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Nature Methods

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https://www.readbyqxmd.com/read/29736000/high-speed-volumetric-imaging-of-neuronal-activity-in-freely-moving-rodents
#1
Oliver Skocek, Tobias Nöbauer, Lukas Weilguny, Francisca Martínez Traub, Chuying Naomi Xia, Maxim I Molodtsov, Abhinav Grama, Masahito Yamagata, Daniel Aharoni, David D Cox, Peyman Golshani, Alipasha Vaziri
Thus far, optical recording of neuronal activity in freely behaving animals has been limited to a thin axial range. We present a head-mounted miniaturized light-field microscope (MiniLFM) capable of capturing neuronal network activity within a volume of 700 × 600 × 360 µm3 at 16 Hz in the hippocampus of freely moving mice. We demonstrate that neurons separated by as little as ~15 µm and at depths up to 360 µm can be discriminated.
May 7, 2018: Nature Methods
https://www.readbyqxmd.com/read/29735999/single-shot-super-resolution-total-internal-reflection-fluorescence-microscopy
#2
Min Guo, Panagiotis Chandris, John Paul Giannini, Adam J Trexler, Robert Fischer, Jiji Chen, Harshad D Vishwasrao, Ivan Rey-Suarez, Yicong Wu, Xufeng Wu, Clare M Waterman, George H Patterson, Arpita Upadhyaya, Justin W Taraska, Hari Shroff
We combined instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM) in an approach referred to as instant TIRF-SIM, thereby improving the lateral spatial resolution of TIRFM to 115 ± 13 nm without compromising speed, and enabling imaging frame rates up to 100 Hz over hundreds of time points. We applied instant TIRF-SIM to multiple live samples and achieved rapid, high-contrast super-resolution imaging close to the coverslip surface.
May 7, 2018: Nature Methods
https://www.readbyqxmd.com/read/29735998/boxcar-acquisition-method-enables-single-shot-proteomics-at-a-depth-of-10-000-proteins-in-100-minutes
#3
Florian Meier, Philipp E Geyer, Sebastian Virreira Winter, Juergen Cox, Matthias Mann
Great advances have been made in sensitivity and acquisition speed on the Orbitrap mass analyzer, enabling increasingly deep proteome coverage. However, these advances have been mainly limited to the MS2 level, whereas ion beam sampling for the MS1 scans remains extremely inefficient. Here we report a data-acquisition method, termed BoxCar, in which filling multiple narrow mass-to-charge segments increases the mean ion injection time more than tenfold as compared to that of a standard full scan. In 1-h analyses, the method provided MS1-level evidence for more than 90% of the proteome of a human cancer cell line that had previously been identified in 24 fractions, and it quantified more than 6,200 proteins in ten of ten replicates...
May 7, 2018: Nature Methods
https://www.readbyqxmd.com/read/29735997/discovery-of-proteins-associated-with-a-predefined-genomic-locus-via-dcas9-apex-mediated-proximity-labeling
#4
Samuel A Myers, Jason Wright, Ryan Peckner, Brian T Kalish, Feng Zhang, Steven A Carr
Regulation of gene expression is primarily controlled by changes in the proteins that occupy genes' regulatory elements. We developed genomic locus proteomics (GLoPro), in which we combine CRISPR-based genome targeting, proximity labeling, and quantitative proteomics to discover proteins associated with a specific genomic locus in native cellular contexts.
May 7, 2018: Nature Methods
https://www.readbyqxmd.com/read/29735996/c-berst-defining-subnuclear-proteomic-landscapes-at-genomic-elements-with-dcas9-apex2
#5
Xin D Gao, Li-Chun Tu, Aamir Mir, Tomás Rodriguez, Yuehe Ding, John Leszyk, Job Dekker, Scott A Shaffer, Lihua Julie Zhu, Scot A Wolfe, Erik J Sontheimer
Mapping proteomic composition at distinct genomic loci in living cells has been a long-standing challenge. Here we report that dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. C-BERST enables the high-throughput identification of proteins associated with specific sequences, thereby facilitating annotation of these factors and their roles.
May 7, 2018: Nature Methods
https://www.readbyqxmd.com/read/29713083/accurate-detection-of-complex-structural-variations-using-single-molecule-sequencing
#6
Fritz J Sedlazeck, Philipp Rescheneder, Moritz Smolka, Han Fang, Maria Nattestad, Arndt von Haeseler, Michael C Schatz
Structural variations are the greatest source of genetic variation, but they remain poorly understood because of technological limitations. Single-molecule long-read sequencing has the potential to dramatically advance the field, although high error rates are a challenge with existing methods. Addressing this need, we introduce open-source methods for long-read alignment (NGMLR; https://github.com/philres/ngmlr ) and structural variant identification (Sniffles; https://github.com/fritzsedlazeck/Sniffles ) that provide unprecedented sensitivity and precision for variant detection, even in repeat-rich regions and for complex nested events that can have substantial effects on human health...
April 30, 2018: Nature Methods
https://www.readbyqxmd.com/read/29713082/self-interference-3d-super-resolution-microscopy-for-deep-tissue-investigations
#7
Pierre Bon, Jeanne Linarès-Loyez, Maxime Feyeux, Kevin Alessandri, Brahim Lounis, Pierre Nassoy, Laurent Cognet
Fluorescence localization microscopy has achieved near-molecular resolution capable of revealing ultra-structures, with a broad range of applications, especially in cellular biology. However, it remains challenging to attain such resolution in three dimensions and inside biological tissues beyond the first cell layer. Here we introduce SELFI, a framework for 3D single-molecule localization within multicellular specimens and tissues. The approach relies on self-interference generated within the microscope's point spread function (PSF) to simultaneously encode equiphase and intensity fluorescence signals, which together provide the 3D position of an emitter...
April 30, 2018: Nature Methods
https://www.readbyqxmd.com/read/29713081/picky-comprehensively-detects-high-resolution-structural-variants-in-nanopore-long-reads
#8
Liang Gong, Chee-Hong Wong, Wei-Chung Cheng, Harianto Tjong, Francesca Menghi, Chew Yee Ngan, Edison T Liu, Chia-Lin Wei
Acquired genomic structural variants (SVs) are major hallmarks of cancer genomes, but they are challenging to reconstruct from short-read sequencing data. Here we exploited the long reads of the nanopore platform using our customized pipeline, Picky ( https://github.com/TheJacksonLaboratory/Picky ), to reveal SVs of diverse architecture in a breast cancer model. We identified the full spectrum of SVs with superior specificity and sensitivity relative to short-read analyses, and uncovered repetitive DNA as the major source of variation...
April 30, 2018: Nature Methods
https://www.readbyqxmd.com/read/29630062/real-time-3d-single-molecule-localization-using-experimental-point-spread-functions
#9
Yiming Li, Markus Mund, Philipp Hoess, Joran Deschamps, Ulf Matti, Bianca Nijmeijer, Vilma Jimenez Sabinina, Jan Ellenberg, Ingmar Schoen, Jonas Ries
We present a real-time fitter for 3D single-molecule localization microscopy using experimental point spread functions (PSFs) that achieves minimal uncertainty in 3D on any microscope and is compatible with any PSF engineering approach. We used this method to image cellular structures and attained unprecedented image quality for astigmatic PSFs. The fitter compensates for most optical aberrations and makes accurate 3D super-resolution microscopy broadly accessible, even on standard microscopes without dedicated 3D optics...
April 9, 2018: Nature Methods
https://www.readbyqxmd.com/read/29630061/fateid-infers-cell-fate-bias-in-multipotent-progenitors-from-single-cell-rna-seq-data
#10
Josip S Herman, Sagar, Dominic Grün
To understand stem cell differentiation along multiple lineages, it is necessary to resolve heterogeneous cellular states and the ancestral relationships between them. We developed a robotic miniaturized CEL-Seq2 implementation to carry out deep single-cell RNA-seq of ∼2,000 mouse hematopoietic progenitors enriched for lymphoid lineages, and used an improved clustering algorithm, RaceID3, to identify cell types. To resolve subtle transcriptome differences indicative of lineage biases, we developed FateID, an iterative supervised learning algorithm for the probabilistic quantification of cell fate bias in progenitor populations...
April 9, 2018: Nature Methods
https://www.readbyqxmd.com/read/29608556/real-time-fluorescence-and-deformability-cytometry
#11
Philipp Rosendahl, Katarzyna Plak, Angela Jacobi, Martin Kraeter, Nicole Toepfner, Oliver Otto, Christoph Herold, Maria Winzi, Maik Herbig, Yan Ge, Salvatore Girardo, Katrin Wagner, Buzz Baum, Jochen Guck
The throughput of cell mechanical characterization has recently approached that of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we developed an approach that combines real-time 1D-imaging fluorescence and deformability cytometry in one instrument (RT-FDC), thus opening many new research avenues. We demonstrated its utility by using subcellular fluorescence localization to identify mitotic cells and test for mechanical changes in those cells in an RNA interference screen...
April 2, 2018: Nature Methods
https://www.readbyqxmd.com/read/29608555/scmap-projection-of-single-cell-rna-seq-data-across-data-sets
#12
Vladimir Yu Kiselev, Andrew Yiu, Martin Hemberg
Single-cell RNA-seq (scRNA-seq) allows researchers to define cell types on the basis of unsupervised clustering of the transcriptome. However, differences in experimental methods and computational analyses make it challenging to compare data across experiments. Here we present scmap (http://bioconductor.org/packages/scmap; web version at http://www.sanger.ac.uk/science/tools/scmap), a method for projecting cells from an scRNA-seq data set onto cell types or individual cells from other experiments.
April 2, 2018: Nature Methods
https://www.readbyqxmd.com/read/29608554/specter-linear-deconvolution-for-targeted-analysis-of-data-independent-acquisition-mass-spectrometry-proteomics
#13
Ryan Peckner, Samuel A Myers, Alvaro Sebastian Vaca Jacome, Jarrett D Egertson, Jennifer G Abelin, Michael J MacCoss, Steven A Carr, Jacob D Jaffe
Mass spectrometry with data-independent acquisition (DIA) is a promising method to improve the comprehensiveness and reproducibility of targeted and discovery proteomics, in theory by systematically measuring all peptide precursors in a biological sample. However, the analytical challenges involved in discriminating between peptides with similar sequences in convoluted spectra have limited its applicability in important cases, such as the detection of single-nucleotide polymorphisms (SNPs) and alternative site localizations in phosphoproteomics data...
April 2, 2018: Nature Methods
https://www.readbyqxmd.com/read/29600992/response-to-unexpected-mutations-after-crispr-cas9-editing-in-vivo
#14
Caleb A Lareau, Kendell Clement, Jonathan Y Hsu, Vikram Pattanayak, J Keith Joung, Martin J Aryee, Luca Pinello
No abstract text is available yet for this article.
March 30, 2018: Nature Methods
https://www.readbyqxmd.com/read/29600991/response-to-unexpected-mutations-after-crispr-cas9-editing-in-vivo
#15
Lauryl M J Nutter, Jason D Heaney, K C Kent Lloyd, Stephen A Murray, John R Seavitt, William C Skarnes, Lydia Teboul, Steve D M Brown, Mark Moore
No abstract text is available yet for this article.
March 30, 2018: Nature Methods
https://www.readbyqxmd.com/read/29600990/response-to-unexpected-mutations-after-crispr-cas9-editing-in-vivo
#16
Sang-Tae Kim, Jeongbin Park, Daesik Kim, Kyoungmi Kim, Sangsu Bae, Matthias Schlesner, Jin-Soo Kim
No abstract text is available yet for this article.
March 30, 2018: Nature Methods
https://www.readbyqxmd.com/read/29600989/response-to-unexpected-mutations-after-crispr-cas9-editing-in-vivo
#17
Christopher J Wilson, Tim Fennell, Anne Bothmer, Morgan L Maeder, Deepak Reyon, Cecilia Cotta-Ramusino, Cecilia A Fernandez, Eugenio Marco, Luis A Barrera, Hariharan Jayaram, Charles F Albright, Gerald F Cox, George M Church, Vic E Myer
No abstract text is available yet for this article.
March 30, 2018: Nature Methods
https://www.readbyqxmd.com/read/29600988/response-to-unexpected-mutations-after-crispr-cas9-editing-in-vivo
#18
Reynald M Lescarbeau, Bradley Murray, Thomas M Barnes, Nessan Bermingham
No abstract text is available yet for this article.
March 30, 2018: Nature Methods
https://www.readbyqxmd.com/read/29578537/photoactivatable-drugs-for-nicotinic-optopharmacology
#19
Sambashiva Banala, Matthew C Arvin, Nicholas M Bannon, Xiao-Tao Jin, John J Macklin, Yong Wang, Can Peng, Guiqing Zhao, John J Marshall, Kyle R Gee, David L Wokosin, Veronica J Kim, J Michael McIntosh, Anis Contractor, Henry A Lester, Yevgenia Kozorovitskiy, Ryan M Drenan, Luke D Lavis
Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.
March 26, 2018: Nature Methods
https://www.readbyqxmd.com/read/29578536/burden-driven-feedback-control-of-gene-expression
#20
Francesca Ceroni, Alice Boo, Simone Furini, Thomas E Gorochowski, Olivier Borkowski, Yaseen N Ladak, Ali R Awan, Charlie Gilbert, Guy-Bart Stan, Tom Ellis
Cells use feedback regulation to ensure robust growth despite fluctuating demands for resources and differing environmental conditions. However, the expression of foreign proteins from engineered constructs is an unnatural burden that cells are not adapted for. Here we combined RNA-seq with an in vivo assay to identify the major transcriptional changes that occur in Escherichia coli when inducible synthetic constructs are expressed. We observed that native promoters related to the heat-shock response activated expression rapidly in response to synthetic expression, regardless of the construct...
March 26, 2018: Nature Methods
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