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158 Publications

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    03/01/09 | Mass selection of ions from beams using waveform isolation in radiofrequency quadrupoles.
    Song Q, Smith SA, Gao L, Xu W, Volný M, Ouyang Z, Cooks RG
    Analytical Chemistry. 2009 Mar 1;81(5):1833-40. doi: 10.1364/AO.50.001792

    A waveform isolation method is described for the mass-selective transmission of ions through quadrupole mass filters, and it is implemented on a new tandem mass analyzer instrument. The method features the application of broad-band waveforms comprising appropriate frequencies to cause mass-selective instability in ions of particular mass-to-charge (m/z) and to transmit all others. The experiment is implemented in a tandem quadrupole system in which the first mass filter is a rectilinear ion trap (RIT) operated in a continuous mass-selective mode to transmit ions of ions of one or more arbitrarily selected m/z value(s). The second analyzer was used to verify the quality of the mass selection achieved using the first analyzer via conventional quadrupole ion trap mass-selective instability scanning. A new subtype of product ion tandem mass spectrometry (MS/MS) scan, termed the summed product ion scan, is demonstrated with a mixture of biological compounds. It is used to characterize product ions arising after simultaneous isolation and collisional activation of multiple precursor species, in this case ions of the same analyte generated in different charge states. The summed product ion scan can be useful for enhancing sensitivity for the analyte of interest or for providing more comprehensive information about an analyte than is available by monitoring a single ionized form of the analyte. The analytical performance of the waveform isolation method is tested using simple drug mixtures, and its potential for increasing overall yields in preparative mass spectrometry is explored briefly. It is shown that efficiencies of ca. 70% of ion transfer to a surface for ion soft landing surface can be achieved. The upper mass range is limited by axial acceleration arising from the stretched geometry, and one solution to this problem is provided.

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    03/01/09 | VANO: a volume-object image annotation system.
    Peng H, Long F, Myers EW
    Bioinformatics. 2009 Mar 1;25:695-7. doi: 10.1093/bioinformatics/btp046

    Volume-object annotation system (VANO) is a cross-platform image annotation system that enables one to conveniently visualize and annotate 3D volume objects including nuclei and cells. An application of VANO typically starts with an initial collection of objects produced by a segmentation computation. The objects can then be labeled, categorized, deleted, added, split, merged and redefined. VANO has been used to build high-resolution digital atlases of the nuclei of Caenorhabditis elegans at the L1 stage and the nuclei of Drosophila melanogaster’s ventral nerve cord at the late embryonic stage. AVAILABILITY: Platform independent executables of VANO, a sample dataset, and a detailed description of both its design and usage are available at research.janelia.org/peng/proj/vano. VANO is open-source for co-development.

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    Grigorieff Lab
    02/27/09 | Abeta(1-40) fibril polymorphism implies diverse interaction patterns in amyloid fibrils.
    Meinhardt J, Sachse C, Hortschansky P, Grigorieff N, Fändrich M
    Journal of Molecular Biology. 2009 Feb 27;386(3):869-77. doi: 10.1016/j.jmb.2008.11.005

    Amyloid fibrils characterize a diverse group of human diseases that includes Alzheimer’s disease, Creutzfeldt-Jakob and type II diabetes. Alzheimer’s amyloid fibrils consist of amyloid-beta (Abeta) peptide and occur in a range of structurally different fibril morphologies. The structural characteristics of 12 single Abeta(1-40) amyloid fibrils, all formed under the same solution conditions, were determined by electron cryo-microscopy and three-dimensional reconstruction. The majority of analyzed fibrils form a range of morphologies that show almost continuously altering structural properties. The observed fibril polymorphism implies that amyloid formation can lead, for the same polypeptide sequence, to many different patterns of inter- or intra-residue interactions. This property differs significantly from native, monomeric protein folding reactions that produce, for one protein sequence, only one ordered conformation and only one set of inter-residue interactions.

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    02/26/09 | Sensory neurons in the Drosophila genital tract regulate female reproductive behavior.
    Häsemeyer M, Yapici N, Heberlein U, Dickson BJ
    Neuron. 2009 Feb 26;61(4):511-8. doi: 10.1016/j.neuron.2009.01.009

    Females of many animal species behave very differently before and after mating. In Drosophila melanogaster, changes in female behavior upon mating are triggered by the sex peptide (SP), a small peptide present in the male's seminal fluid. SP activates a specific receptor, the sex peptide receptor (SPR), which is broadly expressed in the female reproductive tract and nervous system. Here, we pinpoint the action of SPR to a small subset of internal sensory neurons that innervate the female uterus and oviduct. These neurons express both fruitless (fru), a marker for neurons likely to have sex-specific functions, and pickpocket (ppk), a marker for proprioceptive neurons. We show that SPR expression in these fru+ ppk+ neurons is both necessary and sufficient for behavioral changes induced by mating. These neurons project to regions of the central nervous system that have been implicated in the control of reproductive behaviors in Drosophila and other insects.

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    Sternson LabSvoboda Lab
    02/26/09 | The subcellular organization of neocortical excitatory connections.
    Petreanu L, Mao T, Sternson SM, Svoboda K
    Nature. 2009 Feb 26;457:1142-5. doi: 10.1038/nature07709

    Understanding cortical circuits will require mapping the connections between specific populations of neurons, as well as determining the dendritic locations where the synapses occur. The dendrites of individual cortical neurons overlap with numerous types of local and long-range excitatory axons, but axodendritic overlap is not always a good predictor of actual connection strength. Here we developed an efficient channelrhodopsin-2 (ChR2)-assisted method to map the spatial distribution of synaptic inputs, defined by presynaptic ChR2 expression, within the dendritic arborizations of recorded neurons. We expressed ChR2 in two thalamic nuclei, the whisker motor cortex and local excitatory neurons and mapped their synapses with pyramidal neurons in layers 3, 5A and 5B (L3, L5A and L5B) in the mouse barrel cortex. Within the dendritic arborizations of L3 cells, individual inputs impinged onto distinct single domains. These domains were arrayed in an orderly, monotonic pattern along the apical axis: axons from more central origins targeted progressively higher regions of the apical dendrites. In L5 arborizations, different inputs targeted separate basal and apical domains. Input to L3 and L5 dendrites in L1 was related to whisker movement and position, suggesting that these signals have a role in controlling the gain of their target neurons. Our experiments reveal high specificity in the subcellular organization of excitatory circuits.

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    Looger Lab
    02/20/09 | Cofactor engineering of lactobacillus brevis alcohol dehydrogenase by computational design.
    Ronnie Machielsen , Loren L. Looger , John Raedts , Sjoerd Dijkhuizen , Werner Hummel , Hans‐Georg Hennemann , Thomas Daussmann , John van der Oost
    Engineering in Life Sciences. 2009 Feb 20;9(1):38-44. doi: 10.1002/elsc.200800046

    The R‐specific alcohol dehydrogenase from Lactobacillus brevis (Lb‐ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for biotechnological applications. A drawback is its preference for NADP(H) as a cofactor, which is more expensive and labile than NAD(H). Structure‐based computational protein engineering was used to predict mutations to alter the cofactor specificity of Lb‐ADH. Mutations were introduced into Lb‐ADH and tested against the substrate acetophenone, with either NAD(H) or NADP(H) as cofactor. The mutant Arg38Pro showed fourfold increased activity with acetophenone and NAD(H) relative to the wild type. Both Arg38Pro and wild type exhibit a pH optimum of 5.5 with NAD(H) as cofactor, significantly more acidic than with NADP(H). These and related Lb‐ADH mutants may prove useful for the green synthesis of pharmaceutical precursors.

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    02/17/09 | Odor coding by modules of coherent mitral/tufted cells in the vertebrate olfactory bulb.
    Chen T, Lin B, Schild D
    Proceedings of the National Academy of Sciences of the United States of America. 2009 Feb 17;106(7):2401-6. doi: 10.1073/pnas.0810151106

    Odor representation in the olfactory bulb (OB) undergoes a transformation from a combinatorial glomerular map to a distributed mitral/tufted (M/T) cell code. To understand this transformation, we analyzed the odor representation in large populations of individual M/T cells in the Xenopus OB. The spontaneous [Ca(2+)] activities of M/T cells appeared to be irregular, but there were groups of spatially distributed neurons showing synchronized [Ca(2+)] activities. These neurons were always connected to the same glomerulus. Odorants elicited complex spatiotemporal response patterns in M/T cells where nearby neurons generally showed little correlation. But the responses of neurons connected to the same glomerulus were virtually identical, irrespective of whether the responses were excitatory or inhibitory, and independent of the distance between them. Synchronous neurons received correlated EPSCs and were coupled by electrical conductances that could account for the correlated responses. Thus, at the output stage of the OB, odors are represented by modules of distributed and synchronous M/T cells associated with the same glomeruli. This allows for parallel input to higher brain centers.

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    02/11/09 | Endodomain diversity in the Drosophila Dscam and its roles in neuronal morphogenesis.
    Yu H, Yang JS, Wang J, Huang Y, Lee T
    The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 2009 Feb 11;29(6):1904-14. doi: 10.1523/JNEUROSCI.5743-08.2009

    Drosophila Down syndrome cell adhesion molecule (Dscam) can be variably spliced to encode 152,064 distinct single-pass transmembrane proteins. In addition to 19,008 possible ectodomains and two alternative transmembrane segments, it may carry endodomains containing or lacking exons 19 and 23. Here, we determine the role of Dscam endodomain diversity in neural development. Dscam with full-length endodomain is largely restricted to embryogenesis. In contrast, most Dscams lack exons 19 and 23 at postembryonic stages. As implicated from the expression patterns, removal of Dscam exon 19-containing variants disrupts wiring of embryonic neurons while silencing of Dscam transcripts lacking exon 19 or exon 23 effectively blocks postembryonic neuronal morphogenesis. Furthermore, compared with exon 19-containing Dscam, transgenic Dscam without exon 19 is more efficiently targeted to neurites and more potently suppresses axon bifurcation in Dscam mutant neurons. In sum, Dscam with or without exon 19 in its endodomain is used to govern different stage-specific neuronal morphogenetic processes, possibly due to differences in protein targeting.

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    02/06/09 | Is genetic evolution predictable?
    Stern DL, Orgogozo V
    Science. 2009 Feb 6;323:746-51. doi: 10.1126/science.1158997

    Ever since the integration of Mendelian genetics into evolutionary biology in the early 20th century, evolutionary geneticists have for the most part treated genes and mutations as generic entities. However, recent observations indicate that all genes are not equal in the eyes of evolution. Evolutionarily relevant mutations tend to accumulate in hotspot genes and at specific positions within genes. Genetic evolution is constrained by gene function, the structure of genetic networks, and population biology. The genetic basis of evolution may be predictable to some extent, and further understanding of this predictability requires incorporation of the specific functions and characteristics of genes into evolutionary theory.

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    Tjian LabSinger LabTranscription Imaging
    02/03/09 | Imaging transcription in living cells.
    Darzacq X, Yao J, Larson DR, Causse SZ, Bosanac L, de Turris V, Ruda VM, Lionnet T, Zenklusen D, Guglielmi B, Tjian R, Singer RH
    Annual Review of Biophysics. 2009 Feb 3;38:173-96. doi: 10.1073/pnas.1100640108

    The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.

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