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2691 Publications

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    01/13/14 | Skylign: a tool for creating informative, interactive logos representing sequence alignments and profile hidden Markov models.
    Wheeler TJ, Clements J, Finn RD
    BMC Bioinformatics. 2014 Jan 13;15:7. doi: 10.1186/1471-2105-15-7

    BACKGROUND: Logos are commonly used in molecular biology to provide a compact graphical representation of the conservation pattern of a set of sequences. They render the information contained in sequence alignments or profile hidden Markov models by drawing a stack of letters for each position, where the height of the stack corresponds to the conservation at that position, and the height of each letter within a stack depends on the frequency of that letter at that position. RESULTS: We present a new tool and web server, called Skylign, which provides a unified framework for creating logos for both sequence alignments and profile hidden Markov models. In addition to static image files, Skylign creates a novel interactive logo plot for inclusion in web pages. These interactive logos enable scrolling, zooming, and inspection of underlying values. Skylign can avoid sampling bias in sequence alignments by down-weighting redundant sequences and by combining observed counts with informed priors. It also simplifies the representation of gap parameters, and can optionally scale letter heights based on alternate calculations of the conservation of a position. CONCLUSION: Skylign is available as a website, a scriptable web service with a RESTful interface, and as a software package for download. Skylign’s interactive logos are easily incorporated into a web page with just a few lines of HTML markup. Skylign may be found at http://skylign.org.

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    Svoboda Lab
    01/08/14 | Flow of cortical activity underlying a tactile decision in mice.
    Guo ZV, Li N, Huber D, Ophir E, Gutnisky D, Ting JT, Feng G, Svoboda K
    Neuron. 2014 Jan 8;81:179-94. doi: 10.1016/j.neuron.2013.10.020

    Perceptual decisions involve distributed cortical activity. Does information flow sequentially from one cortical area to another, or do networks of interconnected areas contribute at the same time? Here we delineate when and how activity in specific areas drives a whisker-based decision in mice. A short-term memory component temporally separated tactile "sensation" and "action" (licking). Using optogenetic inhibition (spatial resolution, 2 mm; temporal resolution, 100 ms), we surveyed the neocortex for regions driving behavior during specific behavioral epochs. Barrel cortex was critical for sensation. During the short-term memory, unilateral inhibition of anterior lateral motor cortex biased responses to the ipsilateral side. Consistently, barrel cortex showed stimulus-specific activity during sensation, whereas motor cortex showed choice-specific preparatory activity and movement-related activity, consistent with roles in motor planning and movement. These results suggest serial information flow from sensory to motor areas during perceptual decision making.

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    Eddy/Rivas Lab
    01/01/14 | Annotating functional RNAs in genomes using infernal.
    Nawrocki EP
    Methods in Molecular Biology. 2014;1097:163-97. doi: 10.1007/978-1-62703-709-9_9

    Many different types of functional non-coding RNAs participate in a wide range of important cellular functions but the large majority of these RNAs are not routinely annotated in published genomes. Several programs have been developed for identifying RNAs, including specific tools tailored to a particular RNA family as well as more general ones designed to work for any family. Many of these tools utilize covariance models (CMs), statistical models of the conserved sequence, and structure of an RNA family. In this chapter, as an illustrative example, the Infernal software package and CMs from the Rfam database are used to identify RNAs in the genome of the archaeon Methanobrevibacter ruminantium, uncovering some additional RNAs not present in the genome’s initial annotation. Analysis of the results and comparison with family-specific methods demonstrate some important strengths and weaknesses of this general approach.

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    Eddy/Rivas Lab
    01/01/14 | Computational analysis of conserved RNA secondary structure in transcriptomes and genomes.
    Eddy SR
    Annual Review of Biophysics and Biomolecular Structure. 2014;43:433-56. doi: 10.1146/annurev-biophys-051013-022950

    Transcriptomics experiments and computational predictions both enable systematic discovery of new functional RNAs. However, many putative noncoding transcripts arise instead from artifacts and biological noise, and current computational prediction methods have high false positive rates. I discuss prospects for improving computational methods for analyzing and identifying functional RNAs, with a focus on detecting signatures of conserved RNA secondary structure. An interesting new front is the application of chemical and enzymatic experiments that probe RNA structure on a transcriptome-wide scale. I review several proposed approaches for incorporating structure probing data into the computational prediction of RNA secondary structure. Using probabilistic inference formalisms, I show how all these approaches can be unified in a well-principled framework, which in turn allows RNA probing data to be easily integrated into a wide range of analyses that depend on RNA secondary structure inference. Such analyses include homology search and genome-wide detection of new structural RNAs.

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    01/01/14 | Drosophila intermediate neural progenitors produce lineage-dependent related series of diverse neurons.
    Wang Y, Yang JS, Johnston R, Ren Q, Lee Y, Luan H, Brody T, Odenwald WF, Lee T
    Development. 2014 Jan;141:253-8. doi: 10.1242/dev.103069

    Drosophila type II neuroblasts (NBs), like mammalian neural stem cells, deposit neurons through intermediate neural progenitors (INPs) that can each produce a series of neurons. Both type II NBs and INPs exhibit age-dependent expression of various transcription factors, potentially specifying an array of diverse neurons by combinatorial temporal patterning. Not knowing which mature neurons are made by specific INPs, however, conceals the actual variety of neuron types and limits further molecular studies. Here we mapped neurons derived from specific type II NB lineages and found that sibling INPs produced a morphologically similar but temporally regulated series of distinct neuron types. This suggests a common fate diversification program operating within each INP that is modulated by NB age to generate slightly different sets of diverse neurons based on the INP birth order. Analogous mechanisms might underlie the expansion of neuron diversity via INPs in mammalian brain.

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    01/01/14 | Generating mosaics for lineage analysis in flies.
    Lee T
    Wiley Interdisciplinary Reviews - Developmental Biology. 2014 Jan;3(1):69-81. doi: 10.1002/wdev.122

    By generating and studying mosaic organisms, we are learning how intricate tissues form as cells proliferate and diversify through organism development. FLP/FRT-mediated site-specific mitotic recombination permits the generation of mosaic flies with efficiency and control. With heat-inducible or tissue-specific FLP transgenes at our disposal, we can engineer mosaics carrying clones of homozygous cells that come from specific pools of heterozygous precursors. This permits detailed cell lineage analysis followed by mosaic analysis of gene functions in the underlying developmental processes. Expression of transgenes (e.g., reporters) only in the homozygous cells enables mosaic analysis in the complex nervous system. Tracing neuronal lineages by using mosaics revolutionized mechanistic studies of neuronal diversification and differentiation, exemplifying the power of genetic mosaics in developmental biology. WIREs Dev Biol 2014, 3:69–81. doi: 10.1002/wdev.122

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    01/01/14 | Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM.
    Shtengel G, Wang Y, Zhang Z, Goh WI, Hess HF, Kanchanawong P
    Methods in Cell Biology. 2014;123:273-94. doi: 10.1016/B978-0-12-420138-5.00015-X

    Many biomolecules in cells can be visualized with high sensitivity and specificity by fluorescence microscopy. However, the resolution of conventional light microscopy is limited by diffraction to ~200-250nm laterally and >500nm axially. Here, we describe superresolution methods based on single-molecule localization analysis of photoswitchable fluorophores (PALM: photoactivated localization microscopy) as well as our recent three-dimensional (3D) method (iPALM: interferometric PALM) that allows imaging with a resolution better than 20nm in all three dimensions. Considerations for their implementations, applications to multicolor imaging, and a recent development that extend the imaging depth of iPALM to ~750nm are discussed. As the spatial resolution of superresolution fluorescence microscopy converges with that of electron microscopy (EM), direct imaging of the same specimen using both approaches becomes feasible. This could be particularly useful for cross validation of experiments, and thus, we also describe recent methods that were developed for correlative superresolution fluorescence and EM.

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    01/01/14 | iPfam: a database of protein family and domain interactions found in the Protein Data Bank.
    Finn RD, Miller BL, Clements J, Bateman A
    Nucleic acids research. 2014 Jan;42:D364-73. doi: 10.1093/nar/gkt1210

    The database iPfam, available at http://ipfam.org, catalogues Pfam domain interactions based on known 3D structures that are found in the Protein Data Bank, providing interaction data at the molecular level. Previously, the iPfam domain-domain interaction data was integrated within the Pfam database and website, but it has now been migrated to a separate database. This allows for independent development, improving data access and giving clearer separation between the protein family and interactions datasets. In addition to domain-domain interactions, iPfam has been expanded to include interaction data for domain bound small molecule ligands. Functional annotations are provided from source databases, supplemented by the incorporation of Wikipedia articles where available. iPfam (version 1.0) contains >9500 domain-domain and 15 500 domain-ligand interactions. The new website provides access to this data in a variety of ways, including interactive visualizations of the interaction data.

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    01/01/14 | Microfabrication of a platform to measure and manipulate the mechanics of engineered microtissues.
    Ramade A, Legant WR, Picart C, Chen CS, Boudou T
    Methods in Cell Biology. 2014;121:191-211. doi: 10.1016/B978-0-12-800281-0.00013-0

    Engineered tissues can be used to understand fundamental features of biology, develop organotypic in vitro model systems, and as engineered tissue constructs for replacing damaged tissue in vivo. However, a key limitation is an inability to test the wide range of parameters that might impact the engineered tissue in a high-throughput manner and in an environment that mimics the three-dimensional (3D) native architecture. We developed a microfabricated platform to generate arrays of microtissues embedded within 3D micropatterned matrices. Microcantilevers simultaneously constrain microtissue formation and report forces generated by the microtissues in real time, opening the possibility to use high-throughput, low-volume screening for studies on engineered tissues. Thanks to the micrometer scale of the microtissues, this platform is also suitable for high-throughput monitoring of drug-induced effect on architecture and contractility in engineered tissues. Moreover, independent variations of the mechanical stiffness of the cantilevers and collagen matrix allow the measurement and manipulation of the mechanics of the microtissues. Thus, our approach will likely provide valuable opportunities to elucidate how biomechanical, electrical, biochemical, and genetic/epigenetic cues modulate the formation and maturation of 3D engineered tissues. In this chapter, we describe the microfabrication, preparation, and experimental use of such microfabricated tissue gauges.

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    Fetter Lab
    01/01/14 | Nanometer-resolution fluorescence electron microscopy (nano-EM) in cultured cells.
    Watanabe S, Lehmann M, Hujber E, Fetter RD, Richards J, Söhl-Kielczynski B, Felies A, Rosenmund C, Schmoranzer J, Jorgensen EM
    Methods in Molecular Biology. 2014;1117:503-26. doi: 10.1007/978-1-62703-776-1_22

    Nano-resolution fluorescence electron microscopy (nano-fEM) pinpoints the location of individual proteins in electron micrographs. Plastic sections are first imaged using a super-resolution fluorescence microscope and then imaged on an electron microscope. The two images are superimposed to correlate the position of labeled proteins relative to subcellular structures. Here, we describe the method in detail and present five technical advancements: the use of uranyl acetate during the freeze-substitution to enhance the contrast of tissues and reduce the loss of fluorescence, the use of ground-state depletion instead of photoactivation for temporal control of fluorescence, the use of organic fluorophores instead of fluorescent proteins to obtain brighter fluorescence signals, the use of tissue culture cells to broaden the utility of the method, and the use of a transmission electron microscope to achieve sharper images of ultrastructure.

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