Synthetic lethal screen identification of chemosensitizer loci in cancer cells

Abundant evidence suggests that a unifying principle governing the molecular pathology of cancer is the co-dependent aberrant regulation of core machinery driving proliferation and suppressing apoptosis. Anomalous proteins engaged in support of this tumorigenic regulatory environment most probably represent optimal intervention targets in a heterogeneous population of cancer cells. The advent of RNA-mediated interference (RNAi)-based functional genomics provides the opportunity to derive unbiased comprehensive collections of validated gene targets supporting critical biological systems outside the framework of preconceived notions of mechanistic relationships. We have combined a high-throughput cell-based one-well/one-gene screening platform with a genome-wide synthetic library of chemically synthesized small interfering RNAs for systematic interrogation of the molecular underpinnings of cancer cell chemoresponsiveness. NCI-H1155, a human non-small-cell lung cancer line, was employed in a paclitaxel-dependent synthetic lethal screen designed to identify gene targets that specifically reduce cell viability in the presence of otherwise sublethal concentrations of paclitaxel. Using a stringent objective statistical algorithm to reduce false discovery rates below 5%, we isolated a panel of 87 genes that represent major focal points of the autonomous response of cancer cells to the abrogation of microtubule dynamics. Here we show that several of these targets sensitize lung cancer cells to paclitaxel concentrations 1,000-fold lower than otherwise required for a significant response, and we identify mechanistic relationships between cancer-associated aberrant gene expression programmes and the basic cellular machinery required for robust mitotic progression.     

Calcineurin sets the bandwidth for discrimination of signals during thymocyte development

At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the alphabeta T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide-MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection). Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf-MEK-ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to 'weak' positive selecting signals but not in response to 'strong' negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.     

Millennial-scale trends in west Pacific warm pool hydrology since the Last Glacial Maximum

Models and palaeoclimate data suggest that the tropical Pacific climate system plays a key part in the mechanisms underlying orbital-scale and abrupt climate change. Atmospheric convection over the western tropical Pacific is a major source of heat and moisture to extratropical regions, and may therefore influence the global climate response to a variety of forcing factors. The response of tropical Pacific convection to changes in global climate boundary conditions, abrupt climate changes and radiative forcing remains uncertain, however. Here we present three absolutely dated oxygen isotope records from stalagmites in northern Borneo that reflect changes in west Pacific warm pool hydrology over the past 27,000 years. Our results suggest that convection over the western tropical Pacific weakened 18,000-20,000 years ago, as tropical Pacific and Antarctic temperatures began to rise during the early stages of deglaciation. Convective activity, as inferred from oxygen isotopes, reached a minimum during Heinrich event 1 (ref. 10), when the Atlantic meridional overturning circulation was weak, pointing to feedbacks between the strength of the overturning circulation and tropical Pacific hydrology. There is no evidence of the Younger Dryas event in the stalagmite records, however, suggesting that different mechanisms operated during these two abrupt deglacial climate events. During the Holocene epoch, convective activity appears to track changes in spring and autumn insolation, highlighting the sensitivity of tropical Pacific convection to external radiative forcing. Together, these findings demonstrate that the tropical Pacific hydrological cycle is sensitive to high-latitude climate processes in both hemispheres, as well as to external radiative forcing, and that it may have a central role in abrupt climate change events.     

Scale dependence of direct shear tests

Direct shear test has been widely used to measure the shear strength of soils and other particulate materials in industry because of its simplicity. However, the results can be dependent on the specimen size. The ASTM （American Society for Testing and Materials） publications suggest that for testing soils the shear box should be at least ten times the diameter of the largest particle and the height of the box should be no more than half of its diameter. These guidelines are empirically based. A series of two-dimensional numerical direct shear tests are performed to investigate this scaling effect. By analyzing the bulk friction, particle translation and rotation, percentage of sliding, average volume （area） and shear strain and the evolution of the shear band, we find that the traditional guidelines for direct shear tests are questionable. Scaling dependency of bulk friction on the property of granular materials is clearly present. Our current analysis points out that the scaling effects can vary significantly depending on the particle properties other than their sizes. Of all the parameters we observed, particle rotation appears to have a decisive correlation with the bulk friction. Formation of a shear band is universal. As the shearing progresses, particle rotation begins to concentrate near the shear plane. By defining the width of a shear band as the standard deviation of the distribution of translational gradient or the standard deviation of the distribution of particle rotation, quantitative evolutions of shear band are presented. Both measures of the shear band width dropped rapidly during pre-failure stage. After peak stress both measures begin to approach steady state as the bulk friction stabilizes to the residual stage. These observations suggest that structure formation inside the shear band controls the scaling effect.     

Observation of interstellar lithium in the low-metallicity Small Magellanic Cloud

The primordial abundances of light elements produced in the standard theory of Big Bang nucleosynthesis (BBN) depend only on the cosmic ratio of baryons to photons, a quantity inferred from observations of the microwave background. The predicted primordial (7)Li abundance is four times that measured in the atmospheres of Galactic halo stars. This discrepancy could be caused by modification of surface lithium abundances during the stars' lifetimes or by physics beyond the Standard Model that affects early nucleosynthesis. The lithium abundance of low-metallicity gas provides an alternative constraint on the primordial abundance and cosmic evolution of lithium that is not susceptible to the in situ modifications that may affect stellar atmospheres. Here we report observations of interstellar (7)Li in the low-metallicity gas of the Small Magellanic Cloud, a nearby galaxy with a quarter the Sun's metallicity. The present-day (7)Li abundance of the Small Magellanic Cloud is nearly equal to the BBN predictions, severely constraining the amount of possible subsequent enrichment of the gas by stellar and cosmic-ray nucleosynthesis. Our measurements can be reconciled with standard BBN with an extremely fine-tuned depletion of stellar Li with metallicity. They are also consistent with non-standard BBN.     

Crystal structure of the µ-opioid receptor bound to a morphinan antagonist

Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled μ-opioid receptor (μ-OR) in the central nervous system. Here we describe the 2.8?? crystal structure of the mouse μ-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the μ-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.     

MAP and kinesin-dependent nuclear positioning is required for skeletal muscle function

The basic unit of skeletal muscle in all metazoans is the multinucleate myofibre, within which individual nuclei are regularly positioned. The molecular machinery responsible for myonuclear positioning is not known. Improperly positioned nuclei are a hallmark of numerous diseases of muscle, including centronuclear myopathies, but it is unclear whether correct nuclear positioning is necessary for muscle function. Here we identify the microtubule-associated protein ensconsin (Ens)/microtubule-associated protein 7 (MAP7) and kinesin heavy chain (Khc)/Kif5b as essential, evolutionarily conserved regulators of myonuclear positioning in Drosophila and cultured mammalian myotubes. We find that these proteins interact physically and that expression of the Kif5b motor domain fused to the MAP7 microtubule-binding domain rescues nuclear positioning defects in MAP7-depleted cells. This suggests that MAP7 links Kif5b to the microtubule cytoskeleton to promote nuclear positioning. Finally, we show that myonuclear positioning is physiologically important. Drosophila ens mutant larvae have decreased locomotion and incorrect myonuclear positioning, and these phenotypes are rescued by muscle-specific expression of Ens. We conclude that improper nuclear positioning contributes to muscle dysfunction in a cell-autonomous fashion.     

The genomic basis of adaptive evolution in threespine sticklebacks

Marine stickleback fish have colonized and adapted to thousands of streams and lakes formed since the last ice age, providing an exceptional opportunity to characterize genomic mechanisms underlying repeated ecological adaptation in nature. Here we develop a high-quality reference genome assembly for threespine sticklebacks. By sequencing the genomes of twenty additional individuals from a global set of marine and freshwater populations, we identify a genome-wide set of loci that are consistently associated with marine-freshwater divergence. Our results indicate that reuse of globally shared standing genetic variation, including chromosomal inversions, has an important role in repeated evolution of distinct marine and freshwater sticklebacks, and in the maintenance of divergent ecotypes during early stages of reproductive isolation. Both coding and regulatory changes occur in the set of loci underlying marine-freshwater evolution, but regulatory changes appear to predominate in this well known example of repeated adaptive evolution in nature.