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  • Articles  (237)
  • Inaugural Articles  (156)
  • QnAs  (63)
  • Chemical Physics of Protein Folding Special Feature  (18)
  • National Academy of Sciences  (237)
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  • 2010-2014  (237)
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  • 1
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    National Academy of Sciences
    Publication Date: 2013-09-11
    Description: In a classic psychological experiment performed in the late 1960s, animals in one group could stop administered shocks by pressing a lever, whereas those in another group could not stop the shocks despite pressing the lever. Later, when the powerless animals were eventually presented with a working lever, they didn’t...
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  • 2
    Publication Date: 2013-09-25
    Description: Chaperone–usher pathway pili are a widespread family of extracellular, Gram-negative bacterial fibers with important roles in bacterial pathogenesis. Type 1 pili are important virulence factors in uropathogenic Escherichia coli (UPEC), which cause the majority of urinary tract infections (UTI). FimH, the type 1 adhesin, binds mannosylated glycoproteins on the surface...
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  • 3
    Publication Date: 2013-09-11
    Description: Past research found that the ingestion of glucose can enhance self-control. It has been widely assumed that basic physiological processes underlie this effect. We hypothesized that the effect of glucose also depends on people’s theories about willpower. Three experiments, both measuring (experiment 1) and manipulating (experiments 2 and 3) theories...
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  • 4
    Publication Date: 2013-10-02
    Description: It is generally accepted that visual perception results from the activation of a feed-forward hierarchy of areas, leading to increasingly complex representations. Here we present evidence for a fundamental role of backward projections to the occipito-temporal region for understanding conceptual object properties. The evidence is based on two studies. In...
    Keywords: Inaugural Articles
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  • 5
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    National Academy of Sciences
    Publication Date: 2013-06-12
    Description: The factors underlying obesity are multifaceted, but recent research suggests that the brain’s melanocortin circuits, which play a key role in controlling the balance between energy consumption and use, lie at the heart of obesity. According to Roger D. Cone, elected to the National Academy of Sciences in 2010 and...
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  • 6
    Publication Date: 2014-11-05
    Description: The roles of Argonaute proteins in cytoplasmic microRNA and RNAi pathways are well established. However, their implication in small RNA-mediated transcriptional gene silencing in the mammalian cell nucleus is less understood. We have recently shown that intronic siRNAs cause chromatin modifications that inhibit RNA polymerase II elongation and modulate alternative...
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  • 7
    Publication Date: 2011-06-22
    Description: Spatiotemporal changes in gene expression underlie many evolutionary novelties in nature. However, the evolutionary origins of novel expression patterns, and the transcriptional control elements (“enhancers”) that govern them, remain unclear. Here, we sought to explore the molecular genetic mechanisms by which new enhancers arise. We undertook a survey of closely related Drosophila species to identify recently evolved novel gene expression patterns and traced their evolutionary history. Analyses of gene expression in a variety of developing tissues of the Drosophila melanogaster species subgroup revealed high rates of expression pattern divergence, including numerous evolutionary losses, heterochronic shifts, and expansions or contractions of expression domains. However, gains of novel expression patterns were much less frequent. One gain was observed for the Neprilysin-1 (Nep1) gene, which has evolved a unique expression pattern in optic lobe neuroblasts of Drosophila santomea. Dissection of the Nep1 cis-regulatory region localized a newly derived optic lobe enhancer activity to a region of an intron that has accumulated a small number of mutations. The Nep1 optic lobe enhancer overlaps with other enhancer activities, from which the novel activity was co-opted. We suggest that the novel optic lobe enhancer evolved by exploiting the cryptic activity of extant regulatory sequences, and this may reflect a general mechanism whereby new enhancers evolve.
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  • 8
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    National Academy of Sciences
    Publication Date: 2011-06-08
    Description: Molecular biologist Leroy Hood says a transformation in medicine is gingerly afoot. The change, which he calls P4 medicine (predictive, preventive, personalized, and participatory), could turn today’s reactive approach to medicine into a proactive one in the future. Hood, president of the Institute for Systems Biology in Seattle, Washington, is no stranger to scientific revolutions. In the 1980s Hood helped invent automated DNA and protein synthesizers and sequencers, which made the Human Genome Project possible. Those technological feats snagged Hood many laurels, including the Kyoto Prize and the Lemelson-MIT Prize for Invention and Innovation. Hood tells PNAS how systems biology—the...
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  • 9
    Publication Date: 2011-06-15
    Description: The ability to derive meaning from complex sensory input requires the integration of information over space and time, as well as cognitive mechanisms to shape that integration. We studied these processes in the primary visual cortex (V1), where neurons are thought to integrate visual inputs along contours defined by an association field (AF). We recorded extracellularly from single cells in macaque V1 to map the AF, by using an optimization algorithm to find the contours that maximally activated individual cells. We combined the algorithm with a delayed-match-to-sample task, to test how the optimal contours might be molded by the monkey's expectation for particular cue shapes. We found that V1 neurons were selective for complex shapes, a property previously ascribed to higher cortical areas. Furthermore, the shape selectivity was reprogrammed by perceptual task: Over the whole network, the optimal modes of geometric selectivity shifted between distinct subsets of the AF, alternately representing different stimulus features known to predominate in natural scenes. Our results suggest a general model of cortical function, whereby horizontal connections provide a broad domain of potential associations, and top–down inputs dynamically gate these linkages to task switch the function of a network.
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  • 10
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    National Academy of Sciences
    Publication Date: 2011-08-03
    Description: Insects and plants often share a complicated relationship, and University of Illinois, Urbana–Champaign entomologist May Berenbaum has a fine understanding of its chemistry. Berenbaum, a member of the National Academy of Sciences, has long studied how insects and plants evolve chemical arsenals to survive together, pitting cunning defense against toxic offense. Author of a number of popular science books on coevolution, Berenbaum won the 2011 Tyler Prize for Environmental Achievement for her contributions to entomology. Among her many pursuits is an exploration of the likely cause of honey bee die-offs across the United States, an affliction called colony collapse disorder....
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  • 11
    Publication Date: 2011-08-03
    Description: For a daughter cell to receive a complete genomic complement, it is essential that the mitotic spindle be positioned accurately within the cell. In budding yeast, a signaling system known as the spindle position checkpoint (SPOC) monitors spindle position and regulates the activity of the mitotic exit network (MEN), a GTPase signaling pathway that promotes exit from mitosis. The protein kinase Kin4 is a central component of the spindle position checkpoint. Kin4 primarily localizes to the mother cell and associates with spindle pole bodies (SPBs) located in the mother cell to inhibit MEN signaling. In contrast, the kinase does not associate with the SPB in the bud. Thus, only when a MEN bearing SPB leaves the mother cell and the spindle is accurately positioned along the mother–bud axis can MEN signaling occur and cell division proceed. Here, we describe a mechanism ensuring that Kin4 only associates with mother cell-located SPBs. The bud-localized MEN regulator Lte1, whose molecular function has long been unclear, prevents Kin4 that escapes into the bud from associating with SPBs in the daughter cell.
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  • 12
    Publication Date: 2011-10-05
    Description: Synaptic cell adhesion molecules, including the neurexin ligands, neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs), are thought to organize synapse assembly and specify synapse function. To test the synaptic role of these molecules in vivo, we performed lentivirally mediated knockdown of NL3, LRRTM1, and LRRTM2 in CA1 pyramidal cells of WT and NL1 KO mice at postnatal day (P)0 (when synapses are forming) and P21 (when synapses are largely mature). P0 knockdown of NL3 in WT or NL1 KO neurons did not affect excitatory synaptic transmission, whereas P0 knockdown of LRRTM1 and LRRTM2 selectively reduced AMPA receptor-mediated synaptic currents. P0 triple knockdown of NL3 and both LRRTMs in NL1 KO mice yielded greater reductions in AMPA and NMDA receptor-mediated currents, suggesting functional redundancy between NLs and LRRTMs during early synapse development. In contrast, P21 knockdown of LRRTMs did not alter excitatory transmission, whereas NL manipulations supported a role for NL1 in maintaining NMDA receptor-mediated transmission. These results show that neurexin ligands in vivo form a dynamic synaptic cell adhesion network, with compensation between NLs and LRRTMs during early synapse development and functional divergence upon synapse maturation.
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  • 13
    Publication Date: 2011-12-07
    Description: The exceptionally high temperature sensitivity of certain transient receptor potential (TRP) family ion channels is the molecular basis of hot and cold sensation in sensory neurons. The laws of thermodynamics dictate that opening of these specialized TRP channels must involve an unusually large conformational standard-state enthalpy, ΔHo: positive ΔHo for heat-activated and negative ΔHo for cold-activated TRPs. However, the molecular source of such high-enthalpy changes has eluded neurobiologists and biophysicists. Here we offer a general, unifying mechanism for both hot and cold activation that recalls long-appreciated principles of protein folding. We suggest that TRP channel gating is accompanied by large changes in molar heat capacity, ΔCP. This postulate, along with the laws of thermodynamics and independent of mechanistic detail, leads to the conclusion that hot- and cold-sensing TRPs operate by identical conformational changes.
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  • 14
    Publication Date: 2011-12-07
    Description: Listeria monocytogenes has, in 25 y, become a model in infection biology. Through the analysis of both its saprophytic life and infectious process, new concepts in microbiology, cell biology, and pathogenesis have been discovered. This review will update our knowledge on this intracellular pathogen and highlight the most recent breakthroughs. Promising areas of investigation such as the increasingly recognized relevance for the infectious process, of RNA-mediated regulations in the bacterium, and the role of bacterially controlled posttranslational and epigenetic modifications in the host will also be discussed.
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  • 15
    Publication Date: 2012-02-08
    Description: The atmospheric and deep sea reservoirs of carbon dioxide are linked via physical, chemical, and biological processes. The last of these include photosynthesis, particle settling, and organic matter remineralization, and are collectively termed the “biological carbon pump.” Herein, we present results from a 13-y (1992–2004) sediment trap experiment conducted in the permanently oligotrophic North Pacific Subtropical Gyre that document a large, rapid, and predictable summertime (July 15–August 15) pulse in particulate matter export to the deep sea (4,000 m). Peak daily fluxes of particulate matter during the summer export pulse (SEP) average 408, 283, 24.1, 1.1, and 67.5 μmol·m−2·d−1 for total carbon, organic carbon, nitrogen, phosphorus (PP), and biogenic silica, respectively. The SEP is approximately threefold greater than mean wintertime particle fluxes and fuels more efficient carbon sequestration because of low remineralization during downward transit that leads to elevated total carbon/PP and organic carbon/PP particle stoichiometry (371:1 and 250:1, respectively). Our long-term observations suggest that seasonal changes in the microbial assemblage, namely, summertime increases in the biomass and productivity of symbiotic nitrogen-fixing cyanobacteria in association with diatoms, are the main cause of the prominent SEP. The recurrent SEP is enigmatic because it is focused in time despite the absence of any obvious predictable stimulus or habitat condition. We hypothesize that changes in day length (photoperiodism) may be an important environmental cue to initiate aggregation and subsequent export of organic matter to the deep sea.
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  • 16
    Publication Date: 2012-12-19
    Description: Autophagy, a cytoplasmic catabolic process, plays a critical role in defense against intracellular infection. In turn, evasion or inhibition of autophagy has emerged as an important virulence factor for intracellular pathogens. However, Anaplasma phagocytophilum, the obligatory intracellular bacterium that causes human granulocytic anaplasmosis, replicates in the membrane-bound compartment resembling early...
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  • 17
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    National Academy of Sciences
    Publication Date: 2013-01-16
    Description: The moment a bacterium invades a human cell, the immune system triggers a series of events meant to destroy the pathogen and display the remnants like victory flags on the cell’s surface. National Academy of Sciences member Ralph R. Isberg, a professor of molecular biology and microbiology at Tufts University...
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  • 18
    Publication Date: 2013-01-16
    Description: We investigate experimentally and theoretically the sequence of phases that occurs when a self-assembled monolayer of gold nanoparticles supported on a fluid is compressed uniaxially in a Langmuir trough. Uniaxial compression of the monolayer results in the appearance of lines that have been shown to be regions of trilayer. These...
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  • 19
    Publication Date: 2013-02-06
    Description: Using our newly developed explicit three-body (E3B) water model, we simulate the surface of liquid water. We find that the timescale for hydrogen-bond switching dynamics at the surface is about three times slower than that in the bulk. In contrast, with this model rotational dynamics are slightly faster at the...
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  • 20
    Publication Date: 2012-11-08
    Description: During cardiogenesis, Fibroblast Growth Factor (Fgf10) is expressed in the anterior second heart field. Together with Fibroblast growth factor 8 (Fgf8), Fgf10 promotes the proliferation of these cardiac progenitor cells that form the arterial pole of the heart. We have identified a 1.7-kb region in the first intron of Fgf10...
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  • 21
    Publication Date: 2012-10-10
    Description: In a pioneer experiment, Bohlein et al. realized the controlled sliding of two-dimensional colloidal crystals over laser-generated periodic or quasi-periodic potentials. Here we present realistic simulations and arguments that besides reproducing the main experimentally observed features give a first theoretical demonstration of the potential impact of colloid sliding in nanotribology....
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  • 22
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    National Academy of Sciences
    Publication Date: 2012-10-03
    Description: Expertise, transparency, impartiality, appropriateness, confidentiality, and integrity: Those are the guiding principles of scientific merit review espoused by a recent global summit hosted by the National Science Foundation (NSF). Led by the federal agency’s director Subra Suresh, former dean of engineering at Massachusetts Institute of Technology and a newly elected...
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  • 23
    Publication Date: 2012-08-22
    Description: Few terrestrial localities preserve more than a trace lithic record prior to ca. 3.8 Ga greatly limiting our understanding of the first 700 Ma of Earth history, a period inferred to have included a spike in the bolide flux to the inner solar system at ca. 3.85–3.95 Ga (the Late Heavy Bombardment, LHB)....
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  • 24
    Publication Date: 2012-04-11
    Description: Developmental signaling networks are composed of dozens of components whose interactions are very difficult to quantify in an embryo. Geometric reasoning enumerates a discrete hierarchy of phenotypic models with a few composite variables whose parameters may be defined by in vivo data. Vulval development in the nematode Caenorhabditis elegans is a classic model for the integration of two signaling pathways; induction by EGF and lateral signaling through Notch. Existing data for the relative probabilities of the three possible terminal cell types in diverse genetic backgrounds as well as timed ablation of the inductive signal favor one geometric model and suffice to fit most of its parameters. The model is fully dynamic and encompasses both signaling and commitment. It then predicts the correlated cell fate probabilities for a cross between any two backgrounds/conditions. The two signaling pathways are combined additively, without interactions, and epistasis only arises from the nonlinear dynamical flow in the landscape defined by the geometric model. In this way, the model quantitatively fits genetic experiments purporting to show mutual pathway repression. The model quantifies the contributions of extrinsic vs. intrinsic sources of noise in the penetrance of mutant phenotypes in signaling hypomorphs and explains available experiments with no additional parameters. Data for anchor cell ablation fix the parameters needed to define Notch autocrine signaling.
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  • 25
    Publication Date: 2013-02-13
    Description: Recent experiments have shown that spreading epithelial sheets exhibit a long-range coordination of motility forces that leads to a buildup of tension in the tissue, which may enhance cell division and the speed of wound healing. Furthermore, the edges of these epithelial sheets commonly show finger-like protrusions whereas the bulk...
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  • 26
    Publication Date: 2012-09-05
    Description: Genomes of RNA viruses contain multiple functional RNA elements required for translation or RNA replication. We use unique approaches to identify functional RNA elements in the coding sequence of poliovirus (PV), a plus strand RNA virus. The general method is to recode large segments of the genome using synonymous codons,...
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  • 27
    Publication Date: 2012-08-29
    Description: Projections of countries’ future populations, broken down by age and sex, are widely used for planning and research. They are mostly done deterministically, but there is a widespread need for probabilistic projections. We propose a Bayesian method for probabilistic population projections for all countries. The total fertility rate and female...
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  • 28
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    National Academy of Sciences
    Publication Date: 2012-08-22
    Description: As an aspiring young biologist in the 1960s, Baldomero Olivera left Stanford University to return to his native Philippines, finding a laboratory devoid of equipment. While applying for research funding, Olivera decided to study something local and inexpensive, recalling from his childhood that certain cone snails use a harpoon-like tooth to inject prey with a deadly paralyzing venom. On a whim, Olivera decided to purify cone snail venom to see how it works. That off-the-cuff decision spawned a surprisingly successful career. Olivera, now a distinguished professor of biology at the University of Utah (Salt Lake City, UT) and recently elected...
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  • 29
    Publication Date: 2012-07-25
    Description: Rapid advances in DNA sequencing promise to enable new diagnostics and individualized therapies. Achieving personalized medicine, however, will require extensive research on highly reidentifiable, integrated datasets of genomic and health information. To assist with this, participants in the Personal Genome Project choose to forgo privacy via our institutional review board- approved “open consent” process. The contribution of public data and samples facilitates both scientific discovery and standardization of methods. We present our findings after enrollment of more than 1,800 participants, including whole-genome sequencing of 10 pilot participant genomes (the PGP-10). We introduce the Genome-Environment-Trait Evidence (GET-Evidence) system. This tool automatically processes genomes and prioritizes both published and novel variants for interpretation. In the process of reviewing the presumed healthy PGP-10 genomes, we find numerous literature references implying serious disease. Although it is sometimes impossible to rule out a late-onset effect, stringent evidence requirements can address the high rate of incidental findings. To that end we develop a peer production system for recording and organizing variant evaluations according to standard evidence guidelines, creating a public forum for reaching consensus on interpretation of clinically relevant variants. Genome analysis becomes a two-step process: using a prioritized list to record variant evaluations, then automatically sorting reviewed variants using these annotations. Genome data, health and trait information, participant samples, and variant interpretations are all shared in the public domain—we invite others to review our results using our participant samples and contribute to our interpretations. We offer our public resource and methods to further personalized medical research.
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  • 30
    Publication Date: 2012-09-12
    Description: Charles F. Stevens applies the methods of theoretical physics, molecular biology, and anatomy to answer fundamental questions about the brain. A professor of neurobiology at the Salk Institute for Biological Studies and a member of the National Academy of Sciences since 1982, Stevens has helped unravel the molecular details of...
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  • 31
    Publication Date: 2012-09-12
    Description: We summarize, in this review, the evidence that genomic balance influences gene expression, quantitative traits, dosage compensation, aneuploid syndromes, population dynamics of copy number variants and differential evolutionary fate of genes after partial or whole-genome duplication. Gene balance effects are hypothesized to result from stoichiometric differences among members of macromolecular...
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  • 32
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    National Academy of Sciences
    Publication Date: 2012-06-20
    Description: Shu Chien is one of only 13 scholars to belong jointly to the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine—a testament to his expertise in biology, medicine, and engineering and his ability to fuse the three fields in his research. A professor of bioengineering and medicine at the University of California, San Diego (UCSD) and Director of the UCSD Institute of Engineering in Medicine, Chien received the 2011 National Medal of Science, the highest honor bestowed to scientists and engineers by the United States government. His research has contributed greatly to our understanding...
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  • 33
    Publication Date: 2012-06-20
    Description: The theoretical description of the forces that shape ecological communities focuses around two classes of models. In niche theory, deterministic interactions between species, individuals, and the environment are considered the dominant factor, whereas in neutral theory, stochastic forces, such as demographic noise, speciation, and immigration, are dominant. Species abundance distributions predicted by the two classes of theory are difficult to distinguish empirically, making it problematic to deduce ecological dynamics from typical measures of diversity and community structure. Here, we show that the fusion of species abundance data with genome-derived measures of evolutionary distance can provide a clear indication of ecological dynamics, capable of quantifying the relative roles played by niche and neutral forces. We apply this technique to six gastrointestinal microbiomes drawn from three different domesticated vertebrates, using high-resolution surveys of microbial species abundance obtained from carefully curated deep 16S rRNA hypervariable tag sequencing data. Although the species abundance patterns are seemingly well fit by the neutral theory of metacommunity assembly, we show that this theory cannot account for the evolutionary patterns in the genomic data; moreover, our analyses strongly suggest that these microbiomes have, in fact, been assembled through processes that involve a significant nonneutral (niche) contribution. Our results demonstrate that high-resolution genomics can remove the ambiguities of process inference inherent in classic ecological measures and permits quantification of the forces shaping complex microbial communities.
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  • 34
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    National Academy of Sciences
    Publication Date: 2012-06-27
    Description: For his strident advocacy of people-centered conservation, aimed at striking a balance between economic and ecological interests, Peter Kareiva has been often cast as a maverick among environmentalists. Thanks to a growing infusion of science into conservation efforts in the 21st century, Kareiva, elected in 2011 to the National Academy of Sciences, says the environmental movement has come a long way since its birth two centuries ago. As chief scientist at The Nature Conservancy, a bastion for environmental interests, Kareiva helped launch a collaborative endeavor called the Natural Capital Project in 2006 to develop scientific tools to evaluate the costs...
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  • 35
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    National Academy of Sciences
    Publication Date: 2012-05-30
    Description: Nearly half of the world’s population relies on fuels such as wood or dung for cooking and heating. In the 1980s, Kirk R. Smith, a member of the National Academy of Sciences and a professor of global health at the University of California at Berkeley, sounded the alarm that these fuels, when burned in open fires or traditional cook stoves, produce high levels of indoor air pollution that prematurely kill about 2 million people each year—more than either malaria or tuberculosis, according to Smith. Cleaner alternatives to traditional cook stoves exist, but convincing funding agencies and decision makers to invest...
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  • 36
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    National Academy of Sciences
    Publication Date: 2012-06-06
    Description: Rising global demand for food, biofuels, and agricultural commodities such as soybeans and oil palm has driven the clearing of vast swaths of tropical forests to make way for cattle ranches and cropland. According to Columbia University geographer and National Academy of Sciences member Ruth DeFries, intensive high-yield farming is an often-proposed solution to simultaneously conserve tropical forests, increase food production, and reduce greenhouse gas emissions from forest clearing. But whether this strategy works remains unclear. DeFries has long used satellite imagery to examine human transformation of the planet’s landscape. Here, DeFries shares her recent counterintuitive findings from a study...
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  • 37
    Publication Date: 2012-04-18
    Description: Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma.
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  • 38
    Publication Date: 2012-05-30
    Description: At least three pathways control maintenance of DNA cytosine methylation in Arabidopsis thaliana. However, the RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IDN2) as being an RNA-binding RdDM component that is required for DNA methylation establishment. In this study, we describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE 1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis confirms the placement of the IDN proteins in an RdDM pathway that affects DNA methylation and transcriptional control at many sites in the genome. Results from this study identify and describe two unique components of the RdDM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.
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  • 39
    Publication Date: 2012-06-06
    Description: Dendritic cells (DCs) and B cells present antigen-derived peptides bound to MHC class II (MHC II) molecules for recognition by CD4-positive T lymphocytes. DCs control the intracellular traffic of peptide–MHC II complexes by regulating the ubiquitination of MHC II. In resting or “immature” DCs, ubiquitinated MHC II molecules are targeted to lysosomes, but upon pathogen-induced “maturation,” ubiquitination is down-regulated and MHC II can accumulate on the plasma membrane of mature DCs. Although B cells constitutively ubiquitinate their MHC II, it unexpectedly remains at the surface. We find that DCs and B cells differ in MHC II-conjugated ubiquitin (Ub) chain length: four to six Ub in immature DCs vs. two to three in B cells. In both cell types, experimentally increasing Ub chain length led to efficient lysosomal transport of MHC II, whereas MHC II with fewer than two Ubs did not reach lysosomes. Thus, Ub chain length plays a crucial role in regulating the intracellular fate and function of MHC II in DCs and B cells.
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  • 40
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    National Academy of Sciences
    Publication Date: 2012-04-25
    Description: The interplay of light and matter has long preoccupied Stanford University physical chemist W. E. Moerner. As a scientist at IBM Research during the late 1980s, Moerner developed then-novel techniques to configure holograms by altering the light-refracting properties of a polymer. Today, Moerner uses the power of light to probe how individual biological molecules behave within and without the seeming clutter of living cells. Moerner was elected to the National Academy of Sciences in recognition of his work, in particular the optical detection and spectroscopy of single molecules. Those studies opened a window into the nanoscale world of cells, offering...
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  • 41
    Publication Date: 2012-04-25
    Description: The Ca2+/Calmodulin-dependent phosphatase calcineurin is essential for exit from meiotic arrest at metaphases I and II in Drosophila and Xenopus oocytes. We previously found that Sarah, the Drosophila homolog of regulator of calcineurin, acts as a positive regulator of calcineurin and is required to complete anaphase I of female meiosis. Here, we undertook biochemical approaches, including MS and posttranslational modification analyses, to better understand the mechanism by which Sarah regulates calcineurin. A search for phosphorylated residues revealed that Sarah is highly phosphorylated at Ser100, Thr102, and Ser219 in both ovaries and activated eggs and that Ser215 is phosphorylated only in activated eggs. Functional analyses using mutant forms of Sarah showed that phosphorylation at Ser215, a consensus phosphorylation site for glycogen synthase kinase 3β (GSK-3β) and its priming kinase site Ser219, are essential for Sarah function. Furthermore, germ-line clones homozygous for a null allele of shaggy (Drosophila GSK-3β) both fail to complete meiosis and lack phosphorylation of Sarah at Ser215, suggesting that the phosphorylation of Sarah by Shaggy/GSK-3β is required to complete meiosis. Our findings suggest a mechanism in which Shaggy/GSK-3β activates calcineurin through Sarah phosphorylation on egg activation in Drosophila.
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  • 42
    Publication Date: 2012-05-02
    Description: Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein–protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development.
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  • 43
    Publication Date: 2012-05-16
    Description: A rodent model of diet-induced obesity revealed that obesity significantly altered hematopoietic and lymphopoietic functions in the bone marrow and thymus. C57BL/6 mice were fed a mixed high-fat diet (HFD) of 45% fat or 10% fat diet (lean controls) for 180 d. A sustained increase in the numbers of cells found in bone marrow and thymus of HFD mice occurred from day 90 to day 180. However, with the exception of a 10–18% increase in the proportion of lymphocytes, the composition of monocytes, granulocytes, erythrocytes, and mixed progenitor lineages remained normal in the marrow. Likewise, thymuses of HFD mice increased 30–50% in size compared with controls, with analogous increases in thymocyte numbers. The overall thymus cellular composition remained normal. Although increased blood and lymphatic volume in obese mice would play a role in increased hematopoiesis, there were large and disproportionate increases in blood leukocytes of HFD mice, indicating that homeostasis was not maintained. Leptin, which promotes lymphopoiesis and myelopoiesis, reached 100 ng/mL in sera from HFD mice. Moreover, a three- to sixfold increase in adipocytes in marrow resulted in spiked leptin mRNA expression in bones of HFD mice compared with lean controls. Other cytokines and growth factors did not show any increases in obese marrow. The substantial increase in lymphopoietic and hematopoietic processes in HFD mice indicates that the primary tissues are another facet of the immune system dysregulated by obesity, which was perhaps fostered by higher amounts of leptin in marrow and serum.
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  • 44
    Publication Date: 2012-05-09
    Description: The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography–MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.
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  • 45
    Publication Date: 2011-11-23
    Description: In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17β-estradiol (E2) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E2 paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E2-induced apoptosis by analysis of gene expression across time (2–96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E2-induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E2 in 5C cells compared with both WS8 and 2A cells and hence, were associated with E2-induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E2 inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E2-induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E2-inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E2 interacted to superadditively induce apoptosis. Therefore, these data indicate that E2 induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.
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  • 46
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    National Academy of Sciences
    Publication Date: 2011-11-30
    Description: Like good and bad cholesterol, human body fat comes in two varieties: white fat cells, which store excess calories, and brown fat cells, which burn energy to generate body heat. Less well known outside the scientific community, brown fat cells have long been a fascination for National Academy of Sciences member Bruce Spiegelman, a professor of cell biology at Dana–Farber Cancer Institute and Harvard Medical School. Spiegelman’s work on fat metabolism is far-reaching: From finding ways to stimulate brown fat development in the body to unraveling the activity of drugs against diabetes, he has shown how understanding the genetics of...
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  • 47
    Publication Date: 2012-03-07
    Description: For prevention of Streptococcus pneumoniae (pneumococcus) infections in infancy, protein-conjugated capsular polysaccharide vaccines provide serotype-specific, antibody-mediated immunity but do not cover all of the 90+ capsule serotypes. Therefore, microbiologists have sought protective noncapsular antigens common to all strains. Alternatively, we investigated killed cells of a noncapsulated strain, which expose many such common antigens. Given to mice intranasally, this vaccine elicits antibody-independent, CD4+ T lymphocyte-dependent accelerated clearance of pneumococci of various serotypes from the nasopharynx mediated by the cytokine IL-17A. Such immunity may reproduce the natural resistance that develops in infants before capsular antibodies arise. Given by injection, the killed cell vaccine induces bifunctional immunity: plasma antibodies protective against fatal pneumonia challenge, as well as IL-17A–mediated nasopharyngeal clearance. Human testing of this inexpensive candidate vaccine by intramuscular injection is planned. Bacterial cellular vaccines are complex—a challenge for reproducibility. However, when several known protective antigens were deleted, the killed pneumococcal vaccine was still protective. This antigenic redundancy may prevent vaccine escape variants by recombinational loss, which is frequent in pneumococcus. Biochemically defined immunogens with bifunctional activity have also been devised. These immunogens are three-component conjugates in which cell wall teichoic acid (a common antigen capable of T cell activation) is coupled to a genetic fusion of two common pneumococcal proteins: a protective surface antigen and a derivative of pneumolysin, which provides TLR4 agonist activity and induces antitoxic immunity. Such constructs induce accelerated clearance when given intranasally and induce both immune mechanisms when injected. The defined composition permits analysis of structure-function activity.
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  • 48
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    National Academy of Sciences
    Publication Date: 2012-02-29
    Description: In the late 1950s, biochemist Christian Anfinsen demonstrated that the sequence of amino acids in a protein contains all of the information required for the protein to acquire its functional shape. In recent years, researchers have found that many newly minted proteins make an important stop on their journey to the native shape: they bind to one or more helper proteins called molecular chaperones, which prevent the proteins’ exposed surfaces from sticking to one another. Such undesirable molecular association can lead to protein aggregation, a hallmark of many human diseases. Together, German biochemist F. Ulrich Hartl and Yale University geneticist...
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  • 49
    Publication Date: 2012-03-07
    Description: The work of National Academy of Sciences (NAS) member Porter W. Anderson, Jr. has benefitted millions of people around the globe: Anderson has spent his career developing vaccines against some of the leading causes of infection in children. For his contributions to the development and commercialization of the Haemophilus influenzae type b (Hib) vaccine—which has virtually eradicated one of the leading causes of meningitis in preschool-aged children—Anderson shared the 1996 Albert Lasker Clinical Medical Research Award with the late David Smith, Rachel Schneerson, and NAS member John Robbins. Since retiring from the University of Rochester in the late 1990s, Anderson...
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  • 50
    Publication Date: 2012-02-29
    Description: We used in vivo amperometry to monitor changes in synaptic dopamine (DA) release in the striatum induced by overexpression of human wild-type α-synuclein in nigral DA neurons, induced by injection of an adeno-associated virus type 6 (AAV6)–α-synuclein vector unilaterally into the substantia nigra in adult rats. Impairments in DA release evolved in parallel with the development of degenerative changes in the nigrostriatal axons and terminals. The earliest change, seen 10 d after vector injection, was a marked, ≈50%, reduction in DA reuptake, consistent with an early dysfunction of the DA transporter that developed before any overt signs of axonal damage. At 3 wk, when the first signs of axonal damage were observed, the amount of DA released after a KCl pulse was reduced by 70–80%, and peak DA concentration was delayed, indicating an impaired release mechanism. At later time points, 8–16 wk, overall striatal innervation density was reduced by 60–80% and accompanied by abundant signs of axonal damage in the form of α-synuclein aggregates, axonal swellings, and dystrophic axonal profiles. At this stage DA release and reuptake were profoundly reduced, by 80–90%. The early changes in synaptic DA release induced by overexpression of human α-synuclein support the idea that early predegenerative changes in the handling of DA may initiate, and drive, a progressive degenerative process that hits the axons and terminals first. Synaptic dysfunction and axonopathy would thus be the hallmark of presymptomatic and early-stage Parkinson disease, followed by neuronal degeneration and cell loss, characteristic of more advanced stages of the disease.
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  • 51
    Publication Date: 2012-03-07
    Description: The present study explores the dramatic projection of one's own views onto those of Jesus among conservative and liberal American Christians. In a large-scale survey, the relevant views that each group attributed to a contemporary Jesus differed almost as much as their own views. Despite such dissonance-reducing projection, however, conservatives acknowledged the relevant discrepancy with regard to “fellowship” issues (e.g., taxation to reduce economic inequality and treatment of immigrants) and liberals acknowledged the relevant discrepancy with regard to “morality” issues (e.g., abortion and gay marriage). However, conservatives also claimed that a contemporary Jesus would be even more conservative than themselves on the former issues whereas liberals claimed that Jesus would be even more liberal than themselves on the latter issues. Further reducing potential dissonance, liberal and conservative Christians differed markedly in the types of issues they claimed to be more central to their faith. A concluding discussion considers the relationship between individual motivational processes and more social processes that may underlie the present findings, as well as implications for contemporary social and political conflict.
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  • 52
    Publication Date: 2011-11-02
    Description: What are the physical limits to cell behavior? Often, the physical limitations can be dominated by the proteome, the cell’s complement of proteins. We combine known protein sizes, stabilities, and rates of folding and diffusion, with the known protein-length distributions P(N) of proteomes (Escherichia coli, yeast, and worm), to formulate distributions and scaling relationships in order to address questions of cell physics. Why do mesophilic cells die around 50 °C? How can the maximal growth-rate temperature (around 37 °C) occur so close to the cell-death temperature? The model shows that the cell’s death temperature coincides with a denaturation catastrophe of its proteome. The reason cells can function so well just a few degrees below their death temperature is because proteome denaturation is so cooperative. Why are cells so dense-packed with protein molecules (about 20% by volume)? Cells are packed at a density that maximizes biochemical reaction rates. At lower densities, proteins collide too rarely. At higher densities, proteins diffuse too slowly through the crowded cell. What limits cell sizes and growth rates? Cell growth is limited by rates of protein synthesis, by the folding rates of its slowest proteins, and—for large cells—by the rates of its protein diffusion. Useful insights into cell physics may be obtainable from scaling laws that encapsulate information from protein knowledge bases.
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  • 53
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    National Academy of Sciences
    Publication Date: 2011-09-21
    Description: In late November 2011, the National Aeronautics and Space Administration (NASA) plans to launch its robotic explorer to scour Mars for signs of the planet’s ability to support life. The Mars Science Laboratory (MSL) spacecraft is scheduled to lift off from Cape Canaveral Air Force Station in Florida, shuttling Curiosity, an SUV-sized rover with a hefty scientific payload, to the red planet’s surface. John Grotzinger, a member of the National Academy of Sciences and professor of geology at the California Institute of Technology, helps oversee the mission. He became involved in the quest after studying how changes in the Earth’s...
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  • 54
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    National Academy of Sciences
    Publication Date: 2011-03-09
    Description: At Albert Einstein College of Medicine of Yeshiva University in New York City, Vern Schramm, a professor of biochemistry, devises ways to block cellular enzymes. Those enzymes, which catalyze a range of reactions at the heart of normal physiology, might hold keys to treating psoriasis, autoimmune disease, and some forms of cancer. Schramm’s studies on the mechanism of enzyme-catalyzed reactions have led to a handful of drugs now being tested in clinical trials. In 2009, he developed a test for ricin, a deadly toxin found in castor beans, which has the potential to be used by bioterrorists. Schramm tells PNAS...
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  • 55
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    National Academy of Sciences
    Publication Date: 2011-03-30
    Description: Gary King, a professor of social science at Harvard University and a member of the National Academy of Sciences, fashions tools that harness the power of statistics, machine learning, and informatics to make sense of the numbers that matter to society. From evaluating the efficacy of a Mexican health policy reform to predicting the fate of the US Social Security trust fund, King’s sophisticated number crunching has important practical implications for disciplines as diverse as social, political, and health sciences. King tells PNAS how quantitative social science research can extend from academic journals into real-world scenarios.PNAS:You designed a health policy...
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  • 56
    Publication Date: 2011-01-26
    Description: In Saccharomyces cerevisiae, silent chromatin inhibits the expression of genes at the HML, HMR, and telomeric loci. When silent chromatin forms de novo, the rate of its establishment is influenced by different chromatin states. In particular, loss of the enzyme Dot1, an H3 K79 methyltransferase, leads to rapid silencing establishment. We tested whether silencing establishment was antagonized by H3 K79 methylation or by the Dot1 protein itself competing with Sir3 for binding sites on nucleosomes. To do so, we monitored fluorescence activity in cells containing a GFP gene within the HML locus during silencing establishment in a series of dot1 and histone mutant backgrounds. Silencing establishment rate was correlated with Dot1’s enzymatic function rather than with the Dot1 protein itself. In addition, histone mutants that mimicked the conformation of unmethylated H3 K79 increased the rate of silencing establishment, indicating that the H3 K79 residue affected silencing independently of Dot1 abundance. Using fluorophore-based reporters, we confirmed that mother and daughter cells often silence in concert, but in instances where asymmetric silencing occurs, daughter cells established silencing earlier than their mothers. This noninvasive technique enabled us to demonstrate an asymmetry in silencing establishment of a key regulatory locus controlling cell fate.
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  • 57
    Publication Date: 2011-03-02
    Description: This article describes the state and the development of an artificial cell project. We discuss the experimental constraints to synthesize the most elementary cell-sized compartment that can self-reproduce using synthetic genetic information. The original idea was to program a phospholipid vesicle with DNA. Based on this idea, it was shown that in vitro gene expression could be carried out inside cell-sized synthetic vesicles. It was also shown that a couple of genes could be expressed for a few days inside the vesicles once the exchanges of nutrients with the outside environment were adequately introduced. The development of a cell-free transcription/translation toolbox allows the expression of a large number of genes with multiple transcription factors. As a result, the development of a synthetic DNA program is becoming one of the main hurdles. We discuss the various possibilities to enrich and to replicate this program. Defining a program for self-reproduction remains a difficult question as nongenetic processes, such as molecular self-organization, play an essential and complementary role. The synthesis of a stable compartment with an active interface, one of the critical bottlenecks in the synthesis of artificial cell, depends on the properties of phospholipid membranes. The problem of a self-replicating artificial cell is a long-lasting goal that might imply evolution experiments.
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  • 58
    Publication Date: 2011-03-30
    Description: It is widely believed that functional mammalian microRNA (miRNA) recognition sequences are located preferentially in the 3' untranslated region (3'UTR) of target mRNAs. Nonetheless, putative miRNA target sites within coding regions have been found at lower frequency in genome-wide studies, and several have been genetically validated. To account for these findings, it has been proposed that translation may inhibit miRNA access to target sites. Here we identify a naturally occurring viral miRNA target that, owing to the compact nature of the viral transcriptome, is situated naturally in the coding region of one transcript and in the 3′UTR of an overlapping mRNA. Examination of the expression of these mRNAs reveals that the cognate miRNA can inhibit expression in both contexts, but inhibition is more potent when the target site is in the UTR. Similarly, forced translation of the target site in the UTR diminished, but did not abolish, its down-regulation by the miRNA. These data affirm that miRNAs can exert regulatory effects on targets within coding regions; however, the dampening of these effects by translation likely accounts for the observed selection for target sites in the 3′UTRs.
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  • 59
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    National Academy of Sciences
    Publication Date: 2011-03-16
    Description: Of the world’s mathematicians, Gilbert Strang is possibly the most visible—or at least among the most frequently viewed. Millions of students from the Americas, Africa, China, Europe, India, and Singapore have watched Strang’s lectures on linear algebra courtesy of Massachusetts Institute of Technology (MIT)’s OpenCourseWare Web site (1), and many have e-mailed him to ask for one-on-one help. A former president of the Society for Industrial and Applied Mathematics (SIAM), author of several textbooks (2–9), and 2009 electee to the National Academy of Sciences, Strang wrote the book on linear algebra—and his text has changed how the material is taught....
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  • 60
    Publication Date: 2011-02-02
    Description: Imprinted genes are expressed primarily or exclusively from either the maternal or paternal allele, a phenomenon that occurs in flowering plants and mammals. Flowering plant imprinted gene expression has been described primarily in endosperm, a terminal nutritive tissue consumed by the embryo during seed development or after germination. Imprinted expression in Arabidopsis thaliana endosperm is orchestrated by differences in cytosine DNA methylation between the paternal and maternal genomes as well as by Polycomb group proteins. Currently, only 11 imprinted A. thaliana genes are known. Here, we use extensive sequencing of cDNA libraries to identify 9 paternally expressed and 34 maternally expressed imprinted genes in A. thaliana endosperm that are regulated by the DNA-demethylating glycosylase DEMETER, the DNA methyltransferase MET1, and/or the core Polycomb group protein FIE. These genes encode transcription factors, proteins involved in hormone signaling, components of the ubiquitin protein degradation pathway, regulators of histone and DNA methylation, and small RNA pathway proteins. We also identify maternally expressed genes that may be regulated by unknown mechanisms or deposited from maternal tissues. We did not detect any imprinted genes in the embryo. Our results show that imprinted gene expression is an extensive mechanistically complex phenomenon that likely affects multiple aspects of seed development.
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  • 61
    Publication Date: 2011-02-16
    Description: We develop a computer-assisted method for the discovery of insightful conceptualizations, in the form of clusterings (i.e., partitions) of input objects. Each of the numerous fully automated methods of cluster analysis proposed in statistics, computer science, and biology optimize a different objective function. Almost all are well defined, but how to determine before the fact which one, if any, will partition a given set of objects in an “insightful” or “useful” way for a given user is unknown and difficult, if not logically impossible. We develop a metric space of partitions from all existing cluster analysis methods applied to a given dataset (along with millions of other solutions we add based on combinations of existing clusterings) and enable a user to explore and interact with it and quickly reveal or prompt useful or insightful conceptualizations. In addition, although it is uncommon to do so in unsupervised learning problems, we offer and implement evaluation designs that make our computer-assisted approach vulnerable to being proven suboptimal in specific data types. We demonstrate that our approach facilitates more efficient and insightful discovery of useful information than expert human coders or many existing fully automated methods.
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  • 62
    Publication Date: 2011-01-26
    Description: Argonaute-associated siRNAs and Piwi-associated piRNAs have overlapping roles in silencing mobile genetic elements in animals. In Caenorhabditis elegans, mutator (mut) class genes mediate siRNA-guided repression of transposons as well as exogenous RNAi, but their roles in endogenous RNA silencing pathways are not well-understood. To characterize the endogenous small RNAs dependent on mut class genes, small RNA populations from a null allele of mut-16 as well as a regulatory mut-16(mg461) allele that disables only somatic RNAi were subjected to deep sequencing. Additionally, each of the mut class genes was tested for a requirement in 26G siRNA pathways. The results indicate that mut-16 is an essential factor in multiple endogenous germline and somatic siRNA pathways involving several distinct Argonautes and RNA-dependent RNA polymerases. The results also reveal essential roles for mut-2 and mut-7 in the ERGO-1 class 26G siRNA pathway and less critical roles for mut-8, mut-14, and mut-15. We show that transposons are hypersusceptible to mut-16–dependent silencing and identify a requirement for the siRNA machinery in piRNA biogenesis from Tc1 transposons. We also show that the soma-specific mut-16(mg461) mutant allele is present in multiple C. elegans laboratory strains.
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  • 63
    Publication Date: 2011-07-20
    Description: In organisms, cell-fate decisions result from external cues presented by the extracellular microenvironment or the niche. In principle, synthetic niches can be engineered to give rise to patterned cell signaling, an advance that would transform the fields of tissue engineering and regenerative medicine. Biomaterials that display adhesive motifs are critical steps in this direction, but promoting localized signaling remains a major obstacle. We sought to exert precise spatial control over activation of TGF-β signaling. TGF-β signaling, which plays fundamental roles in development, tissue homeostasis, and cancer, is initiated by receptor oligomerization. We therefore hypothesized that preorganizing the transmembrane receptors would potentiate local TGF-β signaling. To generate surfaces that would nucleate the signaling complex, we employed defined self-assembled monolayers that present peptide ligands to TGF-β receptors. These displays of nondiffusible ligands do not compete with the growth factor but rather sensitize bound cells to subpicomolar concentrations of endogenous TGF-β. Cells adhering to the surfaces undergo TGF-β-mediated growth arrest and the epithelial to mesenchymal transition. Gene expression profiles reveal that the surfaces selectively regulate TGF-β responsive genes. This strategy provides access to tailored surfaces that can deliver signals with spatial control.
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  • 64
    Publication Date: 2011-07-27
    Description: Cohesin is a member of the Smc family of protein complexes that mediates higher-order chromosome structure by tethering different regions of chromatin. We present a new in vitro system that assembles cohesin-DNA complexes with in vivo properties. The assembly of these physiological salt-resistant complexes requires the cohesin holo-complex, its ability to bind ATP, the cohesin loader Scc2p and a closed DNA topology. Both the number of cohesin molecules bound to the DNA substrate and their distribution on the DNA substrate are limited. Cohesin and Scc2p bind preferentially to cohesin associated regions (CARs), DNA sequences with enriched cohesin binding in vivo. A subsequence of CARC1 promotes cohesin binding to neighboring sequences within CARC1. The enhancer-like function of this sequence is validated by in vivo deletion analysis. By demonstrating the physiological relevance of these in vitro assembled cohesin-DNA complexes, we establish our in vitro system as a powerful tool to elucidate the mechanism of cohesin and other Smc complexes.
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  • 65
    Publication Date: 2011-07-20
    Description: The metabolism of a river basin is defined as the set of processes through which the basin maintains its structure and responds to its environment. Green (or biotic) metabolism is measured via transpiration and blue (or abiotic) metabolism through runoff. A principle of equal metabolic rate per unit area throughout the basin structure is developed and tested in a river basin characterized by large heterogeneities in precipitation, vegetation, soil, and geomorphology. This principle is suggested to have profound implications for the spatial organization of river basin hydrologic dynamics, including the minimization of energy expenditure known to control the scale-invariant characteristics of river networks over several orders of magnitude. Empirically derived, remarkably constant rates of average transpiration per unit area through the basin structure lead to a power law for the probability distribution of transpiration from a randomly chosen subbasin. The average runoff per unit area, evaluated for subbasins of a wide range of topological magnitudes, is also shown to be remarkably constant independently of size. A similar result is found for the rainfall after accounting for canopy interception. Allometric scaling of metabolic rates with size, variously addressed in the biological literature and network theory under the label of Kleiber’s law, is similarly derived. The empirical evidence suggests that river basin metabolic activity is linked with the spatial organization that takes place around the drainage network and therefore with the mechanisms responsible for the fractal geometry of the network, suggesting a new coevolutionary framework for biological, geomorphological, and hydrologic dynamics.
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  • 66
    Publication Date: 2011-06-30
    Description: It is well established that p53 contacts DNA in a sequence-dependent manner in order to transactivate its myriad target genes. Yet little is known about how p53 interacts with its binding site/response element (RE) within such genes in vivo in the context of nucleosomal DNA. In this study we demonstrate that both distal (5′) and proximal (3′) p53 REs within the promoter of the p21 gene in unstressed HCT116 colon carcinoma cells are localized within a region of relatively high nucleosome occupancy. In the absence of cellular stress, p53 is prebound to both p21 REs within nucleosomal DNA in these cells. Treatment of cells with the DNA-damaging drug doxorubicin or the p53 stabilizing agent Nutlin-3, however, is accompanied by p53-dependent subsequent loss of nucleosomes associated with such p53 REs. We show that in vitro p53 can bind to mononucleosomal DNA containing the distal p21 RE, provided the binding site is not close to the diad center of the nucleosome. In line with this, our data indicate that the p53 distal RE within the p21 gene is located close to the end of the nucleosome. Thus, low- and high-resolution mapping of nucleosome boundaries around p53 REs within the p21 promoter have provided insight into the mechanism of p53 binding to its sites in cells and the consequent changes in nucleosome occupancy at such sites.
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  • 67
    Publication Date: 2011-04-06
    Description: To maximize energy efficiency, gas turbine engines used in airplanes and for power generation operate at very high temperatures, even above the melting point of the metal alloys from which they are comprised. This feat is accomplished in part via the deposition of a multilayer, multicomponent thermal barrier coating (TBC), which lasts up to approximately 40,000 h before failing. Understanding failure mechanisms can aid in designing circumvention strategies. We review results of quantum mechanics calculations used to test hypotheses about impurities that harm TBCs and transition metal (TM) additives that render TBCs more robust. In particular, we discovered a number of roles that Pt and early TMs such as Hf and Y additives play in extending the lifetime of TBCs. Fundamental insight into the nature of the bonding created by such additives and its effect on high-temperature evolution of the TBCs led to design principles that can be used to create materials for even more efficient engines.
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  • 68
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    National Academy of Sciences
    Publication Date: 2011-04-06
    Description: As the director of the Rosenstiel Basic Medical Sciences Research Center at Brandeis University in Waltham, MA, James Haber seeks to understand how cells repair damaged DNA—and what happens when the process goes awry, triggering the onset of cancer and other devastating diseases. Haber’s work on yeast genetics has earned him some of the highest honors in science, including the Thomas Hunt Morgan Medal for lifetime contributions in genetics and election to the National Academy of Sciences in 2010. Haber recently spoke with PNAS about the technologies his laboratory has used to explore a form of DNA damage known as...
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  • 69
    Publication Date: 2011-02-23
    Description: The ability to induce synchronously a single site-specific double-strand break (DSB) in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during DSB repair. Special attention has been paid to the switching of mating-type genes in Saccharomyces cerevisiae, a process initiated by the HO endonuclease by cleaving the MAT locus. A DSB in MATa is repaired by homologous recombination—specifically, by gene conversion—using a heterochromatic donor, HMLα. Repair results in the replacement of the a-specific sequences (Ya) by Yα and switching from MATa to MATα. We report that MAT switching requires the DNA replication factor Dpb11, although it does not require the Cdc7-Dbf4 kinase or the Mcm and Cdc45 helicase components. Using Southern blot, PCR, and ChIP analysis of samples collected every 10 min, we extend previous studies of this process to identify the times for the loading of Rad51 recombinase protein onto the DSB ends at MAT, the subsequent strand invasion by the Rad51 nucleoprotein filament into the donor sequences, the initiation of new DNA synthesis, and the removal of the nonhomologous Y sequences. In addition we report evidence for the transient displacement of well-positioned nucleosomes in the HML donor locus during strand invasion.
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  • 70
    Publication Date: 2011-01-04
    Description: Is there a Muslim disadvantage in economic integration for second-generation immigrants to Europe? Previous research has failed to isolate the effect that religion may have on an immigrant family's labor market opportunities because other factors, such as country of origin or race, confound the result. This paper uses a correspondence test in the French labor market to identify and measure this religious effect. The results confirm that in the French labor market, anti-Muslim discrimination exists: a Muslim candidate is 2.5 times less likely to receive a job interview callback than is his or her Christian counterpart. A high-n survey reveals, consistent with expectations from the correspondence test, that second-generation Muslim households in France have lower income compared with matched Christian households. The paper thereby contributes to both substantive debates on the Muslim experience in Europe and methodological debates on how to measure discrimination. Following the National Academy of Sciences’ 2001 recommendations on combining a variety of methodologies and applying them to real-world situations, this research identifies, measures, and infers consequences of discrimination based on religious affiliation, controlling for potentially confounding factors, such as race and country of origin.
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  • 71
    Publication Date: 2011-01-04
    Description: The mosquito Aedes aegypti is the major vector of arboviral diseases, particularly of Dengue fever, of which there are more than 100 million cases annually. Mosquitoes, such as A. aegypti, serve as vectors for disease pathogens because they require vertebrate blood for their egg production. Pathogen transmission is tightly linked to repeated cycles of obligatory blood feeding and egg maturation. Thus, the understanding of mechanisms governing egg production is necessary to develop approaches that limit the spread of mosquito-borne diseases. Previous studies have identified critical roles of hormonal- and nutrition-based target of rapamycin (TOR) pathways in controlling blood-meal–mediated egg maturation in mosquitoes. In this work, we uncovered another essential regulator of blood-meal–activated processes, the microRNA miR-275. The depletion of this microRNA in A. aegypti females after injection of its specific antagomir resulted in severe defects in blood digestion, fluid excretion, and egg development, clearly demonstrating that miR-275 is indispensable for these physiological processes. miR-275 exhibits an expression profile that suggests its regulation by a steroid hormone, 20-hydroxyecdysone (20E). In vitro organ culture experiments demonstrated that miR-275 is induced by this hormone in the presence of amino acids, indicative of a dual regulation by 20E and TOR. This report has uncovered the critical importance of microRNAs in controlling blood-meal–activated physiological events required for completion of egg development in mosquito disease vectors.
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  • 72
    Publication Date: 2011-01-05
    Description: The two haploid genome sequences that a person inherits from the two parents represent the most fundamentally useful type of genetic information for the study of heritable diseases and the development of personalized medicine. Because of the difficulty in obtaining long-range phase information, current sequencing methods are unable to provide this information. Here, we introduce and show feasibility of a scalable approach capable of generating genomic sequences completely phased across the entire chromosome.
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  • 73
    Publication Date: 2011-01-19
    Description: Argonaute and Piwi proteins are key players in the RNA silencing pathway, with the former interacting with micro-RNAs (miRNAs) and siRNAs, whereas the latter targets piwi-interacting RNAs (piRNAs) that are 2′-O-methylated (2′-OCH3) at their 3′ ends. Germline-specific piRNAs and Piwi proteins play a critical role in genome defense against transposable elements, thereby protecting the genome against transposon-induced defects in gametogenesis and fertility. Humans contain four Piwi family proteins designated Hiwi1, Hiwi2, Hiwi3, and Hili. We report on the structures of Hili-PAZ (Piwi/Argonaute/Zwille) domain in the free state and Hiwi1 PAZ domain bound to self-complementary 14-mer RNAs (12-bp + 2-nt overhang) containing 2′-OCH3 and 2′-OH at their 3′ ends. These structures explain the molecular basis underlying accommodation of the 2′-OCH3 group within a preformed Hiwi1 PAZ domain binding pocket, whose hydrophobic characteristics account for the preferential binding of 2′-OCH3 over 2′-OH 3′ ends. These results contrast with the more restricted binding pocket for the human Ago1 PAZ domain, which exhibits a reverse order, with preferential binding of 2′-OH over 2′-OCH3 3′ ends.
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  • 74
    Publication Date: 2011-04-13
    Description: The post-Newtonian approximation is a method for solving Einstein’s field equations for physical systems in which motions are slow compared to the speed of light and where gravitational fields are weak. Yet it has proven to be remarkably effective in describing certain strong-field, fast-motion systems, including binary pulsars containing dense neutron stars and binary black hole systems inspiraling toward a final merger. The reasons for this effectiveness are largely unknown. When carried to high orders in the post-Newtonian sequence, predictions for the gravitational-wave signal from inspiraling compact binaries will play a key role in gravitational-wave detection by laser-interferometric observatories.
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  • 75
    Publication Date: 2011-04-20
    Description: The SNF1 protein kinase of Saccharomyces cerevisiae is a member of the SNF1/AMP-activated protein kinase family, which is essential for metabolic control, energy homeostasis, and stress responses in eukaryotes. SNF1 is activated in response to glucose limitation by phosphorylation of Thr210 on the activation loop of the catalytic subunit Snf1. The SNF1 β-subunit contains a glycogen-binding domain that has been implicated in glucose inhibition of Snf1 Thr210 phosphorylation. To assess the role of glycogen, we examined Snf1 phosphorylation in strains with altered glycogen metabolism. A reg1Δ mutant, lacking Reg1-Glc7 protein phosphatase 1, exhibits elevated glycogen accumulation and phosphorylation of Snf1 during growth on high levels of glucose. Unexpectedly, mutations that abolished glycogen synthesis also restored Thr210 dephosphorylation in glucose-grown reg1Δ cells, indicating that elevated glycogen synthesis contributes to activation of SNF1 and that another phosphatase acts on Snf1. We present evidence that Sit4, a type 2A-like protein phosphatase, contributes to dephosphorylation of Snf1 Thr210. Finally, evidence that the effects of glycogen are not mediated by binding to the β-subunit raises the possibility that elevated glycogen synthesis alters glucose metabolism and thereby reduces glucose signaling to the SNF1 pathway.
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  • 76
    Publication Date: 2011-04-13
    Description: SK channels underlie important physiological functions by linking calcium signaling with neuronal excitability. Potassium currents through SK channels demonstrate inward rectification, which further reduces their small outward conductance. Although it has been generally attributed to block of outward current by intracellular divalent ions, we find that inward rectification is in fact an intrinsic property of SK channels independent of intracellular blockers. We identified three charged residues in the S6 transmembrane domain of SK channels near the inner mouth of the pore that collectively control the conductance and rectification through an electrostatic mechanism. Additionally, electrostatic contributions from these residues also play an important role in determining the intrinsic open probability of SK channels in the absence of Ca2+, affecting the apparent Ca2+ affinity for activation.
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  • 77
    Publication Date: 2011-03-09
    Description: Diatoms are responsible for a large fraction of CO2 export to deep seawater, a process responsible for low modern-day CO2 concentrations in surface seawater and the atmosphere. Like other photosynthetic organisms, diatoms have adapted to these low ambient concentrations by operating a CO2 concentrating mechanism (CCM) to elevate the concentration of CO2 at the site of fixation. We used mass spectrometric measurements of passive and active cellular carbon fluxes and model simulations of these fluxes to better understand the stoichiometric and energetic efficiency and the physiological architecture of the diatom CCM. The membranes of diatoms are highly permeable to CO2, resulting in a large diffusive exchange of CO2 between the cell and external milieu. An active transport of carbon from the cytoplasm into the chloroplast is the main driver of the diatom CCM. Only one-third of this carbon flux is fixed photosynthetically, and the rest is lost by CO2 diffusion back to the cytoplasm. Both the passive influx of CO2 from the external medium and the recycling of the CO2 leaking out of the chloroplast are achieved by the activity of a carbonic anhydrase enzyme combined with the maintenance of a low concentration of HCO3− in the cytoplasm. To achieve the CO2 concentration necessary to saturate carbon fixation, the CO2 is most likely concentrated within the pyrenoid, an organelle within the chloroplast where the CO2-fixating enzyme is located.
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  • 78
    Publication Date: 2014-01-22
    Description: A major challenge in understanding the origin of terrestrial vertebrates has been knowledge of the pelvis and hind appendage of their closest fish relatives. The pelvic girdle and appendage of tetrapods is dramatically larger and more robust than that of fish and contains a number of structures that provide greater...
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  • 79
    Publication Date: 2013-12-11
    Description: Sec2p is a guanine nucleotide exchange factor that promotes exocytosis by activating the Rab GTPase Sec4p. Sec2p is highly phosphorylated, and we have explored the role of phosphorylation in the regulation of its function. We have identified three phosphosites and demonstrate that phosphorylation regulates the interaction of Sec2p with its...
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  • 80
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    National Academy of Sciences
    Publication Date: 2014-01-22
    Description: From the rust-brown sediments of long-dried streams that once rumbled through the rugged terrain of Canada’s Ellesmere Island, a team of paleontologists unearthed in 2004 fossils of a 375 million-year-old species of fish that may have nearly crossed an evolutionary Rubicon. Named Tiktaalik roseae, the now-extinct animal has come to...
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  • 81
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    National Academy of Sciences
    Publication Date: 2013-12-11
    Description: As electrical signals course through brain cells, a stepwise process of information transfer ensures that the signals are relayed across junctions called synapses. Orchestrating the process on either side of the synaptic gulf is a suite of proteins found on neuronal membranes and on tiny vesicles that ferry signaling molecules,...
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  • 82
    Publication Date: 2013-10-16
    Description: Unique tripodal S-donor capping agents with an attached carboxylate are found to bind tightly to the surface of CdSe nanocrystals (NCs), making the latter water soluble. Unlike that in similarly solubilized CdSe NCs with one-sulfur or two-sulfur capping agents, dissociation from the NC surface is greatly reduced. The impact of...
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  • 83
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    National Academy of Sciences
    Publication Date: 2013-10-09
    Description: William Bialek works at the interface of physics and biology, searching for predictive mathematical theories that can quantify and explain diverse biological phenomena. A member of the National Academy of Sciences, Bialek is the John Archibald Wheeler/Battelle Professor in Physics at Princeton University, a member of Princeton’s multidisciplinary Lewis-Sigler Institute...
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  • 84
    Publication Date: 2013-10-09
    Description: Cells in a developing embryo have no direct way of “measuring” their physical position. Through a variety of processes, however, the expression levels of multiple genes come to be correlated with position, and these expression levels thus form a code for “positional information.” We show how to measure this information,...
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  • 85
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    National Academy of Sciences
    Publication Date: 2013-10-23
    Description: Much of our everyday behavior is guided by habits. By studying the electrical activity in the brains of animals as they acquire a habit, Ann M. Graybiel, a neuroscientist at the Massachusetts Institute of Technology and a National Academy of Sciences member, uncovers the neurobiological basis of how habits form...
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  • 86
    Publication Date: 2014-02-26
    Description: The brain mechanisms of fear have been studied extensively using Pavlovian fear conditioning, a procedure that allows exploration of how the brain learns about and later detects and responds to threats. However, mechanisms that detect and respond to threats are not the same as those that give rise to conscious...
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  • 87
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    National Academy of Sciences
    Publication Date: 2014-02-26
    Description: In his 1933 inaugural address to the American people, President Franklin Roosevelt attempted to rouse the nation out of the stultifying Depression with his famous aphorism on fear as a self-fulfilling end. Decades later, a scientific understanding of fear continues to remain elusive, despite the unraveling of brain circuits triggered...
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  • 88
    Publication Date: 2014-04-02
    Description: Chimaeras, fanciful beasts that drew their force from being composed of parts of disparate animals, have stimulated our collective imagination for centuries. In modern terms, chimaeras are composite animals consisting of genetically distinct cell populations and are called “mosaics” if the different cell types have emerged from the same zygote....
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  • 89
    Publication Date: 2012-03-21
    Description: Transferring lipid antigens from membranes into CD1 antigen-presenting proteins represents a major molecular hurdle necessary for T-cell recognition. Saposins facilitate this process, but the mechanisms used are not well understood. We found that saposin B forms soluble saposin protein–lipid complexes detected by native gel electrophoresis that can directly load CD1 proteins. Because saposin B must bind lipids directly to function, we found it could not accommodate long acyl chain containing lipids. In contrast, saposin C facilitates CD1 lipid loading in a different way. It uses a stable, membrane-associated topology and was capable of loading lipid antigens without forming soluble saposin–lipid antigen complexes. These findings reveal how saposins use different strategies to facilitate transfer of structurally diverse lipid antigens.
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  • 90
    Publication Date: 2012-03-28
    Description: Climate change poses threats to human health, safety, and survival via weather extremes and climatic impacts on food yields, fresh water, infectious diseases, conflict, and displacement. Paradoxically, these risks to health are neither widely nor fully recognized. Historical experiences of diverse societies experiencing climatic changes, spanning multicentury to single-year duration, provide insights into population health vulnerability—even though most climatic changes were considerably less than those anticipated this century and beyond. Historical experience indicates the following. (i) Long-term climate changes have often destabilized civilizations, typically via food shortages, consequent hunger, disease, and unrest. (ii) Medium-term climatic adversity has frequently caused similar health, social, and sometimes political consequences. (iii) Infectious disease epidemics have often occurred in association with briefer episodes of temperature shifts, food shortages, impoverishment, and social disruption. (iv) Societies have often learnt to cope (despite hardship for some groups) with recurring shorter-term (decadal to multiyear) regional climatic cycles (e.g., El Niño Southern Oscillation)—except when extreme phases occur. (v) The drought–famine–starvation nexus has been the main, recurring, serious threat to health. Warming this century is not only likely to greatly exceed the Holocene's natural multidecadal temperature fluctuations but to occur faster. Along with greater climatic variability, models project an increased geographic range and severity of droughts. Modern societies, although larger, better resourced, and more interconnected than past societies, are less flexible, more infrastructure-dependent, densely populated, and hence are vulnerable. Adverse historical climate-related health experiences underscore the case for abating human-induced climate change.
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  • 91
    Publication Date: 2012-02-22
    Description: Richard Durrett’s work lies at the interface of mathematics and biology, where the tools of probability theory are used to study problems in ecology, genetics, and cancer biology. Durrett, a professor of mathematics at Duke University and a recently elected member of the National Academy of Sciences, has devoted his career to developing models for biological questions ranging from the behavior of populations in ecological systems to the effects of mutations and natural selection on genomes. Durrett talks to PNAS about his recent work on the development and spread of cancer and how mathematical approaches can be applied to biological...
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  • 92
    Publication Date: 2012-03-14
    Description: Wnts make up a large family of extracellular signaling molecules that play crucial roles in development and disease. A subset of noncanonical Wnts signal independently of the transcription factor β-catenin by a mechanism that regulates key morphogenetic movements during embryogenesis. The best characterized noncanonical Wnt, Wnt5a, has been suggested to signal via a variety of different receptors, including the Ror family of receptor tyrosine kinases, the Ryk receptor tyrosine kinase, and the Frizzled seven-transmembrane receptors. Whether one or several of these receptors mediates the effects of Wnt5a in vivo is not known. Through loss-of-function experiments in mice, we provide conclusive evidence that Ror receptors mediate Wnt5a-dependent processes in vivo and identify Dishevelled phosphorylation as a physiological target of Wnt5a–Ror signaling. The absence of Ror signaling leads to defects that mirror phenotypes observed in Wnt5a null mutant mice, including decreased branching of sympathetic neuron axons and major defects in aspects of embryonic development that are dependent upon morphogenetic movements, such as severe truncation of the caudal axis, the limbs, and facial structures. These findings suggest that Wnt5a–Ror–Dishevelled signaling constitutes a core noncanonical Wnt pathway that is conserved through evolution and is crucial during embryonic development.
    Keywords: Inaugural Articles
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  • 93
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    Unknown
    National Academy of Sciences
    Publication Date: 2011-07-06
    Description: National Academy of Sciences member Peter Gleick is cofounder and president of the Pacific Institute for Studies in Development, Environment, and Security in Oakland, California, where he explores new ways of thinking about water issues. His creative insights have resulted in the biennial book series The World’s Water, a MacArthur Fellowship award in 2003, multiple appearances as an expert witness before Congress and the courts, and the 2010 book Bottled and Sold: The Story Behind Our Obsession with Bottled Water. Peak water, the concept introduced in his PNAS Inaugural Article (1), became one of the New York Times’ “Words of...
    Keywords: QnAs
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  • 94
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    Unknown
    National Academy of Sciences
    Publication Date: 2011-07-13
    Description: This year marks the 10th anniversary of the sequencing of the human genome. More than two decades after the launch of the Human Genome Project, researchers have made remarkable inroads into unraveling human biology, evolution, and disease. As the tools of genome sequencing and analysis grow more sophisticated, insights into the human genome will slowly shift the terrain in the treatment of disease. To be sure, the shift has already begun. Eric Lander, founding director of the Broad Institute of Harvard and Massachusetts Institute of Technology and a member of the National Academy of Sciences, offers PNAS readers his perspectives...
    Keywords: QnAs
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  • 95
    Publication Date: 2011-09-07
    Description: Legionella pneumophila is a bacterial pathogen of amoebae and humans. Intracellular growth requires a type IVB secretion system that translocates at least 200 different proteins into host cells. To distinguish between proteins necessary for growth in culture and those specifically required for intracellular replication, a screen was performed to identify genes necessary for optimal growth in nutrient-rich medium. Mapping of these genes revealed that the L. pneumophila chromosome has a modular architecture consisting of several large genomic islands that are dispensable for growth in bacteriological culture. Strains lacking six of these regions, and thus 18.5% of the genome, were viable but required secondary point mutations for optimal growth. The simultaneous deletion of five of these genomic loci had no adverse effect on growth of the bacterium in nutrient-rich media. Remarkably, this minimal genome strain, which lacked 31% of the known substrates of the type IVB system, caused only marginal defects in intracellular growth within mouse macrophages. In contrast, deletion of single regions reduced growth within amoebae. The importance of individual islands, however, differed among amoebal species. The host-specific requirements of these genomic islands support a model in which the acquisition of foreign DNA has broadened the L. pneumophila host range.
    Keywords: Inaugural Articles
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  • 96
    Publication Date: 2011-09-14
    Description: The conserved nature of the ATP-binding site of the 〉 500 human kinases renders the development of specific inhibitors a challenging task. A widely used chemical genetic strategy to overcome the specificity challenge exploits a large-to-small mutation of the gatekeeper residue (a conserved hydrophobic amino acid) and the use of a bulky inhibitor to achieve specificity via shape complementarity. However, in a number of cases, introduction of a glycine or alanine gatekeeper results in diminished kinase activity and ATP affinity. A new chemical genetic approach based on covalent complementarity between an engineered gatekeeper cysteine and an electrophilic inhibitor was developed to address these challenges. This strategy was evaluated with Src, a proto-oncogenic tyrosine kinase known to lose some enzymatic activity using the shape complementarity chemical genetic strategy. We found that Src with a cysteine gatekeeper recapitulates wild type activity and can be irreversibly inhibited both in vitro and in cells. A cocrystal structure of T338C c-Src with a vinylsulfonamide-derivatized pyrazolopyrimidine inhibitor was solved to elucidate the inhibitor binding mode. A panel of electrophilic inhibitors was analyzed against 307 kinases and MOK (MAPK/MAK/MRK overlapping kinase), one of only two human kinases known to have an endogenous cysteine gatekeeper. This analysis revealed remarkably few off-targets, making these compounds the most selective chemical genetic inhibitors reported to date. Protein engineering studies demonstrated that it is possible to increase inhibitor potency through secondary-site mutations. These results suggest that chemical genetic strategies based on covalent complementarity should be widely applicable to the study of protein kinases.
    Keywords: Inaugural Articles
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  • 97
    Publication Date: 2011-09-07
    Description: During human CMV infection, there is a preferential expansion of natural killer (NK) cells expressing the activating CD94–NKG2C receptor complex, implicating this receptor in the recognition of CMV-infected cells. We hypothesized that NK cells expanded in response to pathogens will be marked by expression of CD57, a carbohydrate antigen expressed on highly mature cells within the CD56dimCD16+ NK cell compartment. Here we demonstrate the preferential expansion of a unique subset of NK cells coexpressing the activating CD94–NKG2C receptor and CD57 in CMV+ donors. These CD57+NKG2Chi NK cells degranulated in response to stimulation through their NKG2C receptor. Furthermore, CD57+NKG2Chi NK cells preferentially lack expression of the inhibitory NKG2A receptor and the inhibitory KIR3DL1 receptor in individuals expressing its HLA-Bw4 ligand. Moreover, in solid-organ transplant recipients with active CMV infection, the percentage of CD57+NKG2Chi NK cells in the total NK cell population preferentially increased. During acute CMV infection, the NKG2C+ NK cells proliferated, became NKG2Chi, and finally acquired CD57. Thus, we propose that CD57 might provide a marker of “memory” NK cells that have been expanded in response to infection.
    Keywords: Inaugural Articles
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  • 98
    Publication Date: 2011-09-21
    Description: We present a randomized algorithm for the approximate nearest neighbor problem in d-dimensional Euclidean space. Given N points {xj} in , the algorithm attempts to find k nearest neighbors for each of xj, where k is a user-specified integer parameter. The algorithm is iterative, and its running time requirements are proportional to T·N·(d·(log d) + k·(d + log k)·(log N)) + N·k2·(d + log k), with T the number of iterations performed. The memory requirements of the procedure are of the order N·(d + k). A by-product of the scheme is a data structure, permitting a rapid search for the k nearest neighbors among {xj} for an arbitrary point . The cost of each such query is proportional to T·(d·(log d) + log(N/k)·k·(d + log k)), and the memory requirements for the requisite data structure are of the order N·(d + k) + T·(d + N). The algorithm utilizes random rotations and a basic divide-and-conquer scheme, followed by a local graph search. We analyze the scheme’s behavior for certain types of distributions of {xj} and illustrate its performance via several numerical examples.
    Keywords: Inaugural Articles
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  • 99
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    Unknown
    National Academy of Sciences
    Publication Date: 2011-12-14
    Description: Prions defy molecular biology’s central dogma. Misfolded proteins that self-perpetuate, prions were first isolated in the early 1980s as the cause of a fatal sheep disease called scrapie. Since then, prions have been implicated in human neurodegenerative diseases, composing a rogue’s gallery of deadly disease agents. Susan Lindquist, a member of the National Academy of Sciences and a professor of biology at the Massachusetts Institute of Technology’s Whitehead Institute for Biomedical Research, has found that prions may have a little-appreciated positive side. Lindquist casts these seeming biochemical misfits in a surprising evolutionary role: Her studies have revealed that prions might...
    Keywords: QnAs
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  • 100
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    National Academy of Sciences
    Publication Date: 2011-12-21
    Description: At the age of 37, David Baltimore accomplished what many researchers dream of but few achieve: reversing an entrenched dogma, eventually leading to a new view of life. In the early 1970s, Baltimore, a member of the National Academy of Sciences and a professor of biology at the California Institute of Technology, discovered reverse transcriptase—an enzyme found in some tumor viruses whose genetic code is written in the RNA alphabet. He found that reverse transcriptase can copy RNA into DNA, indicating that some viruses replicate via a DNA intermediate. The finding, which won Baltimore and others the 1975 Nobel Prize...
    Keywords: QnAs
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