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Nature Methods

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https://www.readbyqxmd.com/read/28892089/crispr-cas9-based-photoactivatable-transcription-systems-to-induce-neuronal-differentiation
#1
Yuta Nihongaki, Yuichi Furuhata, Takahiro Otabe, Saki Hasegawa, Keitaro Yoshimoto, Moritoshi Sato
Our improved CRISPR-Cas9-based photoactivatable transcription systems, CPTS2.0 and Split-CPTS2.0, enable high blue-light-inducible activation of endogenous target genes in various human cell lines. We achieved reversible activation of target genes with CPTS2.0 and induced neuronal differentiation in induced pluripotent stem cells (iPSCs) by upregulating NEUROD1 with Split-CPTS2.0.
September 11, 2017: Nature Methods
https://www.readbyqxmd.com/read/28892088/pan-neuronal-calcium-imaging-with-cellular-resolution-in-freely-swimming-zebrafish
#2
Dal Hyung Kim, Jungsoo Kim, João C Marques, Abhinav Grama, David G C Hildebrand, Wenchao Gu, Jennifer M Li, Drew N Robson
Calcium imaging with cellular resolution typically requires an animal to be tethered under a microscope, which substantially restricts the range of behaviors that can be studied. To expand the behavioral repertoire amenable to imaging, we have developed a tracking microscope that enables whole-brain calcium imaging with cellular resolution in freely swimming larval zebrafish. This microscope uses infrared imaging to track a target animal in a behavior arena. On the basis of the predicted trajectory of the animal, we applied optimal control theory to a motorized stage system to cancel brain motion in three dimensions...
September 11, 2017: Nature Methods
https://www.readbyqxmd.com/read/28869758/internally-ratiometric-fluorescent-sensors-for-evaluation-of-intracellular-gtp-levels-and-distribution
#3
Anna Bianchi-Smiraglia, Mitra S Rana, Colleen E Foley, Leslie M Paul, Brittany C Lipchick, Sudha Moparthy, Kalyana Moparthy, Emily E Fink, Archis Bagati, Edward Hurley, Hayley C Affronti, Andrei V Bakin, Eugene S Kandel, Dominic J Smiraglia, Maria Laura Feltri, Rui Sousa, Mikhail A Nikiforov
GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels...
September 4, 2017: Nature Methods
https://www.readbyqxmd.com/read/28869757/a-general-method-to-fine-tune-fluorophores-for-live-cell-and-in-vivo-imaging
#4
Jonathan B Grimm, Anand K Muthusamy, Yajie Liang, Timothy A Brown, William C Lemon, Ronak Patel, Rongwen Lu, John J Macklin, Philipp J Keller, Na Ji, Luke D Lavis
Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. We refined and extended this strategy, finding that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties of rhodamine dyes with unprecedented precision...
September 4, 2017: Nature Methods
https://www.readbyqxmd.com/read/28869756/red-shifted-luciferase-luciferin-pairs-for-enhanced-bioluminescence-imaging
#5
Hsien-Wei Yeh, Omran Karmach, Ao Ji, David Carter, Manuela M Martins-Green, Hui-Wang Ai
Red-shifted bioluminescence reporters are desirable for biological imaging. We describe the development of red-shifted luciferins based on synthetic coelenterazine analogs and corresponding mutants of NanoLuc that enable bright bioluminescence. One pair in particular showed superior in vitro and in vivo sensitivity over commonly used bioluminescence reporters. We adapted this pair to develop a bioluminescence resonance-energy-based Antares reporter called Antares2, which offers improved signal from deep tissues...
September 4, 2017: Nature Methods
https://www.readbyqxmd.com/read/28869755/sampling-strategies-to-capture-single-cell-heterogeneity
#6
Satwik Rajaram, Louise E Heinrich, John D Gordan, Jayant Avva, Kathy M Bonness, Agnieszka K Witkiewicz, James S Malter, Chloe E Atreya, Robert S Warren, Lani F Wu, Steven J Altschuler
Advances in single-cell technologies have highlighted the prevalence and biological significance of cellular heterogeneity. A critical question researchers face is how to design experiments that faithfully capture the true range of heterogeneity from samples of cellular populations. Here we develop a data-driven approach, illustrated in the context of image data, that estimates the sampling depth required for prospective investigations of single-cell heterogeneity from an existing collection of samples.
September 4, 2017: Nature Methods
https://www.readbyqxmd.com/read/28846090/an-improved-atac-seq-protocol-reduces-background-and-enables-interrogation-of-frozen-tissues
#7
M Ryan Corces, Alexandro E Trevino, Emily G Hamilton, Peyton G Greenside, Nicholas A Sinnott-Armstrong, Sam Vesuna, Ansuman T Satpathy, Adam J Rubin, Kathleen S Montine, Beijing Wu, Arwa Kathiria, Seung Woo Cho, Maxwell R Mumbach, Ava C Carter, Maya Kasowski, Lisa A Orloff, Viviana I Risca, Anshul Kundaje, Paul A Khavari, Thomas J Montine, William J Greenleaf, Howard Y Chang
We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol generates chromatin accessibility profiles from archival frozen tissue samples and 50-μm sections, revealing the activities of disease-associated DNA elements in distinct human brain structures. The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies...
August 28, 2017: Nature Methods
https://www.readbyqxmd.com/read/28846089/oscillatory-stimuli-differentiate-adapting-circuit-topologies
#8
Sahand Jamal Rahi, Johannes Larsch, Kresti Pecani, Alexander Y Katsov, Nahal Mansouri, Krasimira Tsaneva-Atanasova, Eduardo D Sontag, Frederick R Cross
Biology emerges from interactions between molecules, which are challenging to elucidate with current techniques. An orthogonal approach is to probe for 'response signatures' that identify specific circuit motifs. For example, bistability, hysteresis, or irreversibility are used to detect positive feedback loops. For adapting systems, such signatures are not known. Only two circuit motifs generate adaptation: negative feedback loops (NFLs) and incoherent feed-forward loops (IFFLs). On the basis of computational testing and mathematical proofs, we propose differential signatures: in response to oscillatory stimulation, NFLs but not IFFLs show refractory-period stabilization (robustness to changes in stimulus duration) or period skipping...
August 28, 2017: Nature Methods
https://www.readbyqxmd.com/read/28846088/massively-parallel-single-nucleus-rna-seq-with-dronc-seq
#9
Naomi Habib, Inbal Avraham-Davidi, Anindita Basu, Tyler Burks, Karthik Shekhar, Matan Hofree, Sourav R Choudhury, François Aguet, Ellen Gelfand, Kristin Ardlie, David A Weitz, Orit Rozenblatt-Rosen, Feng Zhang, Aviv Regev
Single-nucleus RNA sequencing (sNuc-seq) profiles RNA from tissues that are preserved or cannot be dissociated, but it does not provide high throughput. Here, we develop DroNc-seq: massively parallel sNuc-seq with droplet technology. We profile 39,111 nuclei from mouse and human archived brain samples to demonstrate sensitive, efficient, and unbiased classification of cell types, paving the way for systematic charting of cell atlases.
August 28, 2017: Nature Methods
https://www.readbyqxmd.com/read/28846087/convolutional-neural-networks-for-automated-annotation-of-cellular-cryo-electron-tomograms
#10
Muyuan Chen, Wei Dai, Stella Y Sun, Darius Jonasch, Cynthia Y He, Michael F Schmid, Wah Chiu, Steven J Ludtke
Cellular electron cryotomography offers researchers the ability to observe macromolecules frozen in action in situ, but a primary challenge with this technique is identifying molecular components within the crowded cellular environment. We introduce a method that uses neural networks to dramatically reduce the time and human effort required for subcellular annotation and feature extraction. Subsequent subtomogram classification and averaging yield in situ structures of molecular components of interest. The method is available in the EMAN2...
August 28, 2017: Nature Methods
https://www.readbyqxmd.com/read/28825706/chromvar-inferring-transcription-factor-associated-accessibility-from-single-cell-epigenomic-data
#11
Alicia N Schep, Beijing Wu, Jason D Buenrostro, William J Greenleaf
Single-cell ATAC-seq (scATAC) yields sparse data that make conventional analysis challenging. We developed chromVAR (http://www.github.com/GreenleafLab/chromVAR), an R package for analyzing sparse chromatin-accessibility data by estimating gain or loss of accessibility within peaks sharing the same motif or annotation while controlling for technical biases. chromVAR enables accurate clustering of scATAC-seq profiles and characterization of known and de novo sequence motifs associated with variation in chromatin accessibility...
August 21, 2017: Nature Methods
https://www.readbyqxmd.com/read/28825705/reversed-graph-embedding-resolves-complex-single-cell-trajectories
#12
Xiaojie Qiu, Qi Mao, Ying Tang, Li Wang, Raghav Chawla, Hannah A Pliner, Cole Trapnell
Single-cell trajectories can unveil how gene regulation governs cell fate decisions. However, learning the structure of complex trajectories with multiple branches remains a challenging computational problem. We present Monocle 2, an algorithm that uses reversed graph embedding to describe multiple fate decisions in a fully unsupervised manner. We applied Monocle 2 to two studies of blood development and found that mutations in the genes encoding key lineage transcription factors divert cells to alternative fates...
August 21, 2017: Nature Methods
https://www.readbyqxmd.com/read/28825703/virtual-reality-for-freely-moving-animals
#13
John R Stowers, Maximilian Hofbauer, Renaud Bastien, Johannes Griessner, Peter Higgins, Sarfarazhussain Farooqui, Ruth M Fischer, Karin Nowikovsky, Wulf Haubensak, Iain D Couzin, Kristin Tessmar-Raible, Andrew D Straw
Standard animal behavior paradigms incompletely mimic nature and thus limit our understanding of behavior and brain function. Virtual reality (VR) can help, but it poses challenges. Typical VR systems require movement restrictions but disrupt sensorimotor experience, causing neuronal and behavioral alterations. We report the development of FreemoVR, a VR system for freely moving animals. We validate immersive VR for mice, flies, and zebrafish. FreemoVR allows instant, disruption-free environmental reconfigurations and interactions between real organisms and computer-controlled agents...
August 21, 2017: Nature Methods
https://www.readbyqxmd.com/read/28805795/m3-an-integrative-framework-for-structure-determination-of-molecular-machines
#14
Ezgi Karaca, João P G L M Rodrigues, Andrea Graziadei, Alexandre M J J Bonvin, Teresa Carlomagno
We present a broadly applicable, user-friendly protocol that incorporates sparse and hybrid experimental data to calculate quasi-atomic-resolution structures of molecular machines. The protocol uses the HADDOCK framework, accounts for extensive structural rearrangements both at the domain and atomic levels and accepts input from all structural and biochemical experiments whose data can be translated into interatomic distances and/or molecular shapes.
August 14, 2017: Nature Methods
https://www.readbyqxmd.com/read/28805794/large-scale-mapping-of-cortical-synaptic-projections-with-extracellular-electrode-arrays
#15
Mark Shein-Idelson, Lorenz Pammer, Mike Hemberger, Gilles Laurent
Understanding circuit computation in the nervous system requires sampling activity over large neural populations and maximizing the number of features that can be extracted. By combining planar arrays of extracellular electrodes with the three-layered cortex of turtles, we show that synaptic signals induced along individual axons as well as action potentials can be easily captured. Two types of information can be extracted from these signals, the neuronal subtype (inhibitory or excitatory)-whose identification is more reliable than with traditional measures such as action potential width-and a (partial) spatial map of functional axonal projections from individual neurons...
August 14, 2017: Nature Methods
https://www.readbyqxmd.com/read/28805793/conformational-landscape-of-a-virus-by-single-particle-x-ray-scattering
#16
Ahmad Hosseinizadeh, Ghoncheh Mashayekhi, Jeremy Copperman, Peter Schwander, Ali Dashti, Reyhaneh Sepehr, Russell Fung, Marius Schmidt, Chun Hong Yoon, Brenda G Hogue, Garth J Williams, Andrew Aquila, Abbas Ourmazd
Using a manifold-based analysis of experimental diffraction snapshots from an X-ray free electron laser, we determine the three-dimensional structure and conformational landscape of the PR772 virus to a detector-limited resolution of 9 nm. Our results indicate that a single conformational coordinate controls reorganization of the genome, growth of a tubular structure from a portal vertex and release of the genome. These results demonstrate that single-particle X-ray scattering has the potential to shed light on key biological processes...
August 14, 2017: Nature Methods
https://www.readbyqxmd.com/read/28858341/multielectrodes-join-the-connectome
#17
Harvey A Swadlow, Jose-Manuel Alonso
No abstract text is available yet for this article.
September 2017: Nature Methods
https://www.readbyqxmd.com/read/28825704/statistical-control-of-peptide-and-protein-error-rates-in-large-scale-targeted-data-independent-acquisition-analyses
#18
George Rosenberger, Isabell Bludau, Uwe Schmitt, Moritz Heusel, Christie L Hunter, Yansheng Liu, Michael J MacCoss, Brendan X MacLean, Alexey I Nesvizhskii, Patrick G A Pedrioli, Lukas Reiter, Hannes L Röst, Stephen Tate, Ying S Ting, Ben C Collins, Ruedi Aebersold
Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the main method for high-throughput identification and quantification of peptides and inferred proteins. Within this field, data-independent acquisition (DIA) combined with peptide-centric scoring, as exemplified by the technique SWATH-MS, has emerged as a scalable method to achieve deep and consistent proteome coverage across large-scale data sets. We demonstrate that statistical concepts developed for discovery proteomics based on spectrum-centric scoring can be adapted to large-scale DIA experiments that have been analyzed with peptide-centric scoring strategies, and we provide guidance on their application...
September 2017: Nature Methods
https://www.readbyqxmd.com/read/28783155/histocat-analysis-of-cell-phenotypes-and-interactions-in-multiplex-image-cytometry-data
#19
Denis Schapiro, Hartland W Jackson, Swetha Raghuraman, Jana R Fischer, Vito R T Zanotelli, Daniel Schulz, Charlotte Giesen, Raúl Catena, Zsuzsanna Varga, Bernd Bodenmiller
Single-cell, spatially resolved omics analysis of tissues is poised to transform biomedical research and clinical practice. We have developed an open-source, computational histology topography cytometry analysis toolbox (histoCAT) to enable interactive, quantitative, and comprehensive exploration of individual cell phenotypes, cell-cell interactions, microenvironments, and morphological structures within intact tissues. We highlight the unique abilities of histoCAT through analysis of highly multiplexed mass cytometry images of human breast cancer tissues...
September 2017: Nature Methods
https://www.readbyqxmd.com/read/28783154/informed-proteomics-open-source-software-package-for-top-down-proteomics
#20
Jungkap Park, Paul D Piehowski, Christopher Wilkins, Mowei Zhou, Joshua Mendoza, Grant M Fujimoto, Bryson C Gibbons, Jared B Shaw, Yufeng Shen, Anil K Shukla, Ronald J Moore, Tao Liu, Vladislav A Petyuk, Nikola Tolić, Ljiljana Paša-Tolić, Richard D Smith, Samuel H Payne, Sangtae Kim
Top-down proteomics, the analysis of intact proteins in their endogenous form, preserves valuable information about post-translation modifications, isoforms and proteolytic processing. The quality of top-down liquid chromatography-tandem MS (LC-MS/MS) data sets is rapidly increasing on account of advances in instrumentation and sample-processing protocols. However, top-down mass spectra are substantially more complex than conventional bottom-up data. New algorithms and software tools for confident proteoform identification and quantification are needed...
September 2017: Nature Methods
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