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Nature Protocols

Arun Sharma, Wesley L McKeithan, Ricardo Serrano, Tomoya Kitani, Paul W Burridge, Juan C Del Álamo, Mark Mercola, Joseph C Wu
Cardiotoxicity has historically been a major cause of drug removal from the pharmaceutical market. Several chemotherapeutic compounds have been noted for their propensities to induce dangerous cardiac-specific side effects such as arrhythmias or cardiomyocyte apoptosis. However, improved preclinical screening methodologies have enabled cardiotoxic compounds to be identified earlier in the drug development pipeline. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be used to screen for drug-induced alterations in cardiac cellular contractility, electrophysiology, and viability...
November 9, 2018: Nature Protocols
Yuriko Osakabe, Zhenchang Liang, Chong Ren, Chikako Nishitani, Keishi Osakabe, Masato Wada, Sadao Komori, Mickael Malnoy, Riccardo Velasco, Michele Poli, Min-Hee Jung, Ok-Jae Koo, Roberto Viola, Chidananda Nagamangala Kanchiswamy
The CRISPR-Cas9 genome-editing tool and the availability of whole-genome sequences from plant species have revolutionized our ability to introduce targeted mutations into important crop plants, both to explore genetic changes and to introduce new functionalities. Here, we describe protocols adapting the CRISPR-Cas9 system to apple and grapevine plants, using both plasmid-mediated genome editing and the direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to achieve efficient DNA-free targeted mutations in apple and grapevine protoplasts...
November 2, 2018: Nature Protocols
Claudio Iacobucci, Michael Götze, Christian H Ihling, Christine Piotrowski, Christian Arlt, Mathias Schäfer, Christoph Hage, Rico Schmidt, Andrea Sinz
Chemical cross-linking in combination with mass spectrometric analysis of the created cross-linked products is an emerging technology aimed at deriving valuable structural information from proteins and protein complexes. The goal of our protocol is to obtain distance constraints for structure determination of proteins and to investigate protein-protein interactions. We present an integrated workflow for cross-linking/mass spectrometry (MS) based on protein cross-linking with MS-cleavable reagents, followed by enzymatic digestion, enrichment of cross-linked peptides by strong cation-exchange chromatography (SCX), and LC/MS/MS analysis...
October 31, 2018: Nature Protocols
Helle Krogh Pedersen, Sofia K Forslund, Valborg Gudmundsdottir, Anders Østergaard Petersen, Falk Hildebrand, Tuulia Hyötyläinen, Trine Nielsen, Torben Hansen, Peer Bork, S Dusko Ehrlich, Søren Brunak, Matej Oresic, Oluf Pedersen, Henrik Bjørn Nielsen
We recently presented a three-pronged association study that integrated human intestinal microbiome data derived from shotgun-based sequencing with untargeted serum metabolome data and measures of host physiology. Metabolome and microbiome data are high dimensional, posing a major challenge for data integration. Here, we present a step-by-step computational protocol that details and discusses the dimensionality-reduction techniques used and methods for subsequent integration and interpretation of such heterogeneous types of data...
October 31, 2018: Nature Protocols
Sonja Bergmann, Sean E Lawler, Yuan Qu, Colin M Fadzen, Justin M Wolfe, Michael S Regan, Bradley L Pentelute, Nathalie Y R Agar, Choi-Fong Cho
In vitro models of the blood-brain barrier (BBB) are critical tools for the study of BBB transport and the development of drugs that can reach the CNS. Brain endothelial cells grown in culture are often used to model the BBB; however, it is challenging to maintain reproducible BBB properties and function. 'BBB organoids' are obtained following coculture of endothelial cells, pericytes and astrocytes under low-adhesion conditions. These organoids reproduce many features of the BBB, including the expression of tight junctions, molecular transporters and drug efflux pumps, and hence can be used to model drug transport across the BBB...
October 31, 2018: Nature Protocols
Jichang Wang, Manvendra Singh, Chuanbo Sun, Daniel Besser, Alessandro Prigione, Zoltán Ivics, Laurence D Hurst, Zsuzsanna Izsvák
In the published version of this paper, the authors omitted a funding source. L.D.H. acknowledges support from the European Research Council (Advanced Grant ERC-2014-ADG 669207). The original article has not been corrected.
October 29, 2018: Nature Protocols
Divya Mohan, Daniel L Wansley, Brandon M Sie, Muhammad S Noon, Alan N Baer, Uri Laserson, H Benjamin Larman
The version of this paper originally published contained typesetter-introduced errors in some of the code commands, consisting of conversion of a closing backslash (\) to a forward slash (/). These errors have been corrected in the HTML and PDF versions of the protocol.
October 25, 2018: Nature Protocols
Ricardo Cruz-Acuña, Miguel Quirós, Sha Huang, Dorothée Siuda, Jason R Spence, Asma Nusrat, Andrés J García
In the version of this protocol originally published, the caption for Fig. 3 was erroneously placed with Fig. 4, and that for Fig. 4 was placed with Fig. 3. This error has been corrected in the HTML and PDF versions of the paper.
October 24, 2018: Nature Protocols
Julia Joung, Silvana Konermann, Jonathan S Gootenberg, Omar O Abudayyeh, Randall J Platt, Mark D Brigham, Neville E Sanjana, Feng Zhang
In the published version of this paper, Step 64 of the Procedure reads, "Refer to Steps 37-39 for NGS analysis of the sgRNA distribution." This step should refer the reader to Steps 35-39. This text has not been corrected in the original paper.
October 22, 2018: Nature Protocols
Nancy Osorio, Patrick Delmas
In the HTML version of this article published online, the abstract contains a typo in the first sentence: "key to understanding intestinal motility anGutn of therapeutic strategies" should read "key to understanding intestinal motility and crucial to the design of theraputic strategies." The PDF version of the article is correct.
October 22, 2018: Nature Protocols
Nick P van Goethem, Britt T J van Hagen, Jos Prickaerts
In the HTML version of this paper originally published online, text in Table 6 was misaligned in a way that made it difficult to determine which entries in the "Problem," "Possible reason," and "Solution" columns corresponded to one another. Additional but less severe alignment problems were also present in the PDF and print articles. These errors have been corrected in the HTML and PDF versions of the paper.
October 10, 2018: Nature Protocols
Florian A Dehmelt, Adam von Daranyi, Claire Leyden, Aristides B Arrenberg
The version of this paper originally published contained the following text errors: (1) In the abstract, "(ii) visual stimulation with moving bars; (ii) eye detection and tracking, as well as general motion detection" should have been "(ii) visual stimulation with moving bars; (iii) eye detection and tracking, as well as general motion detection." (2) In the legend for Table 1, "vertical pixel coordinate; LE, left eye; RE, right eye; x, horizontal pixel coordinate; y" should have read "LE, left eye; RE, right eye; x, horizontal pixel coordinate; y, vertical pixel coordinate...
August 21, 2018: Nature Protocols
Loïc A Royer, William C Lemon, Raghav K Chhetri, Philipp J Keller
We describe the implementation and use of an adaptive imaging framework for optimizing spatial resolution and signal strength in a light-sheet microscope. The framework, termed AutoPilot, comprises hardware and software modules for automatically measuring and compensating for mismatches between light-sheet and detection focal planes in living specimens. Our protocol enables researchers to introduce adaptive imaging capabilities in an existing light-sheet microscope or use our SiMView microscope blueprint to set up a new adaptive multiview light-sheet microscope...
November 2018: Nature Protocols
Yuesong Shi, Beril Polat, Qian Huang, Donald J Sirbuly
There is an ongoing need to develop ultrasensitive nanomechanical instrumentation that has high spatial and force resolution, as well as an ability to operate in various biological environments. Here, we present a compact nanofiber optic force transducer (NOFT) with sub-piconewton force sensitivity and a nanoscale footprint that paves the way to the probing of complex mechanical phenomena inside biomolecular systems. The NOFT platform comprises a SnO2 nanofiber optic equipped with a thin, compressible polymer cladding layer studded with plasmonic nanoparticles (NPs)...
November 2018: Nature Protocols
Areti Tsigkinopoulou, Aliah Hawari, Megan Uttley, Rainer Breitling
Ensemble modeling in molecular systems biology requires the reproducible translation of kinetic parameter data into informative probability distributions (priors), as well as approaches that sample parameters from these distributions without violating the thermodynamic consistency of the overall model. Although a number of pioneering frameworks for ensemble modeling have been published, the issue of generating informative priors has not yet been addressed. Here, we present a protocol that aims to fill this gap...
November 2018: Nature Protocols
Bushra Raj, James A Gagnon, Alexander F Schier
Lineage relationships among the large number of heterogeneous cell types generated during development are difficult to reconstruct in a high-throughput manner. We recently established a method, scGESTALT, that combines cumulative editing of a lineage barcode array by CRISPR-Cas9 with large-scale transcriptional profiling using droplet-based single-cell RNA sequencing (scRNA-seq). The technique generates edits in the barcode array over multiple timepoints using Cas9 and pools of single-guide RNAs (sgRNAs) introduced during early and late zebrafish embryonic development, which distinguishes it from similar Cas9 lineage-tracing methods...
November 2018: Nature Protocols
Yunru Yu, Luoran Shang, Jiahui Guo, Jie Wang, Yuanjin Zhao
This protocol describes the design of capillary microfluidics for spinning bioactive (cell-laden) microfibers for three-dimensional (3D) cell culture and tissue-engineering applications. We describe the assembly of three types of microfluidic systems: (i) simple injection capillary microfluidics for the spinning of uniform microfibers; (ii) hierarchical injection capillary microfluidics for the spinning of core-shell or spindle-knot structured microfibers; and (iii) multi-barrel injection capillary microfluidics for the spinning of microfibers with multiple components...
November 2018: Nature Protocols
Stefania Giacomello, Joakim Lundeberg
Elucidation of the complex processes involved in plant growth requires analysis of the spatial gene expression patterns in all affected tissues. This protocol extension is an adaptation of a protocol that describes how to use barcoded oligo-dT microarrays to evaluate spatial global gene expression profiles in mammalian tissue to enable it to be applied to plant material. Here, we explain the required adjustments for preparing and treating plant tissue sections on the array surface, specifically in regard to how to permeabilize and remove the tissue...
November 2018: Nature Protocols
Fredrik Salmén, Patrik L Ståhl, Annelie Mollbrink, José Fernández Navarro, Sanja Vickovic, Jonas Frisén, Joakim Lundeberg
Spatial resolution of gene expression enables gene expression events to be pinpointed to a specific location in biological tissue. Spatially resolved gene expression in tissue sections is traditionally analyzed using immunohistochemistry (IHC) or in situ hybridization (ISH). These technologies are invaluable tools for pathologists and molecular biologists; however, their throughput is limited to the analysis of only a few genes at a time. Recent advances in RNA sequencing (RNA-seq) have made it possible to obtain unbiased high-throughput gene expression data in bulk...
November 2018: Nature Protocols
Tae-Hyun Shin, Sunghwi Kang, Sohyeon Park, Jin-Sil Choi, Pan Ki Kim, Jinwoo Cheon
Sensors that detect specific molecules of interest in a living organism can be useful tools for studying biological functions and diseases. Here, we provide a protocol for the construction of nanosensors that can noninvasively detect biologically important targets with magnetic resonance imaging (MRI). The key operating principle of these sensors is magnetic resonance tuning (MRET), a distance-dependent phenomenon occurring between a superparamagnetic quencher and a paramagnetic enhancer. The change in distance between the two magnetic components modulates the longitudinal (T1 ) relaxivity of the enhancer...
November 2018: Nature Protocols
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