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4 Janelia Publications

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    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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    Looger Lab
    02/01/09 | A bright and photostable photoconvertible fluorescent protein.
    McKinney SA, Murphy CS, Hazelwood KL, Davidson MW, Looger LL
    Nature Methods. 2009 Feb;6(2):131-3. doi: 10.1038/nmeth.1296

    Photoconvertible fluorescent proteins are potential tools for investigating dynamic processes in living cells and for emerging super-resolution microscopy techniques. Unfortunately, most probes in this class are hampered by oligomerization, small photon budgets or poor photostability. Here we report an EosFP variant that functions well in a broad range of protein fusions for dynamic investigations, exhibits high photostability and preserves the approximately 10-nm localization precision of its parent.

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    02/01/09 | Automatic tracking of Escherichia coli in phase-contrast microscopy video.
    Xie J, Khan S, Shah M
    IEEE Transactions on Bio-Medical Engineering. 2009 Feb;56(2):390-9. doi: 10.1109/TBME.2008.2005956

    In this paper, we present an automatic method for estimating the trajectories of Escherichia coli bacteria from in vivo phase-contrast microscopy. To address the low-contrast boundaries in cellular images, an adaptive kernel-based technique is applied to detect cells in each frame. In addition to intensity features, region homogeneity measure and class uncertainty are also applied in this detection technique. To track cells with complex motion, a novel matching gain measure is introduced to cope with the challenges, particularly the presence of low-contrast boundary, the variations of appearance, and the frequent overlapping and occlusion. For multicell tracking over time, an optimal matching strategy is introduced to improve the handling of cell collision and broken trajectories. The results of successful tracking of Escherichia coli from various phase-contrast sequences are reported and compared with manually determined trajectories, as well as those obtained from existing tracking schemes. The stability of the algorithm with different parameter values is also analyzed and discussed.

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