend for the second half of the 20th century. Associated with the warming, there has been an enhanced atmospheric hydrological cycle in the Southern Ocean that results in an increase of the Antarctic sea ice for the past three decades through the reduced upward ocean heat transport and increased snowfall. The simulated sea surface temperature variability from two global coupled climate models for the second half of the 20th century is dominated by natural internal variability associated with the Antarctic Oscillation, suggesting that the models' internal variability is too strong, leading to a response to anthropogenic forcing that is too weak. With increased loading of greenhouse gases in the atmosphere through the 21st century, the models show an accelerated warming in the Southern Ocean, and indicate that anthropogenic forcing exceeds natural internal variability. The increased heating from below (ocean) and above (atmosphere) and increased liquid precipitation associated with the enhanced hydrological cycle results in a projected decline of the Antarctic sea ice.

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PCNA function in the activation and strand direction of MutL{alpha} endonuclease in mismatch repair.

Publication Date: 2010 Aug 16 PMID: 20713735
Authors: Pluciennik, A. - Dzantiev, L. - Iyer, R. R. - Constantin, N. - Kadyrov, F. A. - Modrich, P.
Journal: Proc Natl Acad Sci U S A

MutLalpha (MLH1-PMS2) is a latent endonuclease that is activated in a mismatch-, MutSalpha-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutLalpha activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLalpha function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutLalpha incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion.

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Surface Chemistry Special Feature: Comparative surface dynamics of amorphous and semicrystalline polymer films.

Publication Date: 2010 Aug 16 PMID: 20713734
Authors: Becker, J. S. - Brown, R. D. - Killelea, D. R. - Yuan, H. - Sibener, S. J.
Journal: Proc Natl Acad Sci U S A

The surface dynamics of amorphous and semicrystalline polymer films have been measured using helium atom scattering. Time-of-flight data were collected to resolve the elastic and inelastic scattering components in the diffuse scattering of neutral helium atoms from the surface of a thin poly(ethylene terephthalate) film. Debye-Waller attenuation was observed for both the amorphous and semicrystalline phases of the polymer by recording the decay of elastically scattered helium atoms with increasing surface temperature. Thermal attenuation measurements in the specular scattering geometry yielded perpendicular mean-square displacements of 2.7*10(-4) A(2) K(-1) and 3.1*10(-4) A(2) K(-1) for the amorphous and semicrystalline surfaces, respectively. The semicrystalline surface was consistently approximately 15% softer than the amorphous across a variety of perpendicular momentum transfers. The Debye-Waller factors were also measured at off-specular angles to characterize the parallel mean-square displacements, which were found to increase by an order of magnitude over the perpendicular mean-square displacements for both surfaces. In contrast to the perpendicular motion, the semicrystalline state was approximately 25% stiffer than the amorphous phase in the surface plane. These results were uniquely accessed through low-energy neutral helium atom scattering due to the highly surface-sensitive and nonperturbative nature of these interactions. The goal of tailoring the chemical and physical properties of complex advanced materials requires an improved understanding of interfacial dynamics, information that is obtainable through atomic beam scattering methods.

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Direct mapping of nanoscale compositional connectivity on intact cell membranes.

Publication Date: 2010 Aug 31 PMID: 20713733
Authors: van Zanten, T. S. - Gomez, J. - Manzo, C. - Cambi, A. - Buceta, J. - Reigada, R. - Garcia-Parajo, M. F.
Journal: Proc Natl Acad Sci U S A

Lateral segregation of cell membranes is accepted as a primary mechanism for cells to regulate a diversity of cellular functions. In this context, lipid rafts have been conceptualized as organizing principle of biological membranes where underlying cholesterol-mediated selective connectivity must exist even at the resting state. However, such a level of nanoscale compositional connectivity has been challenging to prove. Here we used single-molecule near-field scanning optical microscopy to visualize the nanolandscape of raft ganglioside GM1 after tightening by its ligand cholera toxin (CTxB) on intact cell membranes. We show that CTxB tightening of GM1 is sufficient to initiate a minimal raft coalescence unit, resulting in the formation of cholesterol-dependent GM1 nanodomains < 120 nm in size. This particular arrangement appeared independent of cell type and GM1 expression level on the membrane. Simultaneous dual color high-resolution images revealed that GPI anchored and certain transmembrane proteins were recruited to regions proximal (< 150 nm) to CTxB-GM1 nanodomains without physical intermixing. Together with in silico experiments, our high-resolution data conclusively demonstrate the existence of raft-based interconnectivity at the nanoscale. Such a linked state on resting cell membranes constitutes thus an obligatory step toward the hierarchical evolution of large-scale raft coalescence upon cell activation.

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A family of diiron monooxygenases catalyzing amino acid beta-hydroxylation in antibiotic biosynthesis.

Publication Date: 2010 Aug 31 PMID: 20713732
Authors: Makris, T. M. - Chakrabarti, M. - Munck, E. - Lipscomb, J. D.
Journal: Proc Natl Acad Sci U S A

The biosynthesis of chloramphenicol requires a beta-hydroxylation tailoring reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the nonribosomal peptide synthetase CmlP. EPR, Mossbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with nonribosomal peptide synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O(2) is the oxygen source, demonstrating a monooxygenase reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect monooxygenase reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics.

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Dynamical organization of the cytoskeletal cortex probed by micropipette aspiration.

Publication Date: 2010 Aug 31 PMID: 20713731
Authors: Brugues, J. - Maugis, B. - Casademunt, J. - Nassoy, P. - Amblard, F. - Sens, P.
Journal: Proc Natl Acad Sci U S A

Bleb-based cell motility proceeds by the successive inflation and retraction of large spherical membrane protrusions ("blebs") coupled with substrate adhesion. In addition to their role in motility, cellular blebs constitute a remarkable illustration of the dynamical interactions between the cytoskeletal cortex and the plasma membrane. Here we study the bleb-based motions of Entamoeba histolytica in the constrained geometry of a micropipette. We construct a generic theoretical model that combines the polymerization of an actin cortex underneath the plasma membrane with the myosin-generated contractile stress in the cortex and the stress-induced failure of membrane-cortex adhesion. One major parameter dictating the cell response to micropipette suction is the stationary cortex thickness, controlled by actin polymerization and depolymerization. The other relevant physical parameters can be combined into two characteristic cortex thicknesses for which the myosin stress (i) balances the suction pressure and (ii) provokes membrane-cortex unbinding. We propose a general phase diagram for cell motions inside a micropipette by comparing these three thicknesses. In particular, we theoretically predict and experimentally verify the existence of saltatory and oscillatory motions for a well-defined range of micropipette suction pressures.

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New strategy for the synthesis of chemically modified RNA constructs exemplified by hairpin and hammerhead ribozymes.

Publication Date: 2010 Aug 31 PMID: 20713730
Authors: El-Sagheer, A. H. - Brown, T.
Journal: Proc Natl Acad Sci U S A

The CuAAC reaction (click chemistry) has been used in conjunction with solid-phase synthesis to produce catalytically active hairpin ribozymes around 100 nucleotides in length. Cross-strand ligation through neighboring nucleobases was successful in covalently linking presynthesized RNA strands with high efficiency (trans-ligation). In an alternative strategy, intrastrand click ligation was employed to produce a functional hammerhead ribozyme containing a novel nucleic acid backbone mimic at the catalytic site (cis-ligation). The ability to synthesize long RNA strands by a combination of solid-phase synthesis and click ligation is an important addition to RNA chemistry. It is compatible with a plethora of site-specific modifications and is applicable to the synthesis of many biologically important RNA molecules.

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Quantitative phosphoproteomic analysis reveals cAMP/vasopressin-dependent signaling pathways in native renal thick ascending limb cells.

Publication Date: 2010 Aug 31 PMID: 20713729
Authors: Gunaratne, R. - Braucht, D. W. - Rinschen, M. M. - Chou, C. L. - Hoffert, J. D. - Pisitkun, T. - Knepper, M. A.
Journal: Proc Natl Acad Sci U S A

Quantitative mass spectrometry was used to identify hormone-dependent signaling pathways in renal medullary thick ascending limb (mTAL) cells via phosphoproteomic analysis. Active transport of NaCl across the mTAL epithelium is accelerated by hormones that increase cAMP levels (vasopressin, glucagon, parathyroid hormone, and calcitonin). mTAL suspensions from rat kidneys were exposed (15 min) to a mixture of these four hormones. Tryptic phosphopeptides (immobilized metal affinity chromatography-enriched) were identified and quantified by mass spectrometry (LTQ-Orbitrap) using label-free methodology. We quantified a total of 654 phosphopeptides, of which 414 were quantified in three experimental pairs (hormone vs. vehicle). Of these phosphopeptides, 82% were statistically unchanged in abundance in response to the hormone mixture. In contrast, 48 phosphopeptides were significantly increased, whereas 28 were significantly decreased. The population of up-regulated phosphopeptides was highly enriched in basophilic kinase substrate motifs (AGC or calmodulin-sensitive kinase families), whereas the down-regulated sites were dominated by "proline-directed" motifs (cyclin-dependent or MAP kinase families). Bioinformatic classification uncovered overrepresentation of transmembrane transporters, protein phosphatase regulators, and cytoskeletal binding proteins among the regulated proteins. Immunoblotting with phospho-specific antibodies confirmed cAMP/vasopressin-dependent phosphorylation at Thr96, Ser126, and Ser874 of the Na(+):K(+):2Cl(-) cotransporter NKCC2, at Ser552 of the Na(+):H(+) exchanger NHE3, and at Ser552 of beta-catenin. Vasopressin also increased phosphorylation of NKCC2 at both Ser126 (more than fivefold) and Ser874 (more than threefold) in rats in vivo. Both sites were phosphorylated by purified protein kinase A during in vitro assays. These results support the view that, although protein kinase A plays a central role in mTAL signaling, additional kinases, including those that target proline-directed motifs, may be involved.

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Altered mRNA transport, docking, and protein translation in neurons lacking fragile X mental retardation protein.

Publication Date: 2010 Aug 31 PMID: 20713728
Authors: Kao, D. I. - Aldridge, G. M. - Weiler, I. J. - Greenough, W. T.
Journal: Proc Natl Acad Sci U S A

Fragile X syndrome is caused by the absence of functional fragile X mental retardation protein (FMRP), an RNA binding protein. The molecular mechanism of aberrant protein synthesis in fmr1 KO mice is closely associated with the role of FMRP in mRNA transport, delivery, and local protein synthesis. We show that GFP-labeled Fmr1 and CaMKIIalpha mRNAs undergo decelerated motion at 0-40 min after group I mGluR stimulation, and later recover at 40-60 min. Then we investigate targeting of mRNAs associated with FMRP after neuronal stimulation. We find that FMRP is synthesized closely adjacent to stimulated mGluR5 receptors. Moreover, in WT neurons, CaMKIIalpha mRNA can be delivered and translated in dendritic spines within 10 min in response to group I mGluR stimulation, whereas KO neurons fail to show this response. These data suggest that FMRP can mediate spatial mRNA delivery for local protein synthesis in response to synaptic stimulation.

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Loss of lysophosphatidylcholine acyltransferase 1 leads to photoreceptor degeneration in rd11 mice.

Publication Date: 2010 Aug 31 PMID: 20713727
Authors: Friedman, J. S. - Chang, B. - Krauth, D. S. - Lopez, I. - Waseem, N. H. - Hurd, R. E. - Feathers, K. L. - Branham, K. E. - Shaw, M. - Thomas, G. E. - Brooks, M. J. - Liu, C. - Bakeri, H. A. - Campos, M. M. - Maubaret, C. - Webster, A. R. - Rodriguez, I. R. - Thompson, D. A. - Bhattacharya, S. S. - Koenekoop, R. K. - Heckenlively, J. R. - Swaroop, A.
Journal: Proc Natl Acad Sci U S A

Retinal degenerative diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are a leading cause of untreatable blindness with substantive impact on the quality of life of affected individuals and their families. Mouse mutants with retinal dystrophies have provided a valuable resource to discover human disease genes and helped uncover pathways critical for photoreceptor function. Here we show that the rd11 mouse mutant and its allelic strain, B6-JR2845, exhibit rapid photoreceptor dysfunction, followed by degeneration of both rods and cones. Using linkage analysis, we mapped the rd11 locus to mouse chromosome 13. We then identified a one-nucleotide insertion (c.420-421insG) in exon 3 of the Lpcat1 gene. Subsequent screening of this gene in the B6-JR2845 strain revealed a seven-nucleotide deletion (c.14-20delGCCGCGG) in exon 1. Both sequence changes are predicted to result in a frame-shift, leading to premature truncation of the lysophosphatidylcholine acyltransferase-1 (LPCAT1) protein. LPCAT1 (also called AYTL2) is a phospholipid biosynthesis/remodeling enzyme that facilitates the conversion of palmitoyl-lysophosphatidylcholine to dipalmitoylphosphatidylcholine (DPPC). The analysis of retinal lipids from rd11 and B6-JR2845 mice showed substantially reduced DPPC levels compared with C57BL/6J control mice, suggesting a causal link to photoreceptor dysfunction. A follow-up screening of LPCAT1 in retinitis pigmentosa and Leber congenital amaurosis patients did not reveal any obvious disease-causing mutations. Previously, LPCAT1 has been suggested to be critical for the production of lung surfactant phospholipids and biosynthesis of platelet-activating factor in noninflammatory remodeling pathway. Our studies add another dimension to an essential role for LPCAT1 in retinal photoreceptor homeostasis.

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Flecainide increases Kir2.1 currents by interacting with cysteine 311, decreasing the polyamine-induced rectification.

Publication Date: 2010 Aug 31 PMID: 20713726
Authors: Caballero, R. - Dolz-Gaiton, P. - Gomez, R. - Amoros, I. - Barana, A. - Gonzalez de la Fuente, M. - Osuna, L. - Duarte, J. - Lopez-Izquierdo, A. - Moraleda, I. - Galvez, E. - Sanchez-Chapula, J. A. - Tamargo, J. - Delpon, E.
Journal: Proc Natl Acad Sci U S A

Both increase and decrease of cardiac inward rectifier current (I(K1)) are associated with severe cardiac arrhythmias. Flecainide, a widely used antiarrhythmic drug, exhibits ventricular proarrhythmic effects while effectively controlling ventricular arrhythmias associated with mutations in the gene encoding Kir2.1 channels that decrease I(K1) (Andersen syndrome). Here we characterize the electrophysiological and molecular basis of the flecainide-induced increase of the current generated by Kir2.1 channels (I(Kir2.1)) and I(K1) recorded in ventricular myocytes. Flecainide increases outward I(Kir2.1) generated by homotetrameric Kir2.1 channels by decreasing their affinity for intracellular polyamines, which reduces the inward rectification of the current. Flecainide interacts with the HI loop of the cytoplasmic domain of the channel, Cys311 being critical for the effect. This explains why flecainide does not increase I(Kir2.2) and I(Kir2.3), because Kir2.2 and Kir2.3 channels do not exhibit a Cys residue at the equivalent position. We further show that incubation with flecainide increases expression of functional Kir2.1 channels in the membrane, an effect also determined by Cys311. Indeed, flecainide pharmacologically rescues R67W, but not R218W, channel mutations found in Andersen syndrome patients. Moreover, our findings provide noteworthy clues about the structural determinants of the C terminus cytoplasmic domain of Kir2.1 channels involved in the control of gating and rectification.

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Profile of Charles M. Newman.

Publication Date: 2010 Aug 16 PMID: 20713725
Authors: Mossman, K. D.
Journal: Proc Natl Acad Sci U S A

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Public perceptions of energy consumption and savings.

Publication Date: 2010 Aug 16 PMID: 20713724
Authors: Attari, S. Z. - Dekay, M. L. - Davidson, C. I. - Bruine de Bruin, W.
Journal: Proc Natl Acad Sci U S A

In a national online survey, 505 participants reported their perceptions of energy consumption and savings for a variety of household, transportation, and recycling activities. When asked for the most effective strategy they could implement to conserve energy, most participants mentioned curtailment (e.g., turning off lights, driving less) rather than efficiency improvements (e.g., installing more efficient light bulbs and appliances), in contrast to experts' recommendations. For a sample of 15 activities, participants underestimated energy use and savings by a factor of 2.8 on average, with small overestimates for low-energy activities and large underestimates for high-energy activities. Additional estimation and ranking tasks also yielded relatively flat functions for perceived energy use and savings. Across several tasks, participants with higher numeracy scores and stronger proenvironmental attitudes had more accurate perceptions. The serious deficiencies highlighted by these results suggest that well-designed efforts to improve the public's understanding of energy use and savings could pay large dividends.

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TGF-{beta} IL-6 axis mediates selective and adaptive mechanisms of resistance to molecular targeted therapy in lung cancer.

Publication Date: 2010 Aug 31 PMID: 20713723
Authors: Yao, Z. - Fenoglio, S. - Gao, D. C. - Camiolo, M. - Stiles, B. - Lindsted, T. - Schlederer, M. - Johns, C. - Altorki, N. - Mittal, V. - Kenner, L. - Sordella, R.
Journal: Proc Natl Acad Sci U S A

The epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been proven to be highly effective in the treatment of nonsmall cell lung cancer (NSCLC) harboring oncogenic EGFR mutations. The majority of patients, however, will eventually develop resistance and succumb to the disease. Recent studies have identified secondary mutations in the EGFR (EGFR T790M) and amplification of the N-Methyl-N'-nitro-N-nitroso-guanidine (MNNG) HOS transforming gene (MET) oncogene as two principal mechanisms of acquired resistance. Although they can account for approximately 50% of acquired resistance cases together, in the remaining 50%, the mechanism remains unknown. In NSCLC-derived cell lines and early-stage tumors before erlotinib treatment, we have uncovered the existence of a subpopulation of cells that are intrinsically resistant to erlotinib and display features suggestive of epithelial-to-mesenchymal transition (EMT). We showed that activation of TGF-beta-mediated signaling was sufficient to induce these phenotypes. In particular, we determined that an increased TGF-beta-dependent IL-6 secretion unleashed previously addicted lung tumor cells from their EGFR dependency. Because IL-6 and TGF-beta are prominently produced during inflammatory response, we used a mouse model system to determine whether inflammation might impair erlotinib sensitivity. Indeed, induction of inflammation not only stimulated IL-6 secretion but was sufficient to decrease the tumor response to erlotinib. Our data, thus, argue that both tumor cell-autonomous mechanisms and/or activation of the tumor microenvironment could contribute to primary and acquired erlotinib resistance, and as such, treatments based on EGFR inhibition may not be sufficient for the effective treatment of lung-cancer patients harboring mutant EGFR.

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Common genetic variation in Neuregulin 3 (NRG3) influences risk for schizophrenia and impacts NRG3 expression in human brain.

Publication Date: 2010 Aug 31 PMID: 20713722
Authors: Kao, W. T. - Wang, Y. - Kleinman, J. E. - Lipska, B. K. - Hyde, T. M. - Weinberger, D. R. - Law, A. J.
Journal: Proc Natl Acad Sci U S A

Structural and polymorphic variations in Neuregulin 3 (NRG3), 10q22-23 are associated with a broad spectrum of neurodevelopmental disorders including developmental delay, cognitive impairment, autism, and schizophrenia. NRG3 is a member of the neuregulin family of EGF proteins and a ligand for the ErbB4 receptor tyrosine kinase that plays pleotropic roles in neurodevelopment. Several genes in the NRG-ErbB signaling pathway including NRG1 and ErbB4 have been implicated in genetic predisposition to schizophrenia. Previous fine mapping of the 10q22-23 locus in schizophrenia identified genome-wide significant association between delusion severity and polymorphisms in intron 1 of NRG3 (rs10883866, rs10748842, and rs6584400). The biological mechanisms remain unknown. We identified significant association of these SNPs with increased risk for schizophrenia in 350 families with an affected offspring and confirmed association to patient delusion and positive symptom severity. Molecular cloning and cDNA sequencing in human brain revealed that NRG3 undergoes complex splicing, giving rise to multiple structurally distinct isoforms. RNA expression profiling of these isoforms in the prefrontal cortex of 400 individuals revealed that NRG3 expression is developmentally regulated and pathologically increased in schizophrenia. Moreover, we show that rs10748842 lies within a DNA ultraconserved element and homedomain and strongly predicts brain expression of NRG3 isoforms that contain a unique developmentally regulated 5' exon (P = 1.097E(-12) to 1.445E(-15)). Our observations strengthen the evidence that NRG3 is a schizophrenia susceptibility gene, provide quantitative insight into NRG3 transcription traits in the human brain, and reveal a probable mechanistic basis for disease association.

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Creating a new option for online-only research articles: PNAS Plus.

Publication Date: 2010 Aug 31 PMID: 20713721
Authors: Schekman, R.
Journal: Proc Natl Acad Sci U S A

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Myonuclei acquired by overload exercise precede hypertrophy and are not lost on detraining.

Publication Date: 2010 Aug 24 PMID: 20713720
Authors: Bruusgaard, J. C. - Johansen, I. B. - Egner, I. M. - Rana, Z. A. - Gundersen, K.
Journal: Proc Natl Acad Sci U S A

Effects of previous strength training can be long-lived, even after prolonged subsequent inactivity, and retraining is facilitated by a previous training episode. Traditionally, such "muscle memory" has been attributed to neural factors in the absence of any identified local memory mechanism in the muscle tissue. We have used in vivo imaging techniques to study live myonuclei belonging to distinct muscle fibers and observe that new myonuclei are added before any major increase in size during overload. The old and newly acquired nuclei are retained during severe atrophy caused by subsequent denervation lasting for a considerable period of the animal's lifespan. The myonuclei seem to be protected from the high apoptotic activity found in inactive muscle tissue. A hypertrophy episode leading to a lasting elevated number of myonuclei retarded disuse atrophy, and the nuclei could serve as a cell biological substrate for such memory. Because the ability to create myonuclei is impaired in the elderly, individuals may benefit from strength training at an early age, and because anabolic steroids facilitate more myonuclei, nuclear permanency may also have implications for exclusion periods after a doping offense.

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Neuropeptide receptor positive allosteric modulation in epilepsy: galanin modulation revealed.

Publication Date: 2010 Aug 24 PMID: 20713719
Authors: Hoyer, D.
Journal: Proc Natl Acad Sci U S A

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Uterine FK506-binding protein 52 (FKBP52)-peroxiredoxin-6 (PRDX6) signaling protects pregnancy from overt oxidative stress.

Publication Date: 2010 Aug 31 PMID: 20713718
Authors: Hirota, Y. - Acar, N. - Tranguch, S. - Burnum, K. E. - Xie, H. - Kodama, A. - Osuga, Y. - Ustunel, I. - Friedman, D. B. - Caprioli, R. M. - Daikoku, T. - Dey, S. K.
Journal: Proc Natl Acad Sci U S A

Immunophilin FK506-binding protein 52 (FKBP52) is a cochaperone that binds to the progesterone receptor (PR) to optimize progesterone (P(4))-PR signaling. We recently showed that Fkbp52-deficient (Fkbp52(-/-)) mice have reduced uterine PR responsiveness and implantation failure which is rescued by excess P(4) supplementation in a genetic background-dependent manner. This finding led us to hypothesize that FKBP52 has functions in addition to optimizing PR activity. Using proteomics analysis, we found that uterine levels of peroxiredoxin-6 (PRDX6), a unique antioxidant, are significantly lower in Fkbp52(-/-) mice than in WT and PR-null (Pgr(-/-)) mice. We also found that Fkbp52(-/-) mice with reduced uterine PRDX6 levels are susceptible to paraquat-induced oxidative stress (OS), leading to implantation failure even with P(4) supplementation. The same dose of paraquat did not interfere with implantation in WT mice. Moreover, treatment with antioxidants alpha-tocopherol and N-acetylcysteine (NAC) attenuated paraquat-induced implantation failure in P(4)-treated Fkbp52(-/-) mice. Functional analyses using mouse embryonic fibroblasts show that Fkbp52 deficiency associated with reduced PRDX6 levels promotes H(2)O(2)-induced cell death, which is reversed by the addition of NAC or by forced expression of PRDX6, suggesting that Fkbp52 deficiency diminishes the threshold against OS by reducing PRDX6 levels. These findings provide evidence that heightened uterine OS in Fkbp52(-/-) females with reduced PRDX6 levels induces implantation failure even in the presence of excess P(4). This study shows that FKBP52-PRDX6 signaling protects pregnancy from overt OS.

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Short-term meditation induces white matter changes in the anterior cingulate.

Publication Date: 2010 Aug 31 PMID: 20713717
Authors: Tang, Y. Y. - Lu, Q. - Geng, X. - Stein, E. A. - Yang, Y. - Posner, M. I.
Journal: Proc Natl Acad Sci U S A

The anterior cingulate cortex (ACC) is part of a network implicated in the development of self-regulation and whose connectivity changes dramatically in development. In previous studies we showed that 3 h of mental training, based on traditional Chinese medicine (integrative body-mind training, IBMT), increases ACC activity and improves self-regulation. However, it is not known whether changes in white matter connectivity can result from small amounts of mental training. We here report that 11 h of IBMT increases fractional anisotropy (FA), an index indicating the integrity and efficiency of white matter in the corona radiata, an important white-matter tract connecting the ACC to other structures. Thus IBMT could provide a means for improving self-regulation and perhaps reducing or preventing various mental disorders.

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Humanized nonobese diabetic-scid IL2r{gamma}null mice are susceptible to lethal Salmonella Typhi infection.

Publication Date: 2010 Aug 31 PMID: 20713716
Authors: Libby, S. J. - Brehm, M. A. - Greiner, D. L. - Shultz, L. D. - McClelland, M. - Smith, K. D. - Cookson, B. T. - Karlinsey, J. E. - Kinkel, T. L. - Porwollik, S. - Canals, R. - Cummings, L. A. - Fang, F. C.
Journal: Proc Natl Acad Sci U S A

Salmonella enterica serovar Typhi, the cause of typhoid fever, is host-adapted to humans and unable to cause disease in mice. Here, we show that S. Typhi can replicate in vivo in nonobese diabetic (NOD)-scid IL2rgamma(null) mice engrafted with human hematopoietic stem cells (hu-SRC-SCID mice) to cause a lethal infection with pathological and inflammatory cytokine responses resembling human typhoid. In contrast, S. Typhi does not exhibit net replication or cause illness in nonengrafted or immunocompetent control animals. Screening of transposon pools in hu-SRC-SCID mice revealed both known and previously unknown Salmonella virulence determinants, including Salmonella Pathogenicity Islands 1, 2, 3, 4, and 6. Our observations indicate that the presence of human immune cells allows the in vivo replication of S. Typhi in mice. The hu-SRC-SCID mouse provides an unprecedented opportunity to gain insights into S. Typhi pathogenesis and devise strategies for the prevention of typhoid fever.

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Heterodimeric integrin complexes containing {beta}1-integrin promote internalization and lethality of anthrax toxin.

Publication Date: 2010 Aug 31 PMID: 20713715
Authors: Martchenko, M. - Jeong, S. Y. - Cohen, S. N.
Journal: Proc Natl Acad Sci U S A

To kill macrophages, the lethal factor component of Bacillus anthracis toxin binds to a carrier protein (PA), which then interacts with the CMG2 receptor protein on the cell surface and is endocytosed into the cytoplasm. CMG2, as well as TEM8, a second PA receptor not present on macrophages, contain a von Willebrand A domain that is crucial for toxin binding. Here we report that integrin beta1, another cell surface von Willebrand A domain protein, can mediate and potentiate anthrax toxin endocytosis. By using microarray-based analysis to globally correlate gene expression profiles with toxin sensitivity, we associated toxin effects with the integrin-activating proteins osteopontin and CD44. Further study showed that PA binds to alpha4beta1- and alpha5beta1-integrin complexes, leading to their conjoint endocytosis, and also interacts-weakly relative to CMG2 but comparably to TEM8-with purified alpha5beta1 complex in vitro. Monoclonal antibody directed against beta1-integrin or its alpha integrin partners reduced PA/integrin endocytosis and anthrax toxin lethality, and hyaluronic acid-which interferes with CD44-mediated integrin activation-had similar effects. Remarkably, whereas deficiency of CMG2 protected macrophages from rapid killing by large toxin doses (>50 ng/mL), by 24 h the toxin-treated cells were dead. Such late killing of CMG2-deficient cells by high dose toxin as well as the late death observed during exposure of CMG2-producing macrophages to low-dose toxin (<1 ng/mL), was dependent on integrin function. Effects of inactivating both CMG2 and integrin were synergistic. Collectively, our findings argue strongly that beta1-integrin can both potentiate CMG2-mediated endocytosis and serve independently as a low-affinity PA receptor.

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Sucrose nonfermenting AMPK-related kinase (SNARK) mediates contraction-stimulated glucose transport in mouse skeletal muscle.

Publication Date: 2010 Aug 31 PMID: 20713714
Authors: Koh, H. J. - Toyoda, T. - Fujii, N. - Jung, M. M. - Rathod, A. - Middelbeek, R. J. - Lessard, S. J. - Treebak, J. T. - Tsuchihara, K. - Esumi, H. - Richter, E. A. - Wojtaszewski, J. F. - Hirshman, M. F. - Goodyear, L. J.
Journal: Proc Natl Acad Sci U S A

The signaling mechanisms that mediate the important effects of contraction to increase glucose transport in skeletal muscle are not well understood, but are known to occur through an insulin-independent mechanism. Muscle-specific knockout of LKB1, an upstream kinase for AMPK and AMPK-related protein kinases, significantly inhibited contraction-stimulated glucose transport. This finding, in conjunction with previous studies of ablated AMPKalpha2 activity showing no effect on contraction-stimulated glucose transport, suggests that one or more AMPK-related protein kinases are important for this process. Muscle contraction increased sucrose nonfermenting AMPK-related kinase (SNARK) activity, an effect blunted in the muscle-specific LKB1 knockout mice. Expression of a mutant SNARK in mouse tibialis anterior muscle impaired contraction-stimulated, but not insulin-stimulated, glucose transport. Whole-body SNARK heterozygotic knockout mice also had impaired contraction-stimulated glucose transport in skeletal muscle, and knockdown of SNARK in C2C12 muscle cells impaired sorbitol-stimulated glucose transport. SNARK is activated by muscle contraction and is a unique mediator of contraction-stimulated glucose transport in skeletal muscle.

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Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes.

Publication Date: 2010 Aug 31 PMID: 20713713
Authors: Taube, J. H. - Herschkowitz, J. I. - Komurov, K. - Zhou, A. Y. - Gupta, S. - Yang, J. - Hartwell, K. - Onder, T. T. - Gupta, P. B. - Evans, K. W. - Hollier, B. G. - Ram, P. T. - Lander, E. S. - Rosen, J. M. - Weinberg, R. A. - Mani, S. A.
Journal: Proc Natl Acad Sci U S A

The epithelial-to-mesenchymal transition (EMT) produces cancer cells that are invasive, migratory, and exhibit stem cell characteristics, hallmarks of cells that have the potential to generate metastases. Inducers of the EMT include several transcription factors (TFs), such as Goosecoid, Snail, and Twist, as well as the secreted TGF-beta1. Each of these factors is capable, on its own, of inducing an EMT in the human mammary epithelial (HMLE) cell line. However, the interactions between these regulators are poorly understood. Overexpression of each of the above EMT inducers up-regulates a subset of other EMT-inducing TFs, with Twist, Zeb1, Zeb2, TGF-beta1, and FOXC2 being commonly induced. Up-regulation of Slug and FOXC2 by either Snail or Twist does not depend on TGF-beta1 signaling. Gene expression signatures (GESs) derived by overexpressing EMT-inducing TFs reveal that the Twist GES and Snail GES are the most similar, although the Goosecoid GES is the least similar to the others. An EMT core signature was derived from the changes in gene expression shared by up-regulation of Gsc, Snail, Twist, and TGF-beta1 and by down-regulation of E-cadherin, loss of which can also trigger an EMT in certain cell types. The EMT core signature associates closely with the claudin-low and metaplastic breast cancer subtypes and correlates negatively with pathological complete response. Additionally, the expression level of FOXC1, another EMT inducer, correlates strongly with poor survival of breast cancer patients.

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Toll-like receptor 3 inhibits memory retention and constrains adult hippocampal neurogenesis.

Publication Date: 2010 Aug 31 PMID: 20713712
Authors: Okun, E. - Griffioen, K. - Barak, B. - Roberts, N. J. - Castro, K. - Pita, M. A. - Cheng, A. - Mughal, M. R. - Wan, R. - Ashery, U. - Mattson, M. P.
Journal: Proc Natl Acad Sci U S A

Toll-like receptors (TLRs) are innate immune receptors that have recently emerged as regulators of neuronal survival and developmental neuroplasticity. Adult TLR3-deficient mice exhibited enhanced hippocampus-dependent working memory in the Morris water maze, novel object recognition, and contextual fear-conditioning tasks. In contrast, TLR3-deficient mice demonstrated impaired amygdala-related behavior and anxiety in the cued fear-conditioning, open field, and elevated plus maze tasks. Further, TLR3-deficient mice exhibited increased hippocampal CA1 and dentate gyrus volumes, increased hippocampal neurogenesis, and elevated levels of the AMPA receptor subunit GluR1 in the CA1 region of the hippocampus. In addition, levels of activated forms of the kinase ERK and the transcription factor CREB were elevated in the hippocampus of TLR3-deficient mice, suggesting that constitutive TLR3 signaling negatively regulates pathways known to play important roles in hippocampal plasticity. Direct activation of TLR3 by intracerebroventricular infusion of a TLR3 ligand impaired working memory, but not reference memory. Our findings reveal previously undescribed roles for TLR3 as a suppressor of hippocampal cellular plasticity and memory retention.

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Megafaunal meiolaniid horned turtles survived until early human settlement in Vanuatu, Southwest Pacific.

Publication Date: 2010 Aug 31 PMID: 20713711
Authors: White, A. W. - Worthy, T. H. - Hawkins, S. - Bedford, S. - Spriggs, M.
Journal: Proc Natl Acad Sci U S A

Meiolaniid or horned turtles are members of the extinct Pleistocene megafauna of Australia and the southwest Pacific. The timing and causes of their extinction have remained elusive. Here we report the remains of meiolaniid turtles from cemetery and midden layers dating 3,100/3,000 calibrated years before present to approximately 2,900/2,800 calibrated years before present in the Teouma Lapita archaeological site on Efate in Vanuatu. The remains are mainly leg bones; shell fragments are scant and there are no cranial or caudal elements, attesting to off-site butchering of the turtles. The new taxon differs markedly from other named insular terrestrial horned turtles. It is the only member of the family demonstrated to have survived into the Holocene and the first known to have become extinct after encountering humans.

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Noninvasive molecular imaging of c-Myc activation in living mice.

Publication Date: 2010 Aug 16 PMID: 20713710
Authors: Fan-Minogue, H. - Cao, Z. - Paulmurugan, R. - Chan, C. T. - Massoud, T. F. - Felsher, D. W. - Gambhir, S. S.
Journal: Proc Natl Acad Sci U S A

The cytoplasmic Myc protein (c-Myc) regulates various human genes and is dysregulated in many human cancers. Phosphorylation mediates the protein activation of c-Myc and is essential for the function of this transcription factor in normal cell behavior and tumor growth. To date, however, the targeting of Myc as a therapeutic approach for cancer treatment has been achieved primarily at the nonprotein level. We have developed a molecular imaging sensor for noninvasive imaging of c-Myc activity in living subjects using a split Firefly luciferase (FL) complementation strategy to detect and quantify the phosphorylation-mediated interaction between glycogen synthase kinase 3beta (GSK3beta) and c-Myc. This sensor system consists of two fusion proteins, GSK 35-433-CFL and NFL-c-Myc, in which specific fragments of GSK3beta and c-Myc are fused with C-terminal and N-terminal fragments of the split FL, respectively. The sensor detects phosphorylation-specific GSK3beta-c-Myc interaction, the imaging signal of which correlates with the steady-state and temporal regulation of c-Myc phosphorylation in cell culture. The sensor also detects inhibition of c-Myc activity via differential pathways, allowing noninvasive monitoring of c-Myc-targeted drug efficacy in intact cells and living mice. Notably, this drug inhibition is detected before changes in tumor size are apparent in mouse xenograft and liver tumor models. This reporter system not only provides an innovative way to investigate the role of functional c-Myc in normal and cancer-related biological processes, but also facilitates c-Myc-targeted drug development by providing a rapid quantitative approach to assessing cancer response to therapy in living subjects.

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Ghrelin secretion stimulated by {beta}1-adrenergic receptors in cultured ghrelinoma cells and in fasted mice.

Publication Date: 2010 Aug 16 PMID: 20713709
Authors: Zhao, T. J. - Sakata, I. - Li, R. L. - Liang, G. - Richardson, J. A. - Brown, M. S. - Goldstein, J. L. - Zigman, J. M.
Journal: Proc Natl Acad Sci U S A

Ghrelin, an octanoylated peptide hormone produced in the stomach, rises dramatically in mouse plasma during chronic severe calorie deprivation, an event that is essential to maintain life. The mechanism for this increase is not understood. Here, we study the control of ghrelin secretion in tissue culture cells derived from mice bearing ghrelinomas induced by a tissue-specific SV40 T-antigen transgene. We found that the ghrelin-secreting cells express high levels of mRNA encoding beta(1)-adrenergic receptors. Addition of norepinephrine or epinephrine to the culture medium stimulated ghrelin secretion, and this effect was blocked by atenolol, a selective beta(1)-adrenergic antagonist. When WT mice were treated with reserpine to deplete adrenergic neurotransmitters from sympathetic neurons, the fasting-induced increase in plasma ghrelin was blocked. Inhibition was also seen following atenolol administration. We conclude that ghrelin secretion during fasting is induced by adrenergic agents released by sympathetic neurons and acting directly on beta(1) receptors on the ghrelin-secreting cells of the stomach.

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Tracking, tuning, and terminating microbial physiology using synthetic riboregulators.

Publication Date: 2010 Aug 16 PMID: 20713708
Authors: Callura, J. M. - Dwyer, D. J. - Isaacs, F. J. - Cantor, C. R. - Collins, J. J.
Journal: Proc Natl Acad Sci U S A

The development of biomolecular devices that interface with biological systems to reveal new insights and produce novel functions is one of the defining goals of synthetic biology. Our lab previously described a synthetic, riboregulator system that affords for modular, tunable, and tight control of gene expression in vivo. Here we highlight several experimental advantages unique to this RNA-based system, including physiologically relevant protein production, component modularity, leakage minimization, rapid response time, tunable gene expression, and independent regulation of multiple genes. We demonstrate this utility in four sets of in vivo experiments with various microbial systems. Specifically, we show that the synthetic riboregulator is well suited for GFP fusion protein tracking in wild-type cells, tight regulation of toxic protein expression, and sensitive perturbation of stress response networks. We also show that the system can be used for logic-based computing of multiple, orthogonal inputs, resulting in the development of a programmable kill switch for bacteria. This work establishes a broad, easy-to-use synthetic biology platform for microbiology experiments and biotechnology applications.

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Six and Eya promote apoptosis through direct transcriptional activation of the proapoptotic BH3-only gene egl-1 in Caenorhabditis elegans.

Publication Date: 2010 Aug 31 PMID: 20713707
Authors: Hirose, T. - Galvin, B. D. - Horvitz, H. R.
Journal: Proc Natl Acad Sci U S A

The decision of a cell to undergo programmed cell death is tightly regulated during animal development and tissue homeostasis. Here, we show that the Caenorhabditis elegans Six family homeodomain protein C. elegans homeobox (CEH-34) and the Eyes absent ortholog EYA-1 promote the programmed cell death of a specific pharyngeal neuron, the sister of the M4 motor neuron. Loss of either ceh-34 or eya-1 function causes survival of the M4 sister cell, which normally undergoes programmed cell death. CEH-34 physically interacts with the conserved EYA domain of EYA-1 in vitro. We identify an egl-1 5' cis-regulatory element that controls the programmed cell death of the M4 sister cell and show that CEH-34 binds directly to this site. Expression of the proapoptotic gene egl-1 in the M4 sister cell requires ceh-34 and eya-1 function. We conclude that an evolutionarily conserved complex that includes CEH-34 and EYA-1 directly activates egl-1 expression through a 5' cis-regulatory element to promote the programmed cell death of the M4 sister cell. We suggest that the regulation of apoptosis by Six and Eya family members is conserved in mammals and involved in human diseases caused by mutations in Six and Eya.

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Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies.

Publication Date: 2010 Aug 31 PMID: 20713706
Authors: Ettenberg, S. A. - Charlat, O. - Daley, M. P. - Liu, S. - Vincent, K. J. - Stuart, D. D. - Schuller, A. G. - Yuan, J. - Ospina, B. - Green, J. - Yu, Q. - Walsh, R. - Li, S. - Schmitz, R. - Heine, H. - Bilic, S. - Ostrom, L. - Mosher, R. - Hartlepp, K. F. - Zhu, Z. - Fawell, S. - Yao, Y. M. - Stover, D. - Finan, P. M. - Porter, J. A. - Sellers, W. R. - Klagge, I. M. - Cong, F.
Journal: Proc Natl Acad Sci U S A

Disregulated Wnt/beta-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.

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{beta}1-integrin is dispensable for the induction of ErbB2 mammary tumors but plays a critical role in the metastatic phase of tumor progression.

Publication Date: 2010 Aug 31 PMID: 20713705
Authors: Huck, L. - Pontier, S. M. - Zuo, D. M. - Muller, W. J.
Journal: Proc Natl Acad Sci U S A

Cross-talk between integrin receptors and activated growth factor receptors has been hypothesized to play a critical role in the initiation and progression of cancer. Despite in vitro evidence documenting the important role of integrin receptors in the regulation of cancer cell proliferation, the relative contribution of the integrin receptors to the initiation and progression of tumors remains unclear. Previous studies with a polyomavirus middle T mammary tumor model have indicated that targeted disruption of beta1-integrin in the mammary glands of these mice completely blocks tumor induction. To further explore the general significance of these observations, we have crossed these conditional beta1-integrin strains to a strain of mice carrying mouse mammary tumor virus/activated erbB2 (herein referred to as the NIC strain). In contrast to the tumor induction block in the polyomavirus middle T model, tumor onset in the beta1-integrin-deficient NIC mice was delayed by only 30 d and was 100% penetrant. This modest effect on tumor induction was not a result of inefficient excision, as all tumors were confirmed as beta1-integrin-null. Animals bearing beta1-integrin-deficient ErbB2 tumors exhibited significantly reduced tumor volume, which was associated with increased tumor cell apoptosis and a reduction in tumor angiogenesis. In addition, beta1-integrin-deficient tumors were compromised in their capacity to metastasize to the lung, a deficiency associated with abrogation of adhesion signaling. Taken together, these observations suggest that, although beta1-integrin is dispensable for the initiation of ErbB2 tumor induction, it plays a critical role in metastatic phase of tumor progression.

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Targeting the voltage sensor of Kv7.2 voltage-gated K+ channels with a new gating-modifier.

Publication Date: 2010 Aug 31 PMID: 20713704
Authors: Peretz, A. - Pell, L. - Gofman, Y. - Haitin, Y. - Shamgar, L. - Patrich, E. - Kornilov, P. - Gourgy-Hacohen, O. - Ben-Tal, N. - Attali, B.
Journal: Proc Natl Acad Sci U S A

The pore and gate regions of voltage-gated cation channels have been often targeted with drugs acting as channel modulators. In contrast, the voltage-sensing domain (VSD) was practically not exploited for therapeutic purposes, although it is the target of various toxins. We recently designed unique diphenylamine carboxylates that are powerful Kv7.2 voltage-gated K(+) channel openers or blockers. Here we show that a unique Kv7.2 channel opener, NH29, acts as a nontoxin gating modifier. NH29 increases Kv7.2 currents, thereby producing a hyperpolarizing shift of the activation curve and slowing both activation and deactivation kinetics. In neurons, the opener depresses evoked spike discharges. NH29 dampens hippocampal glutamate and GABA release, thereby inhibiting excitatory and inhibitory postsynaptic currents. Mutagenesis and modeling data suggest that in Kv7.2, NH29 docks to the external groove formed by the interface of helices S1, S2, and S4 in a way that stabilizes the interaction between two conserved charged residues in S2 and S4, known to interact electrostatically, in the open state of Kv channels. Results indicate that NH29 may operate via a voltage-sensor trapping mechanism similar to that suggested for scorpion and sea-anemone toxins. Reflecting the promiscuous nature of the VSD, NH29 is also a potent blocker of TRPV1 channels, a feature similar to that of tarantula toxins. Our data provide a structural framework for designing unique gating-modifiers targeted to the VSD of voltage-gated cation channels and used for the treatment of hyperexcitability disorders.

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miR-204 is required for lens and retinal development via Meis2 targeting.

Publication Date: 2010 Aug 31 PMID: 20713703
Authors: Conte, I. - Carrella, S. - Avellino, R. - Karali, M. - Marco-Ferreres, R. - Bovolenta, P. - Banfi, S.
Journal: Proc Natl Acad Sci U S A

MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in the regulation of gene expression. The roles of individual miRNAs in controlling vertebrate eye development remain, however, largely unexplored. Here, we show that a single miRNA, miR-204, regulates multiple aspects of eye development in the medaka fish (Oryzias latipes). Morpholino-mediated ablation of miR-204 expression resulted in an eye phenotype characterized by microphthalmia, abnormal lens formation, and altered dorsoventral (D-V) patterning of the retina, which is associated with optic fissure coloboma. Using a variety of in vivo and in vitro approaches, we identified the transcription factor Meis2 as one of the main targets of miR-204 function. We show that, together with altered regulation of the Pax6 pathway, the abnormally elevated levels of Meis2 resulting from miR-204 inactivation are largely responsible for the observed phenotype. These data provide an example of how a specific miRNA can regulate multiple events in eye formation; at the same time, they uncover an as yet unreported function of Meis2 in the specification of D-V patterning of the retina.

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Cancer-derived mutations in the regulatory subunit p85{alpha} of phosphoinositide 3-kinase function through the catalytic subunit p110{alpha}.

Publication Date: 2010 Aug 31 PMID: 20713702
Authors: Sun, M. - Hillmann, P. - Hofmann, B. T. - Hart, J. R. - Vogt, P. K.
Journal: Proc Natl Acad Sci U S A

Cancer-specific mutations in the iSH2 (inter-SH2) and nSH2 (N-terminal SH2) domains of p85alpha, the regulatory subunit of phosphatidylinositide 3-kinase (PI3K), show gain of function. They induce oncogenic cellular transformation, stimulate cellular proliferation, and enhance PI3K signaling. Quantitative determinations of oncogenic activity reveal large differences between individual mutants of p85alpha. The mutant proteins are still able to bind to the catalytic subunits p110alpha and p110beta. Studies with isoform-specific inhibitors of p110 suggest that expression of p85 mutants in fibroblasts leads exclusively to an activation of p110alpha, and p110alpha is the sole mediator of p85 mutant-induced oncogenic transformation. The characteristics of the p85 mutants are in agreement with the hypothesis that the mutations weaken an inhibitory interaction between p85alpha and p110alpha while preserving the stabilizing interaction between p85alpha iSH2 and the adapter-binding domain of p110alpha.

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Profile of Michael Lynch.

Publication Date: 2010 Aug 16 PMID: 20713701
Authors: Azar, B.
Journal: Proc Natl Acad Sci U S A

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Social interactions, information use, and the evolution of collective migration.

Publication Date: 2010 Aug 16 PMID: 20713700
Authors: Guttal, V. - Couzin, I. D.
Journal: Proc Natl Acad Sci U S A

Migration of organisms (or cells) is typically an adaptive response to spatiotemporal variation in resources that requires individuals to detect and respond to long-range and noisy environmental gradients. Many organisms, from wildebeest to bacteria, migrate en masse in a process that can involve a vast number of individuals. Despite the ubiquity of collective migration, and the key function it plays in the ecology of many species, it is still unclear what role social interactions play in the evolution of migratory strategies. Here, we explore the evolution of migratory behavior using an individual-based spatially explicit model that incorporates the costs and benefits of obtaining directional cues from the environment and evolvable social interactions among migrating individuals. We demonstrate that collective migratory strategies evolve under a wide range of ecological scenarios, even when social encounters are rare. Although collective migration appears to be a shared navigational process, populations typically consist of small proportions of individuals actively acquiring directional information from their environment, whereas the majorities use a socially facilitated movement behavior. Because many migratory species face severe threat through anthropogenic influences, we also explore the microevolutionary response of migratory strategies to environmental pressures. We predict a gradual decline of migration due to increasing habitat destruction and argue that much greater restoration is required to recover lost behaviors (i.e., a strong hysteresis effect). Our results provide insights into both the proximate and ultimate factors that underlie evolved migratory behavior in nature.

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Stabilization of neurotoxic Alzheimer amyloid-{beta} oligomers by protein engineering.

Publication Date: 2010 Aug 31 PMID: 20713699
Authors: Sandberg, A. - Luheshi, L. M. - Sollvander, S. - Pereira de Barros, T. - Macao, B. - Knowles, T. P. - Biverstal, H. - Lendel, C. - Ekholm-Petterson, F. - Dubnovitsky, A. - Lannfelt, L. - Dobson, C. M. - Hard, T.
Journal: Proc Natl Acad Sci U S A

Soluble oligomeric aggregates of the amyloid-beta peptide (Abeta) have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the conformation adopted by Abeta within these aggregates is not known, a beta-hairpin conformation is known to be accessible to monomeric Abeta. Here we show that this beta-hairpin is a building block of toxic Abeta oligomers by engineering a double-cysteine mutant (called Abetacc) in which the beta-hairpin is stabilized by an intramolecular disulfide bond. Abeta(40)cc and Abeta(42)cc both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Abeta aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in Abetacc oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that beta-sheet-containing Abeta(42)cc oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Abeta(42), in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Abeta oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Abeta(42) is more toxic than the shorter Abeta(40), and why certain inherited mutations are linked to protofibril formation and early-onset AD.

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Herbivore physiological response to predation risk and implications for ecosystem nutrient dynamics.

Publication Date: 2010 Aug 31 PMID: 20713698
Authors: Hawlena, D. - Schmitz, O. J.
Journal: Proc Natl Acad Sci U S A

The process of nutrient transfer through an ecosystem is an important determinant of production, food-chain length, and species diversity. The general view is that the rate and efficiency of nutrient transfer up the food chain is constrained by herbivore-specific capacity to secure N-rich compounds for survival and production. Using feeding trials with artificial food, we show, however, that physiological stress-response of grasshopper herbivores to spider predation risk alters the nature of the nutrient constraint. Grasshoppers facing predation risk had higher metabolic rates than control grasshoppers. Elevated metabolism accordingly increased requirements for dietary digestible carbohydrate-C to fuel-heightened energy demands. Moreover, digestible carbohydrate-C comprises a small fraction of total plant tissue-C content, so nutrient transfer between plants and herbivores accordingly becomes more constrained by digestible plant C than by total plant C:N. This shift in herbivore diet to meet the altered nutrient requirement increased herbivore body C:N content, the C:N content of the plant community from which grasshoppers select their diet, and grasshopper fecal C:N content. Chronic predation risk thus alters the quality of animal and plant tissue that eventually enters the detrital pool to become decomposed. Our results demonstrate that herbivore physiology causes C:N requirements and nutrient intake to become flexible, thereby providing a mechanism to explain context dependence in the nature of trophic control over nutrient transfer in ecosystems.

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NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart.

Publication Date: 2010 Aug 31 PMID: 20713697
Authors: Kuroda, J. - Ago, T. - Matsushima, S. - Zhai, P. - Schneider, M. D. - Sadoshima, J.
Journal: Proc Natl Acad Sci U S A

NAD(P)H oxidases (Noxs) produce O(2)(-) and play an important role in cardiovascular pathophysiology. The Nox4 isoform is expressed primarily in the mitochondria in cardiac myocytes. To elucidate the function of endogenous Nox4 in the heart, we generated cardiac-specific Nox4(-/-) (c-Nox4(-/-)) mice. Nox4 expression was inhibited in c-Nox4(-/-) mice in a heart-specific manner, and there was no compensatory up-regulation in other Nox enzymes. These mice exhibited reduced levels of O(2)(-) in the heart, indicating that Nox4 is a significant source of O(2)(-) in cardiac myocytes. The baseline cardiac phenotype was normal in young c-Nox4(-/-) mice. In response to pressure overload (PO), however, increases in Nox4 expression and O(2)(-) production in mitochondria were abolished in c-Nox4(-/-) mice, and c-Nox4(-/-) mice exhibited significantly attenuated cardiac hypertrophy, interstitial fibrosis and apoptosis, and better cardiac function compared with WT mice. Mitochondrial swelling, cytochrome c release, and decreases in both mitochondrial DNA and aconitase activity in response to PO were attenuated in c-Nox4(-/-) mice. On the other hand, overexpression of Nox4 in mouse hearts exacerbated cardiac dysfunction, fibrosis, and apoptosis in response to PO. These results suggest that Nox4 in cardiac myocytes is a major source of mitochondrial oxidative stress, thereby mediating mitochondrial and cardiac dysfunction during PO.

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Temperatures and cyclones strongly associated with economic production in the Caribbean and Central America.

Publication Date: 2010 Aug 31 PMID: 20713696
Authors: Hsiang, S. M.
Journal: Proc Natl Acad Sci U S A

Understanding the economic impact of surface temperatures is an important question for both economic development and climate change policy. This study shows that in 28 Caribbean-basin countries, the response of economic output to increased temperatures is structurally similar to the response of labor productivity to high temperatures, a mechanism omitted from economic models of future climate change. This similarity is demonstrated by isolating the direct influence of temperature from that of tropical cyclones, an important correlate. Notably, output losses occurring in nonagricultural production (-2.4%/+1 degrees C) substantially exceed losses occurring in agricultural production (-0.1%/+1 degrees C). Thus, these results suggest that current models of future climate change that focus on agricultural impacts but omit the response of workers to thermal stress may underestimate the global economic costs of climate change.

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Nucleoside diphosphate kinase Nm23-H1 regulates chromosomal stability by activating the GTPase dynamin during cytokinesis.

Publication Date: 2010 Aug 31 PMID: 20713695
Authors: Conery, A. R. - Sever, S. - Harlow, E.
Journal: Proc Natl Acad Sci U S A

Chromosomal instability and the subsequent genetic mutations are considered to be critical factors in the development of the majority of solid tumors. Here, we describe how the nucleoside diphosphate kinase Nm23-H1, a protein with a known link to cancer progression, regulates a critical step during cytokinesis. Nm23-H1 acts to provide a local source of GTP for the GTPase dynamin. Loss of Nm23-H1 in diploid cells leads to cytokinetic furrow regression, followed by cytokinesis failure and generation of tetraploid cells. Loss of dynamin phenocopies loss of Nm23-H1, and ectopic overexpression of WT dynamin complements the loss of Nm23-H1. In the absence of p53 signaling, the tetraploid cells resulting from loss of Nm23-H1 continue cycling and develop classic hallmarks of tumor cells. We thus provide evidence that the loss of Nm23-H1, an event suspected to promote metastasis, may additionally function at an earlier stage of tumor development to drive the acquisition of chromosomal instability.

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High-throughput screens in diploid cells identify factors that contribute to the acquisition of chromosomal instability.

Publication Date: 2010 Aug 31 PMID: 20713694
Authors: Conery, A. R. - Harlow, E.
Journal: Proc Natl Acad Sci U S A

Chromosomal instability and the subsequent genetic mutations are considered to be critical factors in the development of the majority of solid tumors, but the mechanisms by which a stable diploid cell loses the ability to maintain genomic integrity are not well characterized. We have approached this critical issue through the use of high-throughput screens in untransformed diploid epithelial cells. In a screen of a cDNA library, we identified 13 kinases whose overexpression leads to increased ploidy. In a series of shRNA screens, we identified 16 kinases whose loss leads to increased ploidy. In both cDNA and shRNA screens, the majority of hits have not been linked previously to genomic stability. We further show that sustained loss of the shRNA screening hits leads to multipolar spindles and heterogeneous chromosome content, two characteristics of chromosomal instability. Loss of several of the kinases leads to loss of contact inhibition and to anchorage-independent growth, vital traits acquired during tumor development. We anticipate that this work will serve as a template for the comprehensive identification of pathways whose dysregulation can drive tumorigenesis through impaired karyotypic maintenance.

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Extracellular superoxide dismutase protects against pulmonary emphysema by attenuating oxidative fragmentation of ECM.

Publication Date: 2010 Aug 31 PMID: 20713693
Authors: Yao, H. - Arunachalam, G. - Hwang, J. W. - Chung, S. - Sundar, I. K. - Kinnula, V. L. - Crapo, J. D. - Rahman, I.
Journal: Proc Natl Acad Sci U S A

Extracellular superoxide dismutase (ECSOD or SOD3) is highly expressed in lungs and functions as a scavenger of O(2)(* horizontal line ). ECM fragmentation, which can be triggered by oxidative stress, participates in the pathogenesis of chronic obstructive pulmonary disease (COPD) through attracting inflammatory cells into the lungs. The level of SOD3 is significantly decreased in lungs of patients with COPD. However, the role of endogenous SOD3 in the development/progression of emphysema is unknown. We hypothesized that SOD3 protects against emphysema by attenuating oxidative fragmentation of ECM in mice. To test this hypothesis, SOD3-deficient, SOD3-transgenic, and WT C57BL/6J mice were exposed to cigarette smoke (CS) for 3 d (300 mg total particulate matter/m(3)) to 6 mo (100 mg/m(3) total particulate matter) or by intratracheal elastase injection. Airspace enlargement, lung inflammation, lung mechanical properties, and exercise tolerance were determined at different time points during CS exposure or after elastase administration. CS exposure and elastase administration caused airspace enlargement as well as impaired lung function and exercise capacity in SOD3-null mice, which were improved in mice overexpressing SOD3 and by pharmacological SOD mimetic. These phenomena were associated with SOD3-mediated protection against oxidative fragmentation of ECM, such as heparin sulfate and elastin, thereby attenuating lung inflammatory response. In conclusion, SOD3 attenuates emphysema and reduces oxidative fragmentation of ECM in mouse lung. Thus, pharmacological augmentation of SOD3 in the lung may have a therapeutic potential in the intervention of COPD/emphysema.

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RNA silencing amplification in plants: size matters.

Publication Date: 2010 Aug 24 PMID: 20709960
Authors: Schwab, R. - Voinnet, O.
Journal: Proc Natl Acad Sci U S A

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Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire.

Publication Date: 2010 Aug 31 PMID: 20709959
Authors: Roy, E. - Togbe, D. - Holdorf, A. D. - Trubetskoy, D. - Nabti, S. - Kublbeck, G. - Klevenz, A. - Kopp-Schneider, A. - Leithauser, F. - Moller, P. - Bladt, F. - Hammerling, G. - Arnold, B. - Pawson, T. - Tafuri, A.
Journal: Proc Natl Acad Sci U S A

The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability.

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Patterning by heritage in mouse molar row development.

Publication Date: 2010 Aug 31 PMID: 20709958
Authors: Prochazka, J. - Pantalacci, S. - Churava, S. - Rothova, M. - Lambert, A. - Lesot, H. - Klein, O. - Peterka, M. - Laudet, V. - Peterkova, R.
Journal: Proc Natl Acad Sci U S A

It is known from paleontology studies that two premolars have been lost during mouse evolution. During mouse mandible development, two bud-like structures transiently form that may represent rudimentary precursors of the lost premolars. However, the interpretation of these structures and their significance for mouse molar development are highly controversial because of a lack of molecular data. Here, we searched for typical tooth signaling centers in these two bud-like structures, and followed their fate using molecular markers, 3D reconstructions, and lineage tracing in vitro. Transient signaling centers were indeed found to be located at the tips of both the anterior and posterior rudimentary buds. These centers expressed a similar set of molecular markers as the "primary enamel knot" (pEK), the signaling center of the first molar (M1). These two transient signaling centers were sequentially patterned before and anterior to the M1 pEK. We also determined the dynamics of the M1 pEK, which, slightly later during development, spread up to the field formerly occupied by the posterior transient signaling center. It can be concluded that two rudimentary tooth buds initiate the sequential development of the mouse molars and these have previously been mistaken for early stages of M1 development. Although neither rudiment progresses to form an adult tooth, the posterior one merges with the adjacent M1, which may explain the anterior enlargement of the M1 during mouse family evolution. This study highlights how rudiments of lost structures can stay integrated and participate in morphogenesis of functional organs and help in understanding their evolution, as Darwin suspected long ago.

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Age of quantitative proteomics hits voltage-gated calcium channels.

Publication Date: 2010 Aug 24 PMID: 20705901
Authors: Dolphin, A. C.
Journal: Proc Natl Acad Sci U S A

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Organocatalysis Special Feature: Lewis base catalysis of bromo- and iodolactonization, and cycloetherification.

Publication Date: 2010 Aug 12 PMID: 20705900
Authors: Denmark, S. E. - Burk, M. T.
Journal: Proc Natl Acad Sci U S A

Lewis base catalyzed bromo- and iodolactonization reactions have been developed and the effects of catalyst structure on rate and cyclization selectivity have been systematically explored. The effects of substrate structure on halolactonization reactions and the interaction of those effects with the effects of catalyst structure have been investigated, leading to synthetically useful improvements in cyclization selectivity. The knowledge acquired was applied to the development of Lewis base catalyzed bromo- and iodocycloetherification reactions. The ability of some of the surveyed catalysts to influence the cyclization selectivity of halolactonization reactions demonstrates their presence in the transition structure of the product-determining cyclization step. This observation implies that chiral derivatives of these catalysts have the potential to provide enantioenriched products regardless of the rates or mechanisms of halonium ion racemization.

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Inaugural Article: Fast live simultaneous multiwavelength four-dimensional optical microscopy.

Publication Date: 2010 Aug 12 PMID: 20705899
Authors: Carlton, P. M. - Boulanger, J. - Kervrann, C. - Sibarita, J. B. - Salamero, J. - Gordon-Messer, S. - Bressan, D. - Haber, J. E. - Haase, S. - Shao, L. - Winoto, L. - Matsuda, A. - Kner, P. - Uzawa, S. - Gustafsson, M. - Kam, Z. - Agard, D. A. - Sedat, J. W.
Journal: Proc Natl Acad Sci U S A

Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000x below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading.

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Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing.

Publication Date: 2010 Aug 31 PMID: 20705898
Authors: Cho, M. - Xiao, Y. - Nie, J. - Stewart, R. - Csordas, A. T. - Oh, S. S. - Thomson, J. A. - Soh, H. T.
Journal: Proc Natl Acad Sci U S A

We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with K(d) < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have approximately 3-8-fold higher affinity and approximately 2-4-fold higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions.

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Combined niche and neutral effects in a microbial wastewater treatment community.

Publication Date: 2010 Aug 31 PMID: 20705897
Authors: Ofiteru, I. D. - Lunn, M. - Curtis, T. P. - Wells, G. F. - Criddle, C. S. - Francis, C. A. - Sloan, W. T.
Journal: Proc Natl Acad Sci U S A

It has long been assumed that differences in the relative abundance of taxa in microbial communities reflect differences in environmental conditions. Here we show that in the economically and environmentally important microbial communities in a wastewater treatment plant, the population dynamics are consistent with neutral community assembly, where chance and random immigration play an important and predictable role in shaping the communities. Using dynamic observations, we demonstrate a straightforward calibration of a purely neutral model and a parsimonious method to incorporate environmental influence on the reproduction (or birth) rate of individual taxa. The calibrated model parameters are biologically plausible, with the population turnover and diversity in the heterotrophic community being higher than for the ammonia oxidizing bacteria (AOB) and immigration into AOB community being relatively higher. When environmental factors were incorporated more of the variance in the observations could be explained but immigration and random reproduction and deaths remained the dominant driver in determining the relative abundance of the common taxa. Consequently we suggest that neutral community models should be the foundation of any description of an open biological system.

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Slipknotting upon native-like loop formation in a trefoil knot protein.

Publication Date: 2010 Aug 31 PMID: 20702769
Authors: Noel, J. K. - Sulkowska, J. I. - Onuchic, J. N.
Journal: Proc Natl Acad Sci U S A

Protein knots and slipknots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Recent experimental results show that knotting, starting from a fully extended polypeptide, has not yet been observed. Understanding the nucleation process of folding knots is thus a natural challenge for both experimental and theoretical investigation. In this study, we employ energy landscape theory and molecular dynamics to elucidate the entire folding mechanism. The full free energy landscape of a knotted protein is mapped using an all-atom structure-based protein model. Results show that, due to the topological constraint, the protein folds through a three-state mechanism that contains (i) a precise nucleation site that creates a correctly twisted native loop (first barrier) and (ii) a rate-limiting free energy barrier that is traversed by two parallel knot-forming routes. The main route corresponds to a slipknot conformation, a collapsed configuration where the C-terminal helix adopts a hairpin-like configuration while threading, and the minor route to an entropically limited plug motion, where the extended terminus is threaded as through a needle. Knot formation is a late transition state process and results show that random (nonspecific) knots are a very rare and unstable set of configurations both at and below folding temperature. Our study shows that a native-biased landscape is sufficient to fold complex topologies and presents a folding mechanism generalizable to all known knotted protein topologies: knotting via threading a native-like loop in a preordered intermediate.

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Probing in vivo Mn2+ speciation and oxidative stress resistance in yeast cells with electron-nuclear double resonance spectroscopy.

Publication Date: 2010 Aug 31 PMID: 20702768
Authors: McNaughton, R. L. - Reddi, A. R. - Clement, M. H. - Sharma, A. - Barnese, K. - Rosenfeld, L. - Gralla, E. B. - Valentine, J. S. - Culotta, V. C. - Hoffman, B. M.
Journal: Proc Natl Acad Sci U S A

Manganese is an essential transition metal that, among other functions, can act independently of proteins to either defend against or promote oxidative stress and disease. The majority of cellular manganese exists as low molecular-weight Mn(2+) complexes, and the balance between opposing "essential" and "toxic" roles is thought to be governed by the nature of the ligands coordinating Mn(2+). Until now, it has been impossible to determine manganese speciation within intact, viable cells, but we here report that this speciation can be probed through measurements of (1)H and (31)P electron-nuclear double resonance (ENDOR) signal intensities for intracellular Mn(2+). Application of this approach to yeast (Saccharomyces cerevisiae) cells, and two pairs of yeast mutants genetically engineered to enhance or suppress the accumulation of manganese or phosphates, supports an in vivo role for the orthophosphate complex of Mn(2+) in resistance to oxidative stress, thereby corroborating in vitro studies that demonstrated superoxide dismutase activity for this species.

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Sequence dependence of DNA bending rigidity.

Publication Date: 2010 Aug 31 PMID: 20702767
Authors: Geggier, S. - Vologodskii, A.
Journal: Proc Natl Acad Sci U S A

For many aspects of DNA-protein interaction, it is vital to know how DNA bending rigidity (or persistence length, a) depends on its sequence. We addressed this problem using the method based on cyclization of short DNA fragments, which allows very accurate determination of a. Our approach was based on assigning specific values of a to each of 10 distinct dinucleotide steps. We prepared DNA fragments, each about 200 bp in length, with various quasi-periodic sequences, measured their cyclization efficiencies (j factors), and fitted the data by the theoretical equation to obtain the values of a for each fragment. From these data, we obtained a set of a for the dinucleotide steps. To test this set, we used it to design DNA sequences that should correspond to very low and very high values of a, prepared the corresponding fragments, and determined their values of a experimentally. The measured and calculated values of a were very close to one another, confirming that we have found the correct solution to this long-standing problem. The same experimental data also allowed us to determine the sequence dependence of DNA helical repeat.

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Comprehensive microRNA expression profiling of the hematopoietic hierarchy.

Publication Date: 2010 Aug 31 PMID: 20702766
Authors: Petriv, O. I. - Kuchenbauer, F. - Delaney, A. D. - Lecault, V. - White, A. - Kent, D. - Marmolejo, L. - Heuser, M. - Berg, T. - Copley, M. - Ruschmann, J. - Sekulovic, S. - Benz, C. - Kuroda, E. - Ho, V. - Antignano, F. - Halim, T. - Giambra, V. - Krystal, G. - Takei, C. J. - Weng, A. P. - Piret, J. - Eaves, C. - Marra, M. A. - Humphries, R. K. - Hansen, C. L.
Journal: Proc Natl Acad Sci U S A

The hematopoietic system produces a large number of highly specialized cell types that are derived through a hierarchical differentiation process from a common stem cell population. miRNAs are critical players in orchestrating this differentiation. Here, we report the development and application of a high-throughput microfluidic real-time quantitative PCR (RT-qPCR) approach for generating global miRNA profiles for 27 phenotypically distinct cell populations isolated from normal adult mouse hematopoietic tissues. A total of 80,000 RT-qPCR assays were used to map the landscape of miRNA expression across the hematopoietic hierarchy, including rare progenitor and stem cell populations. We show that miRNA profiles allow for the direct inference of cell lineage relations and functional similarity. Our analysis reveals a close relatedness of the miRNA expression patterns in multipotent progenitors and stem cells, followed by a major reprogramming upon restriction of differentiation potential to a single lineage. The analysis of miRNA expression in single hematopoietic cells further demonstrates that miRNA expression is very tightly regulated within highly purified populations, underscoring the potential of single-cell miRNA profiling for assessing compartment heterogeneity.

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Inositol-requiring enzyme 1{alpha} is a key regulator of angiogenesis and invasion in malignant glioma.

Publication Date: 2010 Aug 31 PMID: 20702765
Authors: Auf, G. - Jabouille, A. - Guerit, S. - Pineau, R. - Delugin, M. - Bouchecareilh, M. - Magnin, N. - Favereaux, A. - Maitre, M. - Gaiser, T. - von Deimling, A. - Czabanka, M. - Vajkoczy, P. - Chevet, E. - Bikfalvi, A. - Moenner, M.
Journal: Proc Natl Acad Sci U S A

Inositol-requiring enzyme 1 (IRE1) is a proximal endoplasmic reticulum (ER) stress sensor and a central mediator of the unfolded protein response. In a human glioma model, inhibition of IRE1alpha correlated with down-regulation of prevalent proangiogenic factors such as VEGF-A, IL-1beta, IL-6, and IL-8. Significant up-regulation of antiangiogenic gene transcripts was also apparent. These transcripts encode SPARC, decorin, thrombospondin-1, and other matrix proteins functionally linked to mesenchymal differentiation and glioma invasiveness. In vivo, using both the chick chorio-allantoic membrane assay and a mouse orthotopic brain model, we observed in tumors underexpressing IRE1: (i) reduction of angiogenesis and blood perfusion, (ii) a decreased growth rate, and (iii) extensive invasiveness and blood vessel cooption. This phenotypic change was consistently associated with increased overall survival in glioma-implanted recipient mice. Ectopic expression of IL-6 in IRE1-deficient tumors restored angiogenesis and neutralized vessel cooption but did not reverse the mesenchymal/infiltrative cell phenotype. The ischemia-responsive IRE1 protein is thus identified as a key regulator of tumor neovascularization and invasiveness.

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Serotonin stimulation of cAMP-dependent plasticity in Aplysia sensory neurons is mediated by calmodulin-sensitive adenylyl cyclase.

Publication Date: 2010 Aug 31 PMID: 20702764
Authors: Lin, A. H. - Cohen, J. E. - Wan, Q. - Niu, K. - Shrestha, P. - Bernstein, S. L. - Abrams, T. W.
Journal: Proc Natl Acad Sci U S A

Calmodulin (CaM)-sensitive adenylyl cyclase (AC) in sensory neurons (SNs) in Aplysia has been proposed as a molecular coincidence detector during conditioning. We identified four putative ACs in Aplysia CNS. CaM binds to a sequence in the C1b region of AC-AplA that resembles the CaM-binding sequence in the C1b region of AC1 in mammals. Recombinant AC-AplA was stimulated by Ca(2+)/CaM. AC-AplC is most similar to the Ca(2+)-inhibited AC5 and AC6 in mammals. Recombinant AC-AplC was directly inhibited by Ca(2+), independent of CaM. AC-AplA and AC-AplC are expressed in SNs, whereas AC-AplB and AC-AplD are not. Knockdown of AC-AplA demonstrated that serotonin stimulation of cAMP-dependent plasticity in SNs is predominantly mediated by this CaM-sensitive AC. We propose that the coexpression of a Ca(2+)-inhibited AC in SNs, together with a Ca(2+)/CaM-stimulated AC, would enhance the associative requirement for coincident Ca(2+) influx and serotonin for effective stimulation of cAMP levels and initiation of plasticity mediated by AC-AplA.

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Analysis of proteome dynamics in the mouse brain.

Publication Date: 2010 Aug 10 PMID: 20699386
Authors: Price, J. C. - Guan, S. - Burlingame, A. - Prusiner, S. B. - Ghaemmaghami, S.
Journal: Proc Natl Acad Sci U S A

Advances in systems biology have allowed for global analyses of mRNA and protein expression, but large-scale studies of protein dynamics and turnover have not been conducted in vivo. Protein turnover is an important metabolic and regulatory mechanism in establishing proteome homeostasis, impacting many physiological and pathological processes. Here, we have used organism-wide isotopic labeling to measure the turnover rates of approximately 2,500 proteins in multiple mouse tissues, spanning four orders of magnitude. Through comparison of the brain with the liver and blood, we show that within the respective tissues, proteins performing similar functions often have similar turnover rates. Proteins in the brain have significantly slower turnover (average lifetime of 9.0 d) compared with those of the liver (3.0 d) and blood (3.5 d). Within some organelles (such as mitochondria), proteins have a narrow range of lifetimes, suggesting a synchronized turnover mechanism. Protein subunits within complexes of variable composition have a wide range of lifetimes, whereas those within well-defined complexes turn over in a coordinated manner. Together, the data represent the most comprehensive in vivo analysis of mammalian proteome turnover to date. The developed methodology can be adapted to assess in vivo proteome homeostasis in any model organism that will tolerate a labeled diet and may be particularly useful in the analysis of neurodegenerative diseases in vivo.

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Mutagenic potency of Helicobacter pylori in the gastric mucosa of mice is determined by sex and duration of infection.

Publication Date: 2010 Aug 24 PMID: 20699385
Authors: Sheh, A. - Lee, C. W. - Masumura, K. - Rickman, B. H. - Nohmi, T. - Wogan, G. N. - Fox, J. G. - Schauer, D. B.
Journal: Proc Natl Acad Sci U S A

Helicobacter pylori is a human carcinogen, but the mechanisms evoked in carcinogenesis during this chronic inflammatory disease remain incompletely characterized. We determined whether chronic H. pylori infection induced mutations in the gastric mucosa of male and female gpt delta C57BL/6 mice infected for 6 or 12 mo. Point mutations were increased in females infected for 12 mo. The mutation frequency in this group was 1.6-fold higher than in uninfected mice of both sexes (P < 0.05). A:T-to-G:C transitions and G:C-to-T:A transversions were 3.8 and 2.0 times, respectively, more frequent in this group than in controls. Both mutations are consistent with DNA damage induced by oxidative stress. No increase in the frequency of deletions was observed. Females had more severe gastric lesions than males at 6 mo postinfection (MPI; P < 0.05), but this difference was absent at 12 MPI. In all mice, infection significantly increased expression of IFNgamma, IL-17, TNFalpha, and iNOS at 6 and 12 mo, as well as H. pylori-specific IgG1 levels at 12 MPI (P < 0.05) and IgG2c levels at 6 and 12 MPI (P < 0.01 and P < 0.001). At 12 MPI, IgG2c levels in infected females were higher than at 6 MPI (P < 0.05) and also than those in infected males at 12 MPI (P < 0.05). Intensity of responses was mediated by sex and duration of infection. Lower H. pylori colonization indicated a more robust host response in females than in males. Earlier onset of severe gastric lesions and proinflammatory, Th1-biased responses in female C57BL/6 mice may have promoted mutagenesis by exposing the stomach to prolonged oxidative stress.

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Conserved vertebrate mir-451 provides a platform for Dicer-independent, Ago2-mediated microRNA biogenesis.

Publication Date: 2010 Aug 24 PMID: 20699384
Authors: Yang, J. S. - Maurin, T. - Robine, N. - Rasmussen, K. D. - Jeffrey, K. L. - Chandwani, R. - Papapetrou, E. P. - Sadelain, M. - O'Carroll, D. - Lai, E. C.
Journal: Proc Natl Acad Sci U S A

Canonical animal microRNAs (miRNAs) are generated by sequential cleavage of precursor substrates by the Drosha and Dicer RNase III enzymes. Several variant pathways exploit other RNA metabolic activities to generate functional miRNAs. However, all of these pathways culminate in Dicer cleavage, suggesting that this is a unifying feature of miRNA biogenesis. Here, we show that maturation of miR-451, a functional miRNA that is perfectly conserved among vertebrates, is independent of Dicer. Instead, structure-function and knockdown studies indicate that Drosha generates a short pre-mir-451 hairpin that is directly cleaved by Ago2 and followed by resection of its 3' terminus. We provide stringent evidence for this model by showing that Dicer knockout cells can generate mature miR-451 but not other miRNAs, whereas Ago2 knockout cells reconstituted with wild-type Ago2, but not Slicer-deficient Ago2, can process miR-451. Finally, we show that the mir-451 backbone is amenable to reprogramming, permitting vector-driven expression of diverse functional miRNAs in the absence of Dicer. Beyond the demonstration of an alternative strategy to direct gene silencing, these observations open the way for transgenic rescue of Dicer conditional knockouts.

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Local prolactin is a target to prevent expansion of basal/stem cells in prostate tumors.

Publication Date: 2010 Aug 24 PMID: 20699217
Authors: Rouet, V. - Bogorad, R. L. - Kayser, C. - Kessal, K. - Genestie, C. - Bardier, A. - Grattan, D. R. - Kelder, B. - Kopchick, J. J. - Kelly, P. A. - Goffin, V.
Journal: Proc Natl Acad Sci U S A

Androgen-independent recurrence is the major limit of androgen ablation therapy for prostate cancer. Identification of alternative pathways promoting prostate tumor growth is thus needed. Stat5 has been recently shown to promote human prostate cancer cell survival/proliferation and to be associated with early prostate cancer recurrence. Stat5 is the main signaling pathway triggered by prolactin (PRL), a growth factor whose local production is also increased in high-grade prostate cancers. The first aim of this study was to use prostate-specific PRL transgenic mice to address the mechanisms by which local PRL induces prostate tumorogenesis. We report that (i) Stat5 is the major signaling cascade triggered by local PRL in the mouse dorsal prostate, (ii) this model recapitulates prostate tumorogenesis from precancer lesions to invasive carcinoma, and (iii) tumorogenesis involves dramatic accumulation and abnormal spreading of p63-positive basal cells, and of stem cell antigen-1-positive cells identified as a stem/progenitor-like subpopulation. Because basal epithelial stem cells are proposed to serve as tumor-initiating cells, we challenged the relevance of local PRL as a previously unexplored therapeutic target. Using a double-transgenic approach, we show that Delta1-9-G129R-hPRL, a competitive PRL-receptor antagonist, prevented early stages of prostate tumorogenesis by reducing or inhibiting Stat5 activation, cell proliferation, abnormal basal-cell pattern, and frequency or grade of intraepithelial neoplasia. This study identifies PRL receptor/Stat5 as a unique pathway, initiating prostate tumorogenesis by altering basal-/stem-like cell subpopulations, and strongly supports the importance of further developing strategies to target locally overexpressed PRL in human prostate cancer.

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Quantifying uncertainty in climate change science through empirical information theory.

Publication Date: 2010 Aug 24 PMID: 20696940
Authors: Majda, A. J. - Gershgorin, B.
Journal: Proc Natl Acad Sci U S A

Quantifying the uncertainty for the present climate and the predictions of climate change in the suite of imperfect Atmosphere Ocean Science (AOS) computer models is a central issue in climate change science. Here, a systematic approach to these issues with firm mathematical underpinning is developed through empirical information theory. An information metric to quantify AOS model errors in the climate is proposed here which incorporates both coarse-grained mean model errors as well as covariance ratios in a transformation invariant fashion. The subtle behavior of model errors with this information metric is quantified in an instructive statistically exactly solvable test model with direct relevance to climate change science including the prototype behavior of tracer gases such as CO(2). Formulas for identifying the most sensitive climate change directions using statistics of the present climate or an AOS model approximation are developed here; these formulas just involve finding the eigenvector associated with the largest eigenvalue of a quadratic form computed through suitable unperturbed climate statistics. These climate change concepts are illustrated on a statistically exactly solvable one-dimensional stochastic model with relevance for low frequency variability of the atmosphere. Viable algorithms for implementation of these concepts are discussed throughout the paper.

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The evolution of electronic structure in few-layer graphene revealed by optical spectroscopy.

Publication Date: 2010 Aug 24 PMID: 20696939
Authors: Mak, K. F. - Sfeir, M. Y. - Misewich, J. A. - Heinz, T. F.
Journal: Proc Natl Acad Sci U S A

The massless Dirac spectrum of electrons in single-layer graphene has been thoroughly studied both theoretically and experimentally. Although a subject of considerable theoretical interest, experimental investigations of the richer electronic structure of few-layer graphene (FLG) have been limited. Here we examine FLG graphene crystals with Bernal stacking of layer thicknesses N = 1,2,3,...8 prepared using the mechanical exfoliation technique. For each layer thickness N, infrared conductivity measurements over the spectral range of 0.2-1.0 eV have been performed and reveal a distinctive band structure, with different conductivity peaks present below 0.5 eV and a relatively flat spectrum at higher photon energies. The principal transitions exhibit a systematic energy-scaling behavior with N. These observations are explained within a unified zone-folding scheme that generates the electronic states for all FLG materials from that of the bulk 3D graphite crystal through imposition of appropriate boundary conditions. Using the Kubo formula, we find that the complete infrared conductivity spectra for the different FLG crystals can be reproduced reasonably well within the framework a tight-binding model.

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Physical and functional interaction between the condensin MukB and the decatenase topoisomerase IV in Escherichia coli.

Publication Date: 2010 Aug 9 PMID: 20696938
Authors: Hayama, R. - Marians, K. J.
Journal: Proc Natl Acad Sci U S A

Proper geometric and topological organization of DNA is essential for all chromosomal processes. Two classes of proteins play major roles in organizing chromosomes: condensin complexes and type II topoisomerases. In Escherichia coli, MukB, a structural maintenance of chromosome-like component of the bacterial condensin, and topoisomerase IV (Topo IV), a type II topoisomerase that decatenates the newly replicated daughter chromosomes, are both essential for chromosome segregation in rapidly growing cells. However, little is known about the interplay between MukB and Topo IV. Here we demonstrate a physical and functional interaction between MukB and ParC, a subunit of Topo IV, in vitro. The site of MukB interaction was located on the C-terminal domain of ParC and a loss-of-interaction mutant, ParC R705E R729A, was isolated. This variant retained full activity as a topoisomerase when reconstituted with ParE to form Topo IV. We show that MukB stimulates the superhelical DNA relaxation activity of wild-type Topo IV, but not that of Topo IV reconstituted with ParC R705E R729A.

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Polar front shift and atmospheric CO2 during the glacial maximum of the Early Paleozoic Icehouse.

Publication Date: 2010 Aug 24 PMID: 20696937
Authors: Vandenbroucke, T. R. - Armstrong, H. A. - Williams, M. - Paris, F. - Zalasiewicz, J. A. - Sabbe, K. - Nolvak, J. - Challands, T. J. - Verniers, J. - Servais, T.
Journal: Proc Natl Acad Sci U S A

Our new data address the paradox of Late Ordovician glaciation under supposedly high pCO(2) (8 to 22x PAL: preindustrial atmospheric level). The paleobiogeographical distribution of chitinozoan ("mixed layer") marine zooplankton biotopes for the Hirnantian glacial maximum (440 Ma) are reconstructed and compared to those from the Sandbian (460 Ma): They demonstrate a steeper latitudinal temperature gradient and an equatorwards shift of the Polar Front through time from 55 degrees -70 degrees S to approximately 40 degrees S. These changes are comparable to those during Pleistocene interglacial-glacial cycles. In comparison with the Pleistocene, we hypothesize a significant decline in mean global temperature from the Sandbian to Hirnantian, proportional with a fall in pCO(2) from a modeled Sandbian level of approximately 8x PAL to approximately 5x PAL during the Hirnantian. Our data suggest that a compression of midlatitudinal biotopes and ecospace in response to the developing glaciation was a likely cause of the end-Ordovician mass extinction.

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Behavioral dynamics and influence in networked coloring and consensus.

Publication Date: 2010 Aug 24 PMID: 20696936
Authors: Judd, S. - Kearns, M. - Vorobeychik, Y.
Journal: Proc Natl Acad Sci U S A

We report on human-subject experiments on the problems of coloring (a social differentiation task) and consensus (a social agreement task) in a networked setting. Both tasks can be viewed as coordination games, and despite their cognitive similarity, we find that within a parameterized family of social networks, network structure elicits opposing behavioral effects in the two problems, with increased long-distance connectivity making consensus easier for subjects and coloring harder. We investigate the influence that subjects have on their network neighbors and the collective outcome, and find that it varies considerably, beyond what can be explained by network position alone. We also find strong correlations between influence and other features of individual subject behavior. In contrast to much of the recent research in network science, which often emphasizes network topology out of the context of any specific problem and places primacy on network position, our findings highlight the potential importance of the details of tasks and individuals in social networks.

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Conformational dynamics of bacteriophage T7 DNA polymerase and its processivity factor, Escherichia coli thioredoxin.

Publication Date: 2010 Aug 24 PMID: 20696935
Authors: Akabayov, B. - Akabayov, S. R. - Lee, S. J. - Tabor, S. - Kulczyk, A. W. - Richardson, C. C.
Journal: Proc Natl Acad Sci U S A

Gene 5 of bacteriophage T7 encodes a DNA polymerase (gp5) responsible for the replication of the phage DNA. Gp5 polymerizes nucleotides with low processivity, dissociating after the incorporation of 1 to 50 nucleotides. Thioredoxin (trx) of Escherichia coli binds tightly (Kd = 5 nM) to a unique segment in the thumb subdomain of gp5 and increases processivity. We have probed the molecular basis for the increase in processivity. A single-molecule experiment reveals differences in rates of enzymatic activity and processivity between gp5 and gp5/trx. Small angle X-ray scattering studies combined with nuclease footprinting reveal two conformations of gp5, one in the free state and one upon binding to trx. Comparative analysis of the DNA binding clefts of DNA polymerases and DNA binding proteins show that the binding surface contains more hydrophobic residues than other DNA binding proteins. The balanced composition between hydrophobic and charged residues of the binding site allows for efficient sliding of gp5/trx on the DNA. We propose a model for trx-induced conformational changes in gp5 that enhance the processivity by increasing the interaction of gp5 with DNA.

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Anisotropy of wave propagation in the heart can be modeled by a Riemannian electrophysiological metric.

Publication Date: 2010 Aug 24 PMID: 20696934
Authors: Young, R. J. - Panfilov, A. V.
Journal: Proc Natl Acad Sci U S A

It is well established that wave propagation in the heart is anisotropic and that the ratio of velocities in the three principal directions may be as large as v(f)v(s)v(n) approximately 4(fibers)2(sheets)1(normal). We develop an alternative view of the heart based on this fact by considering it as a non-Euclidean manifold with an electrophysiological(el-) metric based on wave velocity. This metric is more natural than the Euclidean metric for some applications, because el-distances directly encode wave propagation. We develop a model of wave propagation based on this metric; this model ignores higher-order effects like the curvature of wavefronts and the effect of the boundary, but still gives good predictions of local activation times and replicates many of the observed features of isochrones. We characterize this model for the important case of the rotational orthotropic anisotropy seen in cardiac tissue and perform numerical simulations for a slab of cardiac tissue with rotational orthotropic anisotropy and for a model of the ventricles based on diffusion tensor MRI scans of the canine heart. Even though the metric has many slow directions, we show that the rotation of the fibers leads to fast global activation. In the diffusion tensor MRI-based model, with principal velocities 0.25051 m/s, we find examples of wavefronts that eventually reach speeds up to 0.9 m/s and average velocities of 0.7 m/s. We believe that development of this non-Euclidean approach to cardiac anatomy and electrophysiology could become an important tool for the characterization of the normal and abnormal electrophysiological activity of the heart.

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Scanning ultrafast electron microscopy.

Publication Date: 2010 Aug 24 PMID: 20696933
Authors: Yang, D. S. - Mohammed, O. F. - Zewail, A. H.
Journal: Proc Natl Acad Sci U S A

Progress has been made in the development of four-dimensional ultrafast electron microscopy, which enables space-time imaging of structural dynamics in the condensed phase. In ultrafast electron microscopy, the electrons are accelerated, typically to 200 keV, and the microscope operates in the transmission mode. Here, we report the development of scanning ultrafast electron microscopy using a field-emission-source configuration. Scanning of pulses is made in the single-electron mode, for which the pulse contains at most one or a few electrons, thus achieving imaging without the space-charge effect between electrons, and still in ten(s) of seconds. For imaging, the secondary electrons from surface structures are detected, as demonstrated here for material surfaces and biological specimens. By recording backscattered electrons, diffraction patterns from single crystals were also obtained. Scanning pulsed-electron microscopy with the acquired spatiotemporal resolutions, and its efficient heat-dissipation feature, is now poised to provide in situ 4D imaging and with environmental capability.

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Vitamin K epoxide reductase prefers ER membrane-anchored thioredoxin-like redox partners.

Publication Date: 2010 Aug 24 PMID: 20696932
Authors: Schulman, S. - Wang, B. - Li, W. - Rapoport, T. A.
Journal: Proc Natl Acad Sci U S A

Vitamin K epoxide reductase (VKOR) sustains blood coagulation by reducing vitamin K epoxide to the hydroquinone, an essential cofactor for the gamma-glutamyl carboxylation of many clotting factors. The physiological redox partner of VKOR remains uncertain, but is likely a thioredoxin-like protein. Here, we demonstrate that human VKOR has the same membrane topology as the enzyme from Synechococcus sp., whose crystal structure was recently determined. Our results suggest that, during the redox reaction, Cys43 in a luminal loop of human VKOR forms a transient disulfide bond with a thioredoxin (Trx)-like protein located in the lumen of the endoplasmic reticulum (ER). We screened for redox partners of VKOR among the large number of mammalian Trx-like ER proteins by testing a panel of these candidates for their ability to form this specific disulfide bond with human VKOR. Our results show that VKOR interacts strongly with TMX, an ER membrane-anchored Trx-like protein with a unique CPAC active site. Weaker interactions were observed with TMX4, a close relative of TMX, and ERp18, the smallest Trx-like protein of the ER. We performed a similar screen with Ero1-alpha, an ER-luminal protein that oxidizes the Trx-like protein disulfide isomerase. We found that Ero1-alpha interacts with most of the tested Trx-like proteins, although only poorly with the membrane-anchored members of the family. Taken together, our results demonstrate that human VKOR employs the same electron transfer pathway as its bacterial homologs and that VKORs generally prefer membrane-bound Trx-like redox partners.

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Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli.

Publication Date: 2010 Aug 24 PMID: 20696931
Authors: Bogdanov, M. - Heacock, P. - Guan, Z. - Dowhan, W.
Journal: Proc Natl Acad Sci U S A

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

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Structural basis for high-affinity HER2 receptor binding by an engineered protein.

Publication Date: 2010 Aug 24 PMID: 20696930
Authors: Eigenbrot, C. - Ultsch, M. - Dubnovitsky, A. - Abrahmsen, L. - Hard, T.
Journal: Proc Natl Acad Sci U S A

The human epidermal growth factor receptor 2 (HER2) is specifically overexpressed in tumors of several cancers, including an aggressive form of breast cancer. It is therefore a target for both cancer diagnostics and therapy. The 58 amino acid residue Zher2 affibody molecule was previously engineered as a high-affinity binder of HER2. Here we determined the structure of Zher2 in solution and the crystal structure of Zher2 in complex with the HER2 extracellular domain. Zher2 binds to a conformational epitope on HER2 that is distant from those recognized by the therapeutic antibodies trastuzumab and pertuzumab. Its small size and lack of interference may provide Zher2 with advantages for diagnostic use or even for delivery of therapeutic agents to HER2-expressing tumors when trastuzumab or pertuzumab are already employed. Biophysical characterization shows that Zher2 is thermodynamically stable in the folded state yet undergoing conformational interconversion on a submillisecond time scale. The data suggest that it is the HER2-binding conformation that is formed transiently prior to binding. Still, binding is very strong with a dissociation constant K(D) = 22 pM, and perfect conformational homogeneity is therefore not necessarily required in engineered binding proteins. A comparison of the original Z domain scaffold to free and bound Zher2 structures reveals how high-affinity binding has evolved during selection and affinity maturation and suggests how a compromise between binding surface optimization and stability and dynamics of the unbound state has been reached.

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Imaging in-plane and normal stresses near an interface crack using traction force microscopy.

Publication Date: 2010 Aug 24 PMID: 20696929
Authors: Xu, Y. - Engl, W. C. - Jerison, E. R. - Wallenstein, K. J. - Hyland, C. - Wilen, L. A. - Dufresne, E. R.
Journal: Proc Natl Acad Sci U S A

Colloidal coatings, such as paint, are all around us. However, we know little about the mechanics of the film-forming process because the composition and properties of drying coatings vary dramatically in space and time. To surmount this challenge, we extend traction force microscopy to quantify the spatial distribution of all three components of the stress at the interface of two materials. We apply this approach to image stress near the tip of a propagating interface crack in a drying colloidal coating and extract the stress intensity factor.

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Dysregulation of the mevalonate pathway promotes transformation.

Publication Date: 2010 Aug 24 PMID: 20696928
Authors: Clendening, J. W. - Pandyra, A. - Boutros, P. C. - El Ghamrasni, S. - Khosravi, F. - Trentin, G. A. - Martirosyan, A. - Hakem, A. - Hakem, R. - Jurisica, I. - Penn, L. Z.
Journal: Proc Natl Acad Sci U S A

The importance of cancer metabolism has been appreciated for many years, but the intricacies of how metabolic pathways interconnect with oncogenic signaling are not fully understood. With a clear understanding of how metabolism contributes to tumorigenesis, we will be better able to integrate the targeting of these fundamental biochemical pathways into patient care. The mevalonate (MVA) pathway, paced by its rate-limiting enzyme, hydroxymethylglutaryl coenzyme A reductase (HMGCR), is required for the generation of several fundamental end-products including cholesterol and isoprenoids. Despite years of extensive research from the perspective of cardiovascular disease, the contribution of a dysregulated MVA pathway to human cancer remains largely unexplored. We address this issue directly by showing that dysregulation of the MVA pathway, achieved by ectopic expression of either full-length HMGCR or its novel splice variant, promotes transformation. Ectopic HMGCR accentuates growth of transformed and nontransformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice and, importantly, cooperates with RAS to drive the transformation of primary mouse embryonic fibroblasts cells. We further explore whether the MVA pathway may play a role in the etiology of human cancers and show that high mRNA levels of HMGCR and additional MVA pathway genes correlate with poor prognosis in a meta-analysis of six microarray datasets of primary breast cancer. Taken together, our results suggest that HMGCR is a candidate metabolic oncogene and provide a molecular rationale for further exploring the statin family of HMGCR inhibitors as anticancer agents.

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Structure and function of the polymerase core of TRAMP, a RNA surveillance complex.

Publication Date: 2010 Aug 24 PMID: 20696927
Authors: Hamill, S. - Wolin, S. L. - Reinisch, K. M.
Journal: Proc Natl Acad Sci U S A

The Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex recognizes aberrant RNAs in Saccharomyces cerevisiae and targets them for degradation. A TRAMP subcomplex consisting of a noncanonical poly(A) RNA polymerase in the Pol ss superfamily of nucleotidyl transferases, Trf4p, and a zinc knuckle protein, Air2p, mediates initial substrate recognition. Trf4p and related eukaryotic poly(A) and poly(U) polymerases differ from other characterized enzymes in the Pol ss superfamily both in sequence and in the lack of recognizable nucleic acid binding motifs. Here we report, at 2.7-A resolution, the structure of Trf4p in complex with a fragment of Air2p comprising two zinc knuckle motifs. Trf4p consists of a catalytic and central domain similar in fold to those of other noncanonical Pol beta RNA polymerases, and the two zinc knuckle motifs of Air2p interact with the Trf4p central domain. The interaction surface on Trf4p is highly conserved across eukaryotes, providing evidence that the Trf4p/Air2p complex is conserved in higher eukaryotes as well as in yeast and that the TRAMP complex may also function in RNA surveillance in higher eukaryotes. We show that Air2p, and in particular sequences encompassing a zinc knuckle motif near its N terminus, modulate Trf4p activity, and we present data supporting a role for this zinc knuckle in RNA binding. Finally, we show that the RNA 3' end plays a role in substrate recognition.

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Missense mutations in dystrophin that trigger muscular dystrophy decrease protein stability and lead to cross-beta aggregates.

Publication Date: 2010 Aug 24 PMID: 20696926
Authors: Singh, S. M. - Kongari, N. - Cabello-Villegas, J. - Mallela, K. M.
Journal: Proc Natl Acad Sci U S A

A deficiency of functional dystrophin protein in muscle cells causes muscular dystrophy (MD). More than 50% of missense mutations that trigger the disease occur in the N-terminal actin binding domain (N-ABD or ABD1). We examined the effect of four disease-causing mutations--L54R, A168D, A171P, and Y231N--on the structural and biophysical properties of isolated N-ABD. Our results indicate that N-ABD is a monomeric, well-folded alpha-helical protein in solution, as is evident from its alpha-helical circular dichroism spectrum, blue shift of the native state tryptophan fluorescence, well-dispersed amide crosspeaks in 2D NMR (15)N-(1)H HSQC fingerprint region, and rotational correlation time calculated from NMR longitudinal (T(1)) and transverse (T(2)) relaxation experiments. Compared to WT, three mutants--L54R, A168D, and A171P--show a decreased alpha-helicity and do not show a cooperative sigmoidal melt with temperature, indicating that these mutations exist in a wide range of conformations or in a "molten globule" state. In contrast, Y231N has an alpha-helical content similar to WT and shows a cooperative sigmoidal temperature melt but with a decreased stability. All four mutants experience serious misfolding and aggregation. FT-IR, circular dichroism, increase in thioflavin T fluorescence, and the congo red spectral shift and birefringence show that these aggregates contain intermolecular cross-beta structure similar to that found in amyloid diseases. These results indicate that disease-causing mutants affect N-ABD structure by decreasing its thermodynamic stability and increasing its misfolding, thereby decreasing the net functional dystrophin concentration.

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Bridging the gap between ribosomal and nonribosomal protein synthesis.

Publication Date: 2010 Aug 17 PMID: 20696925
Authors: Roy, H. - Ibba, M.
Journal: Proc Natl Acad Sci U S A

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Economic choices can be made using only stimulus values.

Publication Date: 2010 Aug 24 PMID: 20696924
Authors: Wunderlich, K. - Rangel, A. - O'Doherty, J. P.
Journal: Proc Natl Acad Sci U S A

Decision-making often involves choices between different stimuli, each of which is associated with a different physical action. A growing consensus suggests that the brain makes such decisions by assigning a value to each available option and then comparing them to make a choice. An open question in decision neuroscience is whether the brain computes these choices by comparing the values of stimuli directly in goods space or instead by first assigning values to the associated actions and then making a choice over actions. We used a functional MRI paradigm in which human subjects made choices between different stimuli with and without knowledge of the actions required to obtain the different stimuli. We found neural correlates of the value of the chosen stimulus (a postdecision signal) in ventromedial prefrontal cortex before the actual stimulus-action pairing was revealed. These findings provide support for the hypothesis that the brain is capable of making choices in the space of goods without first transferring values into action space.

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Specific involvement of postsynaptic GluN2B-containing NMDA receptors in the developmental elimination of corticospinal synapses.

Publication Date: 2010 Aug 24 PMID: 20696923
Authors: Ohno, T. - Maeda, H. - Murabe, N. - Kamiyama, T. - Yoshioka, N. - Mishina, M. - Sakurai, M.
Journal: Proc Natl Acad Sci U S A

The GluN2B (GluRepsilon2/NR2B) and GluN2A (GluRepsilon1/NR2A) NMDA receptor (NMDAR) subtypes have been differentially implicated in activity-dependent synaptic plasticity. However, little is known about the respective contributions made by these two subtypes to developmental plasticity, in part because studies of GluN2B KO [Grin2b(-/-) (2b(-/-))] mice are hampered by early neonatal mortality. We previously used in vitro slice cocultures of rodent cerebral cortex (Cx) and spinal cord (SpC) to show that corticospinal (CS) synapses, once present throughout the SpC, are eliminated from the ventral side during development in an NMDAR-dependent manner. To study subtype specificity of NMDAR in this developmental plasticity, we cocultured Cx and SpC slices derived from postnatal day 0 (P0) animals with different genotypes [2b(-/-), Grin2a(-/-) (2a(-/-)), or WT mice]. The distribution of CS synapses was studied electrophysiologically and with a voltage-sensitive dye. Synapse elimination on the ventral side was blocked in WT(Cx)-2b(-/-)(SpC) pairs but not in WT(Cx)-2a(-/-)(SpC) or 2b(-/-)(Cx)-WT(SpC) pairs. CS axonal regression was also observed through live imaging of CS axons labeled with enhanced yellow fluorescent protein (EYFP) through exo utero electroporation. These findings suggest that postsynaptic GluN2B is selectively involved in CS synapse elimination. In addition, the elimination was not blocked in 2a(-/-) SpC slices, where Ca(2+) entry through GluN2B-mediated CS synaptic currents was reduced to the same level as in 2b(-/-) slices, suggesting that the differential effect of GluN2B and GluN2A in CS synapse elimination might not be explained based solely on greater Ca(2+) entry through GluN2B-containing channels.

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Profile of Ivan Izquierdo.

Publication Date: 2010 Aug 24 PMID: 20696922
Authors: Trivedi, B. P.
Journal: Proc Natl Acad Sci U S A

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Molecular basis for the resistance of an insect chymotrypsin to a potato type II proteinase inhibitor.

Publication Date: 2010 Aug 24 PMID: 20696921
Authors: Dunse, K. M. - Kaas, Q. - Guarino, R. F. - Barton, P. A. - Craik, D. J. - Anderson, M. A.
Journal: Proc Natl Acad Sci U S A

Plants produce a variety of proteinase inhibitors (PIs) that have a major function in defense against insect herbivores. In turn, insects have developed strategies to minimize the effect of dietary PIs on digestion. We have discovered that Helicoverpa larvae that survive consumption of a multidomain serine PI from Nicotiana alata (NaPI) contain high levels of a chymotrypsin that is not inhibited by NaPI. Here we describe the isolation of this NaPI-resistant chymotrypsin and an NaPI-susceptible chymotrypsin from Helicoverpa larvae, together with their corresponding cDNAs. We investigated the mechanism of resistance by mutating selected positions of the NaPI-susceptible chymotrypsin using the corresponding amino acids of the NaPI-resistant chymotrypsin. Four critical residues that conferred resistance to NaPI were identified. Molecular modeling revealed that a Phe-->Leu substitution at position 37 in the chymotrypsin results in the loss of important binding contacts with NaPI. Identification of the molecular mechanisms that contribute to PI resistance in insect digestive proteases will enable us to develop better inhibitors for the control of lepidopteran species that are major agricultural pests worldwide.

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Profile of thure e. Cerling.

Publication Date: 2010 Aug 31 PMID: 20696920
Authors: Downey, P.
Journal: Proc Natl Acad Sci U S A

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Fine analysis of spontaneous MAGE-C1/CT7-specific immunity in melanoma patients.

Publication Date: 2010 Aug 24 PMID: 20696919
Authors: Nuber, N. - Curioni-Fontecedro, A. - Matter, C. - Soldini, D. - Tiercy, J. M. - von Boehmer, L. - Moch, H. - Dummer, R. - Knuth, A. - van den Broek, M.
Journal: Proc Natl Acad Sci U S A

Cancer/testis (CT) antigens represent prime candidates for immunotherapy in cancer patients, because their expression is restricted to cancer cells and germ cells of the testis. MAGE-C1/CT7 is a CT antigen that is highly expressed in several types of cancers. Spontaneous occurrence of CT7-specific antibodies was previously detected by SEREX screen in a melanoma patient. However, naturally occurring CT7-specific T-cell responses have thus far not been detected. Peripheral blood mononuclear cells (PBMCs) from 26 metastatic melanoma patients expressing CT7 in their tumor lesions (CT7(+)) were analyzed for CT7-specific T-cell responses using overlapping peptides. CT7-specific CD4(+) T-cell responses were detected in three patients (11.5%). These CT7-specific CD4(+) T-cell responses were detectable in melanoma patients' PBMCs exclusively from preexisting CD45RA(-) memory CD4(+) T-cell pool. Additional CT7-specific memory CD4(+) T-cell responses were detected in CT7(+) melanoma patients after depletion of CD4(+)CD25high Treg cells showing that Treg cells impact on CT7-specific CD4(+) T cells in melanoma patients. CT7-specific CD4(+) T-cell clones were generated and used to define minimal epitopes, restriction elements, and confirm the recognition of naturally processed antigen. Surprisingly, these clones were able to secrete perforin and exert cytotoxicity. This study shows that CT7 can induce specific cellular immunity in melanoma patients. Based on these findings, CT7 will be further explored as a potential vaccine for melanoma immunotherapy.

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T-cell receptor complex is essential for Fas signal transduction.

Publication Date: 2010 Aug 24 PMID: 20696918
Authors: Akimzhanov, A. M. - Wang, X. - Sun, J. - Boehning, D.
Journal: Proc Natl Acad Sci U S A

The Fas receptor (also known as CD95 and APO-1) is a member of the tumor necrosis factor alpha-family of death receptors that mediate T-cell responses. Here, we show that Fas receptor signaling requires a functional T-cell receptor (TCR) complex. Fas receptor directly binds to and activates TCR components in a stimulus-dependent manner. Fas receptor stimulation does not activate canonical downstream TCR pathways, but instead the TCR complex is required specifically for Fas-mediated calcium release. Importantly, null mutations in Lck, ZAP70, and the TCR alpha- and beta-chains abrogate Fas signaling. Our results reveal a direct role for the TCR complex in mediating Fas-specific signaling events critical for T-cell homeostasis.

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Proangiogenic scaffolds as functional templates for cardiac tissue engineering.

Publication Date: 2010 Aug 24 PMID: 20696917
Authors: Madden, L. R. - Mortisen, D. J. - Sussman, E. M. - Dupras, S. K. - Fugate, J. A. - Cuy, J. L. - Hauch, K. D. - Laflamme, M. A. - Murry, C. E. - Ratner, B. D.
Journal: Proc Natl Acad Sci U S A

We demonstrate here a cardiac tissue-engineering strategy addressing multicellular organization, integration into host myocardium, and directional cues to reconstruct the functional architecture of heart muscle. Microtemplating is used to shape poly(2-hydroxyethyl methacrylate-co-methacrylic acid) hydrogel into a tissue-engineering scaffold with architectures driving heart tissue integration. The construct contains parallel channels to organize cardiomyocyte bundles, supported by micrometer-sized, spherical, interconnected pores that enhance angiogenesis while reducing scarring. Surface-modified scaffolds were seeded with human ES cell-derived cardiomyocytes and cultured in vitro. Cardiomyocytes survived and proliferated for 2 wk in scaffolds, reaching adult heart densities. Cardiac implantation of acellular scaffolds with pore diameters of 30-40 microm showed angiogenesis and reduced fibrotic response, coinciding with a shift in macrophage phenotype toward the M2 state. This work establishes a foundation for spatially controlled cardiac tissue engineering by providing discrete compartments for cardiomyocytes and stroma in a scaffold that enhances vascularization and integration while controlling the inflammatory response.

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AIDS-protective HLA-B27/B57 and chimpanzee MHC class I molecules target analogous conserved areas of HIV-1/SIVcpz.

Publication Date: 2010 Aug 24 PMID: 20696916
Authors: de Groot, N. G. - Heijmans, C. M. - Zoet, Y. M. - de Ru, A. H. - Verreck, F. A. - van Veelen, P. A. - Drijfhout, J. W. - Doxiadis, G. G. - Remarque, E. J. - Doxiadis, I. I. - van Rood, J. J. - Koning, F. - Bontrop, R. E.
Journal: Proc Natl Acad Sci U S A

In the absence of treatment, most HIV-1-infected humans develop AIDS. However, a minority are long-term nonprogressors, and resistance is associated with the presence of particular HLA-B*27/B*57 molecules. In contrast, most HIV-1-infected chimpanzees do not contract AIDS. In comparison with humans, chimpanzees experienced an ancient selective sweep affecting the MHC class I repertoire. We have determined the peptide-binding properties of frequent chimpanzee MHC class I molecules, and show that, like HLA-B*27/B*57, they target similar conserved areas of HIV-1/SIV(cpz). In addition, many animals appear to possess multiple molecules targeting various conserved areas of the HIV-1/SIV(cpz) Gag protein, a quantitative aspect of the immune response that may further minimize the chance of viral escape. The functional characteristics of the contemporary chimpanzee MHC repertoire suggest that the selective sweep was caused by a lentiviral pandemic.

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Severe anemia in the Nan mutant mouse caused by sequence-selective disruption of erythroid Kruppel-like factor.

Publication Date: 2010 Aug 24 PMID: 20696915
Authors: Siatecka, M. - Sahr, K. E. - Andersen, S. G. - Mezei, M. - Bieker, J. J. - Peters, L. L.
Journal: Proc Natl Acad Sci U S A

Studies of mouse models of anemia have long provided fundamental insights into red blood cell formation and function. Here we show that the semidominant mouse mutation Nan ("neonatal anemia") carries a single amino acid change (E339D) within the second zinc finger of the erythroid Kruppel-like factor (EKLF), a critical erythroid regulatory transcription factor. The mutation alters the DNA-binding specificity of EKLF so that it no longer binds promoters of a subset of its DNA targets. Remarkably, even when mutant Nan and wild-type EKLF alleles are expressed at equivalent levels, the mutant form selectively interferes with expression of EKLF target genes whose promoter elements it no longer binds. This interference yields a distorted genetic output and selective protein deficiencies that differ from those seen in EKLF-heterozygous and EKLF-null red blood cells and presents a unique and unexpected mechanism of inherited disease.

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Defective feedback regulation of NF-kappaB underlies Sjogren's syndrome in mice with mutated kappaB enhancers of the IkappaBalpha promoter.

Publication Date: 2010 Aug 24 PMID: 20696914
Authors: Peng, B. - Ling, J. - Lee, A. J. - Wang, Z. - Chang, Z. - Jin, W. - Kang, Y. - Zhang, R. - Shim, D. - Wang, H. - Fleming, J. B. - Zheng, H. - Sun, S. C. - Chiao, P. J.
Journal: Proc Natl Acad Sci U S A

Feedback regulation of transcription factor NF-kappaB by its inhibitor IkappaBalpha plays an essential role in control of NF-kappaB activity. To understand the biological significance of IkappaBalpha-mediated feedback regulation of NF-kappaB, we generated mice harboring mutated kappaB enhancers in the promoter of the IkappaBalpha gene (IkappaBalpha(M/M)) to inhibit NF-kappaB-regulated IkappaBalpha expression. Here, we report that these mutant mice are defective in NF-kappaB-induced expression of IkappaBalpha. This defective feedback regulation of NF-kappaB by IkappaBalpha not only altered activity of NF-kappaB, but also the expression of NF-kappaB-regulated genes. As a result, IkappaBalpha(M/M), the homozygous knock-in mice with mutated kappaB enhancers in the IkappaBalpha promoter, acquire shorten life span, hypersensitivity to septic shock, abnormal T-cell development and activation, and Sjogren's Syndrome. These findings therefore demonstrate that the IkappaBalpha-mediated feedback regulation of NF-kappaB has an essential role in controlling T-cell development and functions, provide mechanistic insight into the development of Sjogren's Syndrome, and suggest the potential of NF-kappaB signaling as a therapeutic target for Sjogren's Syndrome and other autoimmune diseases.

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Influence of climate on malaria transmission depends on daily temperature variation.

Publication Date: 2010 Aug 24 PMID: 20696913
Authors: Paaijmans, K. P. - Blanford, S. - Bell, A. S. - Blanford, J. I. - Read, A. F. - Thomas, M. B.
Journal: Proc Natl Acad Sci U S A

Malaria transmission is strongly influenced by environmental temperature, but the biological drivers remain poorly quantified. Most studies analyzing malaria-temperature relations, including those investigating malaria risk and the possible impacts of climate change, are based solely on mean temperatures and extrapolate from functions determined under unrealistic laboratory conditions. Here, we present empirical evidence to show that, in addition to mean temperatures, daily fluctuations in temperature affect parasite infection, the rate of parasite development, and the essential elements of mosquito biology that combine to determine malaria transmission intensity. In general, we find that, compared with rates at equivalent constant mean temperatures, temperature fluctuation around low mean temperatures acts to speed up rate processes, whereas fluctuation around high mean temperatures acts to slow processes down. At the extremes (conditions representative of the fringes of malaria transmission, where range expansions or contractions will occur), fluctuation makes transmission possible at lower mean temperatures than currently predicted and can potentially block transmission at higher mean temperatures. If we are to optimize control efforts and develop appropriate adaptation or mitigation strategies for future climates, we need to incorporate into predictive models the effects of daily temperature variation and how that variation is altered by climate change.

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AtsPLA2-alpha nuclear relocalization by the Arabidopsis transcription factor AtMYB30 leads to repression of the plant defense response.

Publication Date: 2010 Aug 24 PMID: 20696912
Authors: Froidure, S. - Canonne, J. - Daniel, X. - Jauneau, A. - Briere, C. - Roby, D. - Rivas, S.
Journal: Proc Natl Acad Sci U S A

The hypersensitive response (HR), characterized by a rapid and localized cell death at the inoculation site, is one of the most efficient resistance reactions to pathogen attack in plants. The transcription factor AtMYB30 was identified as a positive regulator of the HR and resistance responses during interactions between Arabidopsis and bacteria. Here, we show that AtMYB30 and the secreted phospholipase AtsPLA(2)-alpha physically interact in vivo, following the AtMYB30-mediated specific relocalization of AtsPLA(2)-alpha from cytoplasmic vesicles to the plant cell nucleus. This protein interaction leads to repression of AtMYB30 transcriptional activity and negative regulation of plant HR. Moreover, Atspla(2)-alpha mutant plants are more resistant to bacterial inoculation, whereas AtsPLA(2)-alpha overexpression leads to decreased resistance, confirming that AtsPLA(2)-alpha is a negative regulator of AtMYB30-mediated defense. These data underline the importance of cellular dynamics and, particularly, protein translocation to the nucleus, for defense-associated gene regulation in plants.

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Evidence of intense ongoing endemic transmission of hepatitis C virus in Egypt.

Publication Date: 2010 Aug 17 PMID: 20696911
Authors: Miller, F. D. - Abu-Raddad, L. J.
Journal: Proc Natl Acad Sci U S A

Egypt has the highest prevalence of antibodies to hepatitis C virus (HCV) in the world, estimated nationally at 14.7%. An estimated 9.8% are chronically infected. Numerous HCV prevalence studies in Egypt have published various estimates from different Egyptian communities, suggesting that Egypt, relative to the other nations of the world, might be experiencing intense ongoing HCV transmission. More importantly, a new national study provided an opportunity to apply established epidemiologic models to estimate incidence. Validated mathematical models for estimating incidence from age-specific prevalence were used. All previous prevalence studies of HCV in Egypt were reviewed and used to estimate incidence provided that there was sufficient age-specific data required by the models. All reports of anti-HCV antibody prevalence were much higher than any single other national estimate. Age was the strongest and most consistently associated factor to HCV prevalence and HCV RNA positivity. It was not possible to establish a prior reference point for HCV prevalence or incidence to compare with the 2009 incidence estimates. The modeled incidence from the national study and collectively from the modeled incidence from the previous community studies was 6.9/1,000 [95% confidence interval (CI), 5.5-7.4] per person per year and 6.6/1,000 (95% CI, 5.1-7.0) per person per year, respectively. Projected to the age structure of the Egyptian population, more than 500,000 new HCV infections per year were estimated. Iatrogenic transmission is the most likely, underlining exposure to the ongoing transmission. The study demonstrates the urgency to reduce HCV transmission in Egypt.

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A small RNA promotes siderophore production through transcriptional and metabolic remodeling.

Publication Date: 2010 Aug 24 PMID: 20696910
Authors: Salvail, H. - Lanthier-Bourbonnais, P. - Sobota, J. M. - Caza, M. - Benjamin, J. A. - Mendieta, M. E. - Lepine, F. - Dozois, C. M. - Imlay, J. - Masse, E.
Journal: Proc Natl Acad Sci U S A

Siderophores are essential factors for iron (Fe) acquisition in bacteria during colonization and infection of eukaryotic hosts, which restrain iron access through iron-binding protein, such as lactoferrin and transferrin. The synthesis of siderophores by Escherichia coli is considered to be fully regulated at the transcriptional level by the Fe-responsive transcriptional repressor Fur. Here we characterized two different pathways that promote the production of the siderophore enterobactin via the action of the small RNA RyhB. First, RyhB is required for normal expression of an important enterobactin biosynthesis polycistron, entCEBAH. Second, RyhB directly represses the translation of cysE, which encodes a serine acetyltransferase that uses serine as a substrate for cysteine biosynthesis. Reduction of CysE activity by RyhB allows serine to be used as building blocks for enterobactin synthesis through the nonribosomal peptide synthesis pathway. Thus, RyhB plays an essential role in siderophore production and may modulate bacterial virulence through optimization of siderophore production.

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Ectopic vesicular neurotransmitter release along sensory axons mediates neurovascular coupling via glial calcium signaling.

Publication Date: 2010 Aug 24 PMID: 20696909
Authors: Thyssen, A. - Hirnet, D. - Wolburg, H. - Schmalzing, G. - Deitmer, J. W. - Lohr, C.
Journal: Proc Natl Acad Sci U S A

Neurotransmitter release generally is considered to occur at active zones of synapses, and ectopic release of neurotransmitters has been demonstrated in a few instances. However, the mechanism of ectopic neurotransmitter release is poorly understood. We took advantage of the intimate morphological and functional proximity of olfactory receptor axons and specialized glial cells, olfactory ensheathing cells (OECs), to study ectopic neurotransmitter release. Axonal stimulation evoked purinergic and glutamatergic Ca(2+) responses in OECs, indicating ATP and glutamate release. In axons expressing synapto-pHluorin, stimulation evoked an increase in synapto-pHluorin fluorescence, indicative of vesicle fusion. Transmitter release was dependent on Ca(2+) and could be inhibited by bafilomycin A1 and botulinum toxin A. Ca(2+) transients in OECs evoked by ATP, axonal stimulation, and laser photolysis of NP-EGTA resulted in constriction of adjacent blood vessels. Our results indicate that ATP and glutamate are released ectopically by vesicles along axons and mediate neurovascular coupling via glial Ca(2+) signaling.

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Rice yields in tropical/subtropical Asia exhibit large but opposing sensitivities to minimum and maximum temperatures.

Publication Date: 2010 Aug 17 PMID: 20696908
Authors: Welch, J. R. - Vincent, J. R. - Auffhammer, M. - Moya, P. F. - Dobermann, A. - Dawe, D.
Journal: Proc Natl Acad Sci U S A

Data from farmer-managed fields have not been used previously to disentangle the impacts of daily minimum and maximum temperatures and solar radiation on rice yields in tropical/subtropical Asia. We used a multiple regression model to analyze data from 227 intensively managed irrigated rice farms in six important rice-producing countries. The farm-level detail, observed over multiple growing seasons, enabled us to construct farm-specific weather variables, control for unobserved factors that either were unique to each farm but did not vary over time or were common to all farms at a given site but varied by season and year, and obtain more precise estimates by including farm- and site-specific economic variables. Temperature and radiation had statistically significant impacts during both the vegetative and ripening phases of the rice plant. Higher minimum temperature reduced yield, whereas higher maximum temperature raised it; radiation impact varied by growth phase. Combined, these effects imply that yield at most sites would have grown more rapidly during the high-yielding season but less rapidly during the low-yielding season if observed temperature and radiation trends at the end of the 20th century had not occurred, with temperature trends being more influential. Looking ahead, they imply a net negative impact on yield from moderate warming in coming decades. Beyond that, the impact would likely become more negative, because prior research indicates that the impact of maximum temperature becomes negative at higher levels. Diurnal temperature variation must be considered when investigating the impacts of climate change on irrigated rice in Asia.

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Identification of RING finger protein 4 (RNF4) as a modulator of DNA demethylation through a functional genomics screen.

Publication Date: 2010 Aug 24 PMID: 20696907
Authors: Hu, X. V. - Rodrigues, T. M. - Tao, H. - Baker, R. K. - Miraglia, L. - Orth, A. P. - Lyons, G. E. - Schultz, P. G. - Wu, X.
Journal: Proc Natl Acad Sci U S A

DNA methylation is an important epigenetic modification involved in transcriptional regulation, nuclear organization, development, aging, and disease. Although DNA methyltransferases have been characterized, the mechanisms for DNA demethylation remain poorly understood. Using a cell-based reporter assay, we performed a functional genomics screen to identify genes involved in DNA demethylation. Here we show that RNF4 (RING finger protein 4), a SUMO-dependent ubiquitin E3-ligase previously implicated in maintaining genome stability, plays a key role in active DNA demethylation. RNF4 reactivates methylation-silenced reporters and promotes global DNA demethylation. Rnf4 deficiency is embryonic lethal with higher levels of methylation in genomic DNA. Mechanistic studies show that RNF4 interacts with and requires the base excision repair enzymes TDG and APE1 for active demethylation. This activity appears to occur by enhancing the enzymatic activities that repair DNA G:T mismatches generated from methylcytosine deamination. Collectively, our study reveals a unique function for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells.

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Cullin 4-ring finger-ligase plays a key role in the control of endoreplication cycles in Arabidopsis trichomes.

Publication Date: 2010 Aug 24 PMID: 20696906
Authors: Roodbarkelari, F. - Bramsiepe, J. - Weinl, C. - Marquardt, S. - Novak, B. - Jakoby, M. J. - Lechner, E. - Genschik, P. - Schnittger, A.
Journal: Proc Natl Acad Sci U S A

One of the predominant cell-cycle programs found in mature tissues is endoreplication, also known as endoreduplication, that leads to cellular polyploidy. A key question for the understanding of endoreplication cycles is how oscillating levels of cyclin-dependent kinase activity are generated that control repeated rounds of DNA replication. The APC/C performs a pivotal function in the mitotic cell cycle by promoting anaphase and paving the road for a new round of DNA replication. However, using marker lines and plants in which APC/C components are knocked down, we show here that outgrowing and endoreplicating Arabidopsis leaf hairs display no or very little APC/C activity. Instead we find that RBX1-containing Cullin-RING E3 ubiquitin-Ligases (CRLs) are of central importance for the progression through endoreplication cycles; in particular, we have identified CULLIN4 as a major regulator of endoreplication in Arabidopsis trichomes. We have incorporated our findings into a bio-mathematical simulation presenting a robust two-step model of endoreplication control with one type of cyclin-dependent kinase inhibitor function for entry and a CRL-dependent oscillation of cyclin-dependent kinase activity via degradation of a second type of CDK inhibitor during endoreplication cycles.

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Caloric restriction or catalase inactivation extends yeast chronological lifespan by inducing H2O2 and superoxide dismutase activity.

Publication Date: 2010 Aug 24 PMID: 20696905
Authors: Mesquita, A. - Weinberger, M. - Silva, A. - Sampaio-Marques, B. - Almeida, B. - Leao, C. - Costa, V. - Rodrigues, F. - Burhans, W. C. - Ludovico, P.
Journal: Proc Natl Acad Sci U S A

The free radical theory of aging posits oxidative damage to macromolecules as a primary determinant of lifespan. Recent studies challenge this theory by demonstrating that in some cases, longevity is enhanced by inactivation of oxidative stress defenses or is correlated with increased, rather than decreased reactive oxygen species and oxidative damage. Here we show that, in Saccharomyces cerevisiae, caloric restriction or inactivation of catalases extends chronological lifespan by inducing elevated levels of the reactive oxygen species hydrogen peroxide, which activate superoxide dismutases that inhibit the accumulation of superoxide anions. Increased hydrogen peroxide in catalase-deficient cells extends chronological lifespan despite parallel increases in oxidative damage. These findings establish a role for hormesis effects of hydrogen peroxide in promoting longevity that have broad implications for understanding aging and age-related diseases.

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Cortical depth-specific microvascular dilation underlies laminar differences in blood oxygenation level-dependent functional MRI signal.

Publication Date: 2010 Aug 24 PMID: 20696904
Authors: Tian, P. - Teng, I. C. - May, L. D. - Kurz, R. - Lu, K. - Scadeng, M. - Hillman, E. M. - De Crespigny, A. J. - D'Arceuil, H. E. - Mandeville, J. B. - Marota, J. J. - Rosen, B. R. - Liu, T. T. - Boas, D. A. - Buxton, R. B. - Dale, A. M. - Devor, A.
Journal: Proc Natl Acad Sci U S A

Changes in neuronal activity are accompanied by the release of vasoactive mediators that cause microscopic dilation and constriction of the cerebral microvasculature and are manifested in macroscopic blood oxygenation level-dependent (BOLD) functional MRI (fMRI) signals. We used two-photon microscopy to measure the diameters of single arterioles and capillaries at different depths within the rat primary somatosensory cortex. These measurements were compared with cortical depth-resolved fMRI signal changes. Our microscopic results demonstrate a spatial gradient of dilation onset and peak times consistent with "upstream" propagation of vasodilation toward the cortical surface along the diving arterioles and "downstream" propagation into local capillary beds. The observed BOLD response exhibited the fastest onset in deep layers, and the "initial dip" was most pronounced in layer I. The present results indicate that both the onset of the BOLD response and the initial dip depend on cortical depth and can be explained, at least in part, by the spatial gradient of delays in microvascular dilation, the fastest response being in the deep layers and the most delayed response in the capillary bed of layer I.

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Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle.

Publication Date: 2010 Aug 24 PMID: 20696903
Authors: Roepke, J. - Salim, V. - Wu, M. - Thamm, A. M. - Murata, J. - Ploss, K. - Boland, W. - De Luca, V.
Journal: Proc Natl Acad Sci U S A

The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) continue to be the most important source of natural drugs in chemotherapy treatments for a range of human cancers. These anticancer drugs are derived from the coupling of catharanthine and vindoline to yield powerful dimeric MIAs that prevent cell division. However the precise mechanisms for their assembly within plants remain obscure. Here we report that the complex development-, environment-, organ-, and cell-specific controls involved in expression of MIA pathways are coupled to secretory mechanisms that keep catharanthine and vindoline separated from each other in living plants. Although the entire production of catharanthine and vindoline occurs in young developing leaves, catharanthine accumulates in leaf wax exudates of leaves, whereas vindoline is found within leaf cells. The spatial separation of these two MIAs provides a biological explanation for the low levels of dimeric anticancer drugs found in the plant that result in their high cost of commercial production. The ability of catharanthine to inhibit the growth of fungal zoospores at physiological concentrations found on the leaf surface of Catharanthus leaves, as well as its insect toxicity, provide an additional biological role for its secretion. We anticipate that this discovery will trigger a broad search for plants that secrete alkaloids, the biological mechanisms involved in their secretion to the plant surface, and the ecological roles played by them.

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Carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects.

Publication Date: 2010 Aug 24 PMID: 20696902
Authors: Herve, C. - Rogowski, A. - Blake, A. W. - Marcus, S. E. - Gilbert, H. J. - Knox, J. P.
Journal: Proc Natl Acad Sci U S A

Cell wall degrading enzymes have a complex molecular architecture consisting of catalytic modules and noncatalytic carbohydrate-binding modules (CBMs). The function of CBMs in cell wall degrading processes is poorly understood. Here, we have evaluated the potential enzyme-targeting function of CBMs in the context of intact primary and secondary cell wall deconstruction. The capacity of a pectate lyase to degrade pectic homogalacturonan in primary cell walls was potentiated by cellulose-directed CBMs but not by xylan-directed CBMs. Conversely, the arabinofuranosidase-mediated removal of side chains from arabinoxylan in xylan-rich and cellulose-poor wheat grain endosperm cell walls was enhanced by a xylan-binding CBM but less so by a crystalline cellulose-specific module. The capacity of xylanases to degrade xylan in secondary cell walls was potentiated by both xylan- and cellulose-directed CBMs. These studies demonstrate that CBMs can potentiate the action of a cognate catalytic module toward polysaccharides in intact cell walls through the recognition of nonsubstrate polysaccharides. The targeting actions of CBMs therefore have strong proximity effects within cell wall structures, explaining why cellulose-directed CBMs are appended to many noncellulase cell wall hydrolases.

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Small-molecule inducers of insulin expression in pancreatic alpha-cells.

Publication Date: 2010 Aug 24 PMID: 20696901
Authors: Fomina-Yadlin, D. - Kubicek, S. - Walpita, D. - Dancik, V. - Hecksher-Sorensen, J. - Bittker, J. A. - Sharifnia, T. - Shamji, A. - Clemons, P. A. - Wagner, B. K. - Schreiber, S. L.
Journal: Proc Natl Acad Sci U S A

High-content screening for small-molecule inducers of insulin expression identified the compound BRD7389, which caused alpha-cells to adopt several morphological and gene expression features of a beta-cell state. Assay-performance profile analysis suggests kinase inhibition as a mechanism of action, and we show that biochemical and cellular inhibition of the RSK kinase family by BRD7389 is likely related to its ability induce a beta-cell-like state. BRD7389 also increases the endocrine cell content and function of donor human pancreatic islets in culture.

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PARK2 deletions occur frequently in sporadic colorectal cancer and accelerate adenoma development in Apc mutant mice.

Publication Date: 2010 Aug 24 PMID: 20696900
Authors: Poulogiannis, G. - McIntyre, R. E. - Dimitriadi, M. - Apps, J. R. - Wilson, C. H. - Ichimura, K. - Luo, F. - Cantley, L. C. - Wyllie, A. H. - Adams, D. J. - Arends, M. J.
Journal: Proc Natl Acad Sci U S A

In 100 primary colorectal carcinomas, we demonstrate by array comparative genomic hybridization (aCGH) that 33% show DNA copy number (DCN) loss involving PARK2, the gene encoding PARKIN, the E3 ubiquitin ligase whose deficiency is responsible for a form of autosomal recessive juvenile parkinsonism. PARK2 is located on chromosome 6 (at 6q25-27), a chromosome with one of the lowest overall frequencies of DNA copy number alterations recorded in colorectal cancers. The PARK2 deletions are mostly focal (31% approximately 0.5 Mb on average), heterozygous, and show maximum incidence in exons 3 and 4. As PARK2 lies within FRA6E, a large common fragile site, it has been argued that the observed DCN losses in PARK2 in cancer may represent merely the result of enforced replication of locally vulnerable DNA. However, we show that deficiency in expression of PARK2 is significantly associated with adenomatous polyposis coli (APC) deficiency in human colorectal cancer. Evidence of some PARK2 mutations and promoter hypermethylation is described. PARK2 overexpression inhibits cell proliferation in vitro. Moreover, interbreeding of Park2 heterozygous knockout mice with Apc(Min) mice resulted in a dramatic acceleration of intestinal adenoma development and increased polyp multiplicity. We conclude that PARK2 is a tumor suppressor gene whose haploinsufficiency cooperates with mutant APC in colorectal carcinogenesis.

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TCF4 and CDX2, major transcription factors for intestinal function, converge on the same cis-regulatory regions.

Publication Date: 2010 Aug 24 PMID: 20696899
Authors: Verzi, M. P. - Hatzis, P. - Sulahian, R. - Philips, J. - Schuijers, J. - Shin, H. - Freed, E. - Lynch, J. P. - Dang, D. T. - Brown, M. - Clevers, H. - Liu, X. S. - Shivdasani, R. A.
Journal: Proc Natl Acad Sci U S A

Surprisingly few pathways signal between cells, raising questions about mechanisms for tissue-specific responses. In particular, Wnt ligands signal in many mammalian tissues, including the intestinal epithelium, where constitutive signaling causes cancer. Genome-wide analysis of DNA cis-regulatory regions bound by the intestine-restricted transcription factor CDX2 in colonic cells uncovered highly significant overrepresentation of sequences that bind TCF4, a transcriptional effector of intestinal Wnt signaling. Chromatin immunoprecipitation confirmed TCF4 occupancy at most such sites and co-occupancy of CDX2 and TCF4 across short distances. A region spanning the single nucleotide polymorphism rs6983267, which lies within a MYC enhancer and confers colorectal cancer risk in humans, represented one of many co-occupied sites. Co-occupancy correlated with intestine-specific gene expression and CDX2 loss reduced TCF4 binding. These results implicate CDX2 in directing TCF4 binding in intestinal cells. Co-occupancy of regulatory regions by signal-effector and tissue-restricted transcription factors may represent a general mechanism for ubiquitous signaling pathways to achieve tissue-specific outcomes.

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Sleep homeostasis in the rat is preserved during chronic sleep restriction.

Publication Date: 2010 Aug 9 PMID: 20696898
Authors: Leemburg, S. - Vyazovskiy, V. V. - Olcese, U. - Bassetti, C. L. - Tononi, G. - Cirelli, C.
Journal: Proc Natl Acad Sci U S A

Sleep is homeostatically regulated in all animal species that have been carefully studied so far. The best characterized marker of sleep homeostasis is slow wave activity (SWA), the EEG power between 0.5 and 4 Hz during nonrapid eye movement (NREM) sleep. SWA reflects the accumulation of sleep pressure as a function of duration and/or intensity of prior wake: it increases after spontaneous wake and short-term (3-24 h) sleep deprivation and decreases during sleep. However, recent evidence suggests that during chronic sleep restriction (SR) sleep may be regulated by both allostatic and homeostatic mechanisms. Here, we performed continuous, almost completely artifact-free EEG recordings from frontal, parietal, and occipital cortex in freely moving rats (n = 11) during and after 5 d of SR. During SR, rats were allowed to sleep during the first 4 h of the light period (4S(+)) but not during the following 20 h (20S(-)). During the daily 20S(-) most sleep was prevented, whereas the number of short (<20 s) sleep attempts increased. Low-frequency EEG power (1-6 Hz) in both sleep and wake also increased during 20S(-), most notably in the occipital cortex. In all animals NREM SWA increased above baseline levels during the 4S(+) periods and in post-SR recovery. The SWA increase was more pronounced in frontal cortex, and its magnitude was determined by the efficiency of SR. Analysis of cumulative slow wave energy demonstrated that the loss of SWA during SR was compensated by the end of the second recovery day. Thus, the homeostatic regulation of sleep is preserved under conditions of chronic SR.

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Magnetic resonance monitoring of focused ultrasound/magnetic nanoparticle targeting delivery of therapeutic agents to the brain.

Publication Date: 2010 Aug 24 PMID: 20696897
Authors: Liu, H. L. - Hua, M. Y. - Yang, H. W. - Huang, C. Y. - Chu, P. C. - Wu, J. S. - Tseng, I. C. - Wang, J. J. - Yen, T. C. - Chen, P. Y. - Wei, K. C.
Journal: Proc Natl Acad Sci U S A

The superparamagnetic properties of magnetic nanoparticles (MNPs) allow them to be guided by an externally positioned magnet and also provide contrast for MRI. However, their therapeutic use in treating CNS pathologies in vivo is limited by insufficient local accumulation and retention resulting from their inability to traverse biological barriers. The combined use of focused ultrasound and magnetic targeting synergistically delivers therapeutic MNPs across the blood-brain barrier to enter the brain both passively and actively. Therapeutic MNPs were characterized and evaluated both in vitro and in vivo, and MRI was used to monitor and quantify their distribution in vivo. The technique could be used in normal brains or in those with tumors, and significantly increased the deposition of therapeutic MNPs in brains with intact or compromised blood-brain barriers. Synergistic targeting and image monitoring are powerful techniques for the delivery of macromolecular chemotherapeutic agents into the CNS under the guidance of MRI.

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Tracing the protectors path from the germ line to the genome.

Publication Date: 2010 Aug 31 PMID: 20696896
Authors: Coutandin, D. - Der Ou, H. - Lohr, F. - Dotsch, V.
Journal: Proc Natl Acad Sci U S A

One of the basic principles that nature uses in evolution is to recycle successful concepts and create new functions by modifying existing units. This conservatism in evolution has resulted in an astonishingly high sequence identity of genes, even between evolutionarily distant species such as the nematode Caenorhabditis elegans and Homo sapiens. The recycling of successful concepts in conjunction with gene duplication events has also led to the existence of highly homologous proteins within the genome of many species. Often, these homologous proteins show similar, yet distinct functions that, in combination with their individual tissue distribution, define their specific physiological role. One prominent example is the p53 protein family, which consists of p53, p63, and p73. Recent advances in understanding the specific biological functions of these members have shed some light onto the evolution of this crucial protein family, from a germ line-specific quality-control factor to a somatic tumor suppressor. Furthermore, structures of the oligomerization domains of the mammalian paralogs, p53 and p73, and invertebrate orthologs, CEP-1 and DMP53, have delineated evolutionary changes and revealed that the oligomerization domain of p53 lacks additional stabilizing structural elements present in all other p53 family members. This suggests that p53 is the most recent evolutionary member of this protein family and predicts a mechanism for p53 activation.

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Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field.

Publication Date: 2010 Aug 24 PMID: 20696895
Authors: Dunse, K. M. - Stevens, J. A. - Lay, F. T. - Gaspar, Y. M. - Heath, R. L. - Anderson, M. A.
Journal: Proc Natl Acad Sci U S A

Potato type I and II serine protease inhibitors are produced by solanaceous plants as a defense mechanism against insects and microbes. Nicotiana alata proteinase inhibitor (NaPI) is a multidomain potato type II inhibitor (pin II) that is produced at high levels in the female reproductive tissues of the ornamental tobacco, Nicotiana alata. The individual inhibitory domains of NaPI target the major classes of digestive enzymes, trypsin and chymotrypsin, in the gut of lepidopteran larval pests. Although consumption of NaPI dramatically reduced the growth and development of a major insect pest, Helicoverpa punctigera, we discovered that surviving larvae had high levels of chymotrypsin activity resistant to inhibition by NaPI. We found a potato type I inhibitor, Solanum tuberosum potato type I inhibitor (StPin1A), was a strong inhibitor of the NaPI-resistant chymotrypsin activity. The combined inhibitory effect of NaPI and StPin1A on H. armigera larval growth in the laboratory was reflected in the increased yield of cotton bolls in field trials of transgenic plants expressing both inhibitors. Better crop protection thus is achieved using combinations of inhibitors in which one class of proteinase inhibitor is used to match the genetic capacity of an insect to adapt to a second class of proteinase inhibitor.

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Clustering of dispersed ribosomal DNA and its role in gene regulation and chromosome-end associations in malaria parasites.

Publication Date: 2010 Aug 24 PMID: 20696894
Authors: Mancio-Silva, L. - Zhang, Q. - Scheidig-Benatar, C. - Scherf, A.
Journal: Proc Natl Acad Sci U S A

Dynamic changes in gene positioning contribute to differential expression of virulence-related gene families in protozoan pathogens; however, the role of nuclear architecture in gene expression in the human malaria parasite Plasmodium falciparum remains poorly understood. Here we investigated the developmentally regulated ribosomal RNA (rRNA) gene family in P. falciparum, which, unlike that in most eukaryotes, contains only a few unlinked copies of rRNA genes scattered over the subtelomeric regions of several chromosomes. We show that active and silent members of this gene family cluster in a single perinuclear nucleolus. This rDNA nuclear confinement is DNA sequence dependent, as plasmids carrying rDNA fragments are targeted to the nucleolus. Likewise, insertion of an rDNA sequence into a subtelomere from a chromosome lacking rRNA genes leads to repositioning in the nucleolus. Furthermore, we observed that rDNA spatial organization restricted interchromosomal interactions, as chromosome end-bearing rRNA genes were found to be preferentially juxtaposed, demonstrating nonrandom association of telomeres. Using Br-UTP incorporation, we observed two alpha-amanitin-resistant nucleolar transcription sites that disappeared when the rDNA cluster broke up in the replicative blood stages. Taken together, our results provide conceptual insights into functionally differentiated nuclear territories and their role in gene expression in malaria parasites.

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From the Cover: Roles for the transcription elongation factor NusA in both DNA repair and damage tolerance pathways in Escherichia coli.

Publication Date: 2010 Aug 31 PMID: 20696893
Authors: Cohen, S. E. - Lewis, C. A. - Mooney, R. A. - Kohanski, M. A. - Collins, J. J. - Landick, R. - Walker, G. C.
Journal: Proc Natl Acad Sci U S A

We report observations suggesting that the transcription elongation factor NusA promotes a previously unrecognized class of transcription-coupled repair (TCR) in addition to its previously proposed role in recruiting translesion synthesis (TLS) DNA polymerases to gaps encountered during transcription. Earlier, we reported that NusA physically and genetically interacts with the TLS DNA polymerase DinB (DNA pol IV). We find that Escherichia coli nusA11(ts) mutant strains, at the permissive temperature, are highly sensitive to nitrofurazone (NFZ) and 4-nitroquinolone-1-oxide but not to UV radiation. Gene expression profiling suggests that this sensitivity is unlikely to be due to an indirect effect on gene expression affecting a known DNA repair or damage tolerance pathway. We demonstrate that an N(2)-furfuryl-dG (N(2)-f-dG) lesion, a structural analog of the principal lesion generated by NFZ, blocks transcription by E. coli RNA polymerase (RNAP) when present in the transcribed strand, but not when present in the nontranscribed strand. Our genetic analysis suggests that NusA participates in a nucleotide excision repair (NER)-dependent process to promote NFZ resistance. We provide evidence that transcription plays a role in the repair of NFZ-induced lesions through the isolation of RNAP mutants that display altered ability to survive NFZ exposure. We propose that NusA participates in an alternative class of TCR involved in the identification and removal of a class of lesion, such as the N(2)-f-dG lesion, which are accurately and efficiently bypassed by DinB in addition to recruiting DinB for TLS at gaps encountered by RNAP.

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Hypothesis-driven structural connectivity analysis supports network over hierarchical model of brain architecture.

Publication Date: 2010 Aug 24 PMID: 20696892
Authors: Thompson, R. H. - Swanson, L. W.
Journal: Proc Natl Acad Sci U S A

The brain is usually described as hierarchically organized, although an alternative network model has been proposed. To help distinguish between these two fundamentally different structure-function hypotheses, we developed an experimental circuit-tracing strategy that can be applied to any starting point in the nervous system and then systematically expanded, and applied it to a previously obscure dorsomedial corner of the nucleus accumbens identified functionally as a "hedonic hot spot." A highly topographically organized set of connections involving expected and unexpected gray matter regions was identified that prominently features regions associated with appetite, stress, and clinical depression. These connections are arranged as a longitudinal series of circuits (closed loops). Thus, the results do not support a rigidly hierarchical model of nervous system organization but instead indicate a network model of organization. In principle, the double-coinjection circuit tracing strategy can be applied systematically to the rest of the nervous system to establish the architecture of the global structural wiring diagram, and its abstraction, the connectome.

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Mechanisms of estrogen receptor antagonism toward p53 and its implications in breast cancer therapeutic response and stem cell regulation.

Publication Date: 2010 Aug 24 PMID: 20696891
Authors: Konduri, S. D. - Medisetty, R. - Liu, W. - Kaipparettu, B. A. - Srivastava, P. - Brauch, H. - Fritz, P. - Swetzig, W. M. - Gardner, A. E. - Khan, S. A. - Das, G. M.
Journal: Proc Natl Acad Sci U S A

Estrogen receptor alpha (ERalpha) plays an important role in the onset and progression of breast cancer, whereas p53 functions as a major tumor suppressor. We previously reported that ERalpha binds to p53, resulting in inhibition of transcriptional regulation by p53. Here, we report on the molecular mechanisms by which ERalpha suppresses p53's transactivation function. Sequential ChIP assays demonstrated that ERalpha represses p53-mediated transcriptional activation in human breast cancer cells by recruiting nuclear receptor corepressors (NCoR and SMRT) and histone deacetylase 1 (HDAC1). RNAi-mediated down-regulation of NCoR resulted in increased endogenous expression of the cyclin-dependent kinase (CDK)-inhibitor p21(Waf1/Cip1) (CDKN1A) gene, a prototypic transcriptional target of p53. While 17beta-estradiol (E2) enhanced ERalpha binding to p53 and inhibited p21 transcription, antiestrogens decreased ERalpha recruitment and induced transcription. The effects of estrogen and antiestrogens on p21 transcription were diametrically opposite to their known effects on the conventional ERE-containing ERalpha target gene, pS2/TFF1. These results suggest that ERalpha uses dual strategies to promote abnormal cellular proliferation: enhancing the transcription of ERE-containing proproliferative genes and repressing the transcription of p53-responsive antiproliferative genes. Importantly, ERalpha binds to p53 and inhibits transcriptional activation by p53 in stem/progenitor cell-containing murine mammospheres, suggesting a potential role for the ER-p53 interaction in mammary tissue homeostasis and cancer formation. Furthermore, retrospective studies analyzing response to tamoxifen therapy in a subset of patients with ER-positive breast cancer expressing either wild-type or mutant p53 suggest that the presence of wild-type p53 is an important determinant of positive therapeutic response.

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