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6 Janelia Publications

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    08/01/17 | Genetically encoded biosensors.
    Marvin JS, Looger LL, Lee RT, Schreiter ER
    USPTO. 2017 Aug 01;B2:

    The present disclosure provides, inter alia, genetically encoded recombinant peptide biosensors comprising analyte-binding framework portions and signaling portions, wherein the signaling portions are present within the framework portions at sites or amino acid positions that undergo a conformational change upon interaction of the framework portion with an analyte.

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    06/12/17 | Neural signatures of dynamic stimulus selection in Drosophila.
    Sun Y, Nern A, Franconville R, Dana H, Schreiter ER, Looger LL, Svoboda K, Kim DS, Hermundstad AM, Jayaraman V
    Nature Neuroscience. 2017 Jun 12;20(8):1104-13. doi: 10.1038/nn.4581

    Many animals orient using visual cues, but how a single cue is selected from among many is poorly understood. Here we show that Drosophila ring neurons—central brain neurons implicated in navigation—display visual stimulus selection. Using in vivo two-color two-photon imaging with genetically encoded calcium indicators, we demonstrate that individual ring neurons inherit simple-cell-like receptive fields from their upstream partners. Stimuli in the contralateral visual field suppressed responses to ipsilateral stimuli in both populations. Suppression strength depended on when and where the contralateral stimulus was presented, an effect stronger in ring neurons than in their upstream inputs. This history-dependent effect on the temporal structure of visual responses, which was well modeled by a simple biphasic filter, may determine how visual references are selected for the fly's internal compass. Our approach highlights how two-color calcium imaging can help identify and localize the origins of sensory transformations across synaptically connected neural populations.

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    04/06/17 | Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples.
    Kopek BG, Paez-Segala MG, Shtengel G, Sochacki KA, Sun MG, Wang Y, Xu CS, Van Engelenburg SB, Taraska JW, Looger LL, Hess HF
    Nature Protocols. 2017 May;12(5):916-946. doi: 10.1038/nprot.2017.017

    Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replication creates high-contrast, 3D images of the cytoplasmic surface of the plasma membrane but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (∼10-50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2-7 d, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.

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    03/15/17 | Confirmation of five novel susceptibility loci for systemic lupus erythematosus (SLE) and integrated network analysis of 82 SLE susceptibility loci.
    Molineros JE, Yang W, Zhou X, Sun C, Okada Y, Zhang H, Heng Chua K, Lau Y, Kochi Y, Suzuki A, Yamamoto K, Ma J, Bang S, Lee H, Kim K, Bae S, Zhang H, Shen N, Looger LL, Nath SK
    Human Molecular Genetics. 2017 Mar 15;26(6):1205-1216. doi: 10.1093/hmg/ddx026

    We recently identified ten novel SLE susceptibility loci in Asians and uncovered several additional suggestive loci requiring further validation. This study aimed to replicate five of these suggestive loci in a Han Chinese cohort from Hong Kong, followed by meta-analysis (11,656 cases and 23,968 controls) on previously reported Asian and European populations, and to perform bioinformatic analyses on all 82 reported SLE loci to identify shared regulatory signatures. We performed a battery of analyses for these five loci, as well as joint analyses on all 82 SLE loci. All five loci passed genome-wide significance: MYNN (rs10936599, Pmeta = 1.92 × 10-13, OR = 1.14), ATG16L2 (rs11235604, Pmeta = 8.87 × 10 -12, OR = 0.78), CCL22 (rs223881, Pmeta = 5.87 × 10-16, OR = 0.87), ANKS1A (rs2762340, Pmeta = 4.93 × 10-15, OR = 0.87) and RNASEH2C (rs1308020, Pmeta = 2.96 × 10-19, OR = 0.84) and co-located with annotated gene regulatory elements. The novel loci share genetic signatures with other reported SLE loci, including effects on gene expression, transcription factor binding, and epigenetic characteristics. Most (56%) of the correlated (r2 > 0.8) SNPs from the 82 SLE loci were implicated in differential expression (9.81 × 10-198 < P < 5 × 10-3) of cis-genes. Transcription factor binding sites for p53, MEF2A and E2F1 were significantly (P < 0.05) over-represented in SLE loci, consistent with apoptosis playing a critical role in SLE. Enrichment analysis revealed common pathways, gene ontology, protein domains, and cell type-specific expression. In summary, we provide evidence of five novel SLE susceptibility loci. Integrated bioinformatics using all 82 loci revealed that SLE susceptibility loci share many gene regulatory features, suggestive of conserved mechanisms of SLE etiopathogenesis.

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    03/01/17 | All-optical functional synaptic connectivity mapping in acute brain slices using CaMPARI.
    Zolnik TA, Sha F, Johenning F, Schreiter ER, Looger LL, Larkum ME, Sachdev RN
    The Journal of Physiology. 2017 Mar 01;595(5):1465-77. doi: 10.1113/JP273116

    The calcium-modulated photoactivatable ratiometric integrator CaMPARI (Fosque et al., 2015) facilitates the study of neural circuits by permanently marking cells active during user-specified temporal windows. Permanent marking enables measurement of signals from large swathes of tissue and easy correlation of activity with other structural or functional labels. One potential application of CaMPARI is labeling neurons postsynaptic to specific populations targeted for optogenetic stimulation, giving rise to all-optical functional connectivity mapping. Here, we characterized the response of CaMPARI to several common types of neuronal calcium signals in mouse acute cortical brain slices. Our experiments show that CaMPARI is effectively converted by both action potentials and sub-threshold synaptic inputs, and that conversion level is correlated to synaptic strength. Importantly, we found that conversion rate can be tuned: it is linearly related to light intensity. At low photoconversion light levels CaMPARI offers a wide dynamic range due to slower conversion rate; at high light levels conversion is more rapid and more sensitive to activity. Finally, we employed CaMPARI and optogenetics for functional circuit mapping in ex vivo acute brain slices, which preserve in vivo-like connectivity of axon terminals. With a single light source, we stimulated channelrhodopsin-2-expressing long-range posteromedial (POm) thalamic axon terminals in cortex and induced CaMPARI conversion in recipient cortical neurons. We found that POm stimulation triggers robust photoconversion of layer 5 cortical neurons and weaker conversion of layer 2/3 neurons. Thus, CaMPARI enables network-wide, tunable, all-optical functional circuit mapping that captures supra- and sub-threshold depolarization. This article is protected by copyright. All rights reserved.

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    01/01/17 | Solution of the structure of a calmodulin-peptide complex in a novel configuration from a variably twinned data set.
    Keller JP
    Acta Crystallographica. Section D, Structural Biology. 2017 Jan 01;73(Pt 1):22-31. doi: 10.1107/S2059798316019318

    Structure determination of conformationally variable proteins can prove challenging even when many possible molecular-replacement (MR) search models of high sequence similarity are available. Calmodulin (CaM) is perhaps the best-studied archetype of these flexible proteins: while there are currently ∼450 structures of significant sequence similarity available in the Protein Data Bank (PDB), novel conformations of CaM and complexes thereof continue to be reported. Here, the details of the solution of a novel peptide-CaM complex structure by MR are presented, in which only one MR solution of marginal quality was found despite the use of 120 different search models, an exclusivity enhanced by the presence of a high degree of hemihedral twinning (overall refined twin fraction = 0.43). Ambiguities in the initial MR electron-density maps were overcome by using MR-SAD: phases from the MR partial model were used to identify weak anomalous scatterers (calcium, sulfur and chloride), which were in turn used to improve the phases, automatically rebuild the structure and resolve sequence ambiguities. Retrospective analysis of consecutive wedges of the original data sets showed twin fractions ranging from 0.32 to 0.55, suggesting that the data sets were variably twinned. Despite these idiosyncrasies and obstacles, the data themselves and the final model were of high quality and indeed showed a novel, nearly right-angled conformation of the bound peptide.

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