2,714 results match your criteria Nature protocols[Journal]


Purification of tubulin with controlled post-translational modifications by polymerization-depolymerization cycles.

Nat Protoc 2019 Apr 17. Epub 2019 Apr 17.

Institut Curie, PSL Research University, CNRS UMR 3348, Orsay, France.

In vitro reconstitutions of microtubule assemblies have provided essential mechanistic insights into the molecular bases of microtubule dynamics and their interactions with associated proteins. The tubulin code has emerged as a regulatory mechanism for microtubule functions, which suggests that tubulin isotypes and post-translational modifications (PTMs) play important roles in controlling microtubule functions. To investigate the tubulin code mechanism, it is essential to analyze different tubulin variants in vitro. Read More

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http://dx.doi.org/10.1038/s41596-019-0153-7DOI Listing

R-ChIP for genome-wide mapping of R-loops by using catalytically inactive RNASEH1.

Nat Protoc 2019 Apr 17. Epub 2019 Apr 17.

Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA.

Nascent RNA may form a three-stranded structure with DNA, called an R-loop, which has been linked to fundamental biological processes such as transcription, replication and genome instability. Here, we provide a detailed protocol for a newly developed strategy, named R-ChIP, for robust capture of R-loops genome-wide. Distinct from R-loop-mapping methods based on the monoclonal antibody S9. Read More

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http://dx.doi.org/10.1038/s41596-019-0154-6DOI Listing

High-speed imaging of glutamate release with genetically encoded sensors.

Nat Protoc 2019 Apr 15. Epub 2019 Apr 15.

Institute for Synaptic Physiology, Center for Molecular Neurobiology Hamburg, Hamburg, Germany.

The strength of an excitatory synapse depends on its ability to release glutamate and on the density of postsynaptic receptors. Genetically encoded glutamate indicators (GEGIs) allow eavesdropping on synaptic transmission at the level of cleft glutamate to investigate properties of the release machinery in detail. Based on the sensor iGluSnFR, we recently developed accelerated versions of GEGIs that allow investigation of synaptic release during 100-Hz trains. Read More

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http://www.nature.com/articles/s41596-019-0143-9
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http://dx.doi.org/10.1038/s41596-019-0143-9DOI Listing
April 2019
1 Read

Facile F labeling of non-activated arenes via a spirocyclic iodonium(III) ylide method and its application in the synthesis of the mGluR PET radiopharmaceutical [F]FPEB.

Nat Protoc 2019 Apr 12. Epub 2019 Apr 12.

Division of Nuclear Medicine and Molecular Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.

Non-activated (electron-rich and/or sterically hindered) arenes are prevalent chemical scaffolds in pharmaceuticals and positron emission tomography (PET) diagnostics. Despite substantial efforts to develop a general method to introduce F into these moieties for molecular imaging by PET, there is an urgent and unmet need for novel radiofluorination strategies that result in sufficiently labeled tracers to enable human imaging. Herein, we describe an efficient method that relies on spirocyclic iodonium ylide (SCIDY) precursors for one-step and regioselective radiofluorination, as well as proof-of-concept translation to the radiosynthesis of a clinically useful PET tracer, 3-[F]fluoro-5-[(pyridin-3-yl)ethynyl] benzonitrile ([F]FPEB). Read More

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http://www.nature.com/articles/s41596-019-0149-3
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http://dx.doi.org/10.1038/s41596-019-0149-3DOI Listing
April 2019
5 Reads

Nongenetic optical neuromodulation with silicon-based materials.

Nat Protoc 2019 Apr 12. Epub 2019 Apr 12.

Department of Chemistry, The University of Chicago, Chicago, IL, USA.

Optically controlled nongenetic neuromodulation represents a promising approach for the fundamental study of neural circuits and the clinical treatment of neurological disorders. Among the existing material candidates that can transduce light energy into biologically relevant cues, silicon (Si) is particularly advantageous due to its highly tunable electrical and optical properties, ease of fabrication into multiple forms, ability to absorb a broad spectrum of light, and biocompatibility. This protocol describes a rational design principle for Si-based structures, general procedures for material synthesis and device fabrication, a universal method for evaluating material photoresponses, detailed illustrations of all instrumentation used, and demonstrations of optically controlled nongenetic modulation of cellular calcium dynamics, neuronal excitability, neurotransmitter release from mouse brain slices, and brain activity in the mouse brain in vivo using the aforementioned Si materials. Read More

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http://www.nature.com/articles/s41596-019-0135-9
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http://dx.doi.org/10.1038/s41596-019-0135-9DOI Listing
April 2019
2 Reads

Author Correction: Using the tube test to measure social hierarchy in mice.

Nat Protoc 2019 Apr 11. Epub 2019 Apr 11.

Interdisciplinary Institute of Neuroscience and Technology, Qiushi Academy for Advanced Studies, Zhejiang University, Hangzhou, China.

In the version of this paper originally published, an affiliation for Zhengxiao Fan was omitted. In addition, the Reporting Summary incorrectly indicated that human research participants had been used in the study, instead of animal subjects. These errors have been corrected in the PDF and HTML versions of the protocol. Read More

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http://dx.doi.org/10.1038/s41596-019-0158-2DOI Listing

Author Correction: Design, execution, and analysis of CRISPR-Cas9-based deletions and genetic interaction networks in the fungal pathogen Candida albicans.

Nat Protoc 2019 Apr 11. Epub 2019 Apr 11.

Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.

The version of this paper originally published contained reference errors. The sentence "To dissect complex genetic interactions in C. albicans, a CRISPR-Cas9-based Gene Drive Array (GDA) was developed" incorrectly cited ref. Read More

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http://dx.doi.org/10.1038/s41596-019-0157-3DOI Listing

Capturing 5' and 3' native ends of mRNAs concurrently with Akron sequencing.

Nat Protoc 2019 Apr 10. Epub 2019 Apr 10.

Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Advances in RNA-sequencing methods have uncovered many aspects of RNA metabolism but are limited to surveying either the 3' or 5' terminus of RNAs, thus missing mechanistic aspects that could be revealed if both ends were captured. We developed Akron sequencing (Akron-seq), a method that captures in parallel the native 5' ends of uncapped, polyadenylated mRNAs and 3' ends of capped mRNAs from the same input RNA. Thus, Akron-seq uniquely enables assessment of full-length and truncated mRNAs at single-nucleotide resolution. Read More

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http://dx.doi.org/10.1038/s41596-019-0151-9DOI Listing

Guidelines for using sigQC for systematic evaluation of gene signatures.

Nat Protoc 2019 Apr 10. Epub 2019 Apr 10.

Computational Biology and Integrative Genomics Lab, MRC/CRUK Oxford Institute and Department of Oncology, University of Oxford, Oxford, UK.

With the increased use of next-generation sequencing generating large amounts of genomic data, gene expression signatures are becoming critically important tools for the interpretation of these data, and are poised to have a substantial effect on diagnosis, management, and prognosis for a number of diseases. It is becoming crucial to establish whether the expression patterns and statistical properties of sets of genes, or gene signatures, are conserved across independent datasets. Conversely, it is necessary to compare established signatures on the same dataset to better understand how they capture different clinical or biological characteristics. Read More

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http://www.nature.com/articles/s41596-019-0136-8
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http://dx.doi.org/10.1038/s41596-019-0136-8DOI Listing
April 2019
5 Reads

Regeneration of a bioengineered 3D integumentary organ system from iPS cells.

Nat Protoc 2019 Apr 8. Epub 2019 Apr 8.

Laboratory for Organ Regeneration, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.

Organ systems play essential roles in the physiological functions required for homeostasis. A 3D integumentary organ system (3D-IOS) comprises the skin and skin appendages such as hair follicles and sebaceous glands. This protocol describes how to induce the differentiation of murine induced pluripotent stem (iPS) cells into a 3D-IOS. Read More

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http://dx.doi.org/10.1038/s41596-019-0124-zDOI Listing

Whole-brain block-face serial microscopy tomography at subcellular resolution using FAST.

Nat Protoc 2019 Apr 8. Epub 2019 Apr 8.

Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.

Here, we describe an optimized and detailed protocol for block-face serial microscopy tomography (FAST). FAST enables high-speed serial section fluorescence imaging of fixed brains at an axonal spatial resolution and subsequent image data processing. It renders brain-wide anatomical and functional analyses, including structural profiling of nuclear-stained brain at the single-cell level, cell-type-specific mapping with reporter animal brains and neuronal tracing with anterograde/retrograde labeling. Read More

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http://dx.doi.org/10.1038/s41596-019-0148-4DOI Listing

RNase A treatment and reconstitution with DNA damage response RNA in living cells as a tool to study the role of non-coding RNA in the formation of DNA damage response foci.

Nat Protoc 2019 Apr 8. Epub 2019 Apr 8.

IFOM-The FIRC Institute of Molecular Oncology, Milan, Italy.

Non-coding RNA (ncRNA) molecules have been shown to play a variety of cellular roles; however, the contributions of different types of RNA to specific phenomena are often hard to dissect. To study the role of RNA in the assembly of DNA damage response (DDR) foci, we developed the RNase A treatment and reconstitution (RATaR) method, in which cells are mildly permeabilized, incubated with recombinant RNase A and subsequently reconstituted with different RNA species, under conditions of RNase A inactivation and inhibition of endogenous transcription. The block of transcription right after RNase A removal represents a key innovation of RATaR, preventing potential contributions of endogenously neo-synthesized transcripts to the phenotypes studied. Read More

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http://dx.doi.org/10.1038/s41596-019-0147-5DOI Listing

Microfabricated blood vessels for modeling the vascular transport barrier.

Nat Protoc 2019 Apr 5. Epub 2019 Apr 5.

The Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.

The vascular endothelium forms the inner lining of blood vessels and actively regulates vascular permeability in response to chemical and physical stimuli. Understanding the molecular pathways and mechanisms that regulate the permeability of blood vessels is of critical importance for developing therapies for cardiovascular dysfunction and disease. Recently, we developed a novel microfluidic human engineered microvessel (hEMV) platform to enable controlled blood flow through a human endothelial lumen within a physiologic 3D extracellular matrix (ECM) into which pericytes and other stromal cells can be introduced to recapitulate tissue-specific microvascular physiology. Read More

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http://dx.doi.org/10.1038/s41596-019-0144-8DOI Listing

Quantitative imaging of sleep behavior in Caenorhabditis elegans and larval Drosophila melanogaster.

Nat Protoc 2019 Apr 5. Epub 2019 Apr 5.

Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Sleep is nearly universal among animals, yet remains poorly understood. Recent work has leveraged simple model organisms, such as Caenorhabditis elegans and Drosophila melanogaster larvae, to investigate the genetic and neural bases of sleep. However, manual methods of recording sleep behavior in these systems are labor intensive and low in throughput. Read More

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http://dx.doi.org/10.1038/s41596-019-0146-6DOI Listing

Standardization of complex biologically derived spectrochemical datasets.

Nat Protoc 2019 Apr 5. Epub 2019 Apr 5.

School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston, UK.

Spectroscopic techniques such as Fourier-transform infrared (FTIR) spectroscopy are used to study interactions of light with biological materials. This interaction forms the basis of many analytical assays used in disease screening/diagnosis, microbiological studies, and forensic/environmental investigations. Advantages of spectrochemical analysis are its low cost, minimal sample preparation, non-destructive nature and substantially accurate results. Read More

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http://dx.doi.org/10.1038/s41596-019-0150-xDOI Listing
April 2019
1 Read

Nanoscale chemical imaging using tip-enhanced Raman spectroscopy.

Nat Protoc 2019 04 25;14(4):1169-1193. Epub 2019 Mar 25.

National Physical Laboratory, Teddington, UK.

Confocal and surface-enhanced Raman spectroscopy (SERS) are powerful techniques for molecular characterization; however, they suffer from the drawback of diffraction-limited spatial resolution. Tip-enhanced Raman spectroscopy (TERS) overcomes this limitation and provides chemical information at length scales in the tens of nanometers. In contrast to alternative approaches to nanoscale chemical analysis, TERS is label free, is non-destructive, and can be performed in both air and liquid environments, allowing its use in a diverse range of applications. Read More

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http://www.nature.com/articles/s41596-019-0132-z
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http://dx.doi.org/10.1038/s41596-019-0132-zDOI Listing
April 2019
6 Reads

Bioluminescence resonance energy transfer-based imaging of protein-protein interactions in living cells.

Nat Protoc 2019 04 25;14(4):1084-1107. Epub 2019 Mar 25.

Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, QC, Canada.

Bioluminescence resonance energy transfer (BRET) is a transfer of energy between a luminescence donor and a fluorescence acceptor. Because BRET occurs when the distance between the donor and acceptor is <10 nm, and its efficiency is inversely proportional to the sixth power of distance, it has gained popularity as a proximity-based assay to monitor protein-protein interactions and conformational rearrangements in live cells. In such assays, one protein of interest is fused to a bioluminescent energy donor (luciferases from Renilla reniformis or Oplophorus gracilirostris), and the other protein is fused to a fluorescent energy acceptor (such as GFP or YFP). Read More

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http://www.nature.com/articles/s41596-019-0129-7
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http://dx.doi.org/10.1038/s41596-019-0129-7DOI Listing
April 2019
3 Reads

Derivation of enteric neuron lineages from human pluripotent stem cells.

Nat Protoc 2019 04 25;14(4):1261-1279. Epub 2019 Mar 25.

Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, CA, USA.

The enteric nervous system (ENS) represents a vast network of neuronal and glial cell types that develops entirely from migratory neural crest (NC) progenitor cells. Considerable improvements in the understanding of the molecular mechanisms underlying NC induction and regional specification have recently led to the development of a robust method to re-create the process in vitro using human pluripotent stem cells (hPSCs). Directing the fate of hPSCs toward the enteric NC (ENC) results in an accessible and scalable in vitro model of ENS development. Read More

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http://www.nature.com/articles/s41596-019-0141-y
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http://dx.doi.org/10.1038/s41596-019-0141-yDOI Listing
April 2019
10 Reads

Fabrication and practical applications of molybdenum disulfide nanopores.

Nat Protoc 2019 04 22;14(4):1130-1168. Epub 2019 Mar 22.

Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL, Lausanne, Switzerland.

Among the different developed solid-state nanopores, nanopores constructed in a monolayer of molybdenum disulfide (MoS) stand out as powerful devices for single-molecule analysis or osmotic power generation. Because the ionic current through a nanopore is inversely proportional to the thickness of the pore, ultrathin membranes have the advantage of providing relatively high ionic currents at very small pore sizes. This increases the signal generated during translocation of biomolecules and improves the nanopores' efficiency when used for desalination or reverse electrodialysis applications. Read More

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http://dx.doi.org/10.1038/s41596-019-0131-0DOI Listing

High-resolution 3D imaging and analysis of axon regeneration in unsectioned spinal cord with or without tissue clearing.

Nat Protoc 2019 04 22;14(4):1235-1260. Epub 2019 Mar 22.

Laboratory for Axon Growth and Regeneration, German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany.

Here we present a protocol for analyses of axon regeneration and density in unsectioned adult mouse spinal cord. This includes methods for injury and tracing of dorsal column sensory and corticospinal axons; clearing and staining of unsectioned spinal cord; visualization of axon degeneration and regeneration in cleared and uncleared specimens using two-photon microscopy; and either manual or semi-automatic analysis of axon density and regeneration in 3D space using Imaris and ImageJ software. This protocol can be used to elucidate the molecular and cellular mechanisms underlying nervous system degeneration and regeneration and to establish the therapeutic efficacy of candidate neuroregenerative treatments. Read More

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http://dx.doi.org/10.1038/s41596-019-0140-zDOI Listing

Synthetic chemistry in water: applications to peptide synthesis and nitro-group reductions.

Nat Protoc 2019 04 22;14(4):1108-1129. Epub 2019 Mar 22.

Department of Chemistry & Biochemistry, University of California, Santa Barbara, Santa Barbara, CA, USA.

Amide bond formation and aromatic/heteroaromatic nitro-group reductions represent two of the most commonly used transformations in organic synthesis. Unfortunately, such processes can be especially wasteful and hence environmentally harmful, and may present safety hazards as well, given the reaction conditions involved. The two protocols herein describe alternative technologies that offer solutions to these issues. Read More

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http://dx.doi.org/10.1038/s41596-019-0130-1DOI Listing
April 2019
3 Reads

Temperature imaging using a cationic linear fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy.

Nat Protoc 2019 04 22;14(4):1293-1321. Epub 2019 Mar 22.

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.

Temperature is one of the most important of the physiological parameters that determine the biological status of living organisms. However, intracellular temperature was not imaged at the single-cell level until recently because of the lack of a molecular thermometer that can be applied to living cells. We have recently developed a method for imaging intracellular temperature using a cationic linear fluorescent polymeric thermometer (FPT) and fluorescence lifetime imaging microscopy (FLIM). Read More

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http://dx.doi.org/10.1038/s41596-019-0145-7DOI Listing
April 2019
4 Reads

RNA sequencing and swarm intelligence-enhanced classification algorithm development for blood-based disease diagnostics using spliced blood platelet RNA.

Nat Protoc 2019 04 20;14(4):1206-1234. Epub 2019 Mar 20.

Department of Neurosurgery, Cancer Center Amsterdam, Amsterdam UMC, VU University Medical Center, Amsterdam, the Netherlands.

Blood-based diagnostics tests, using individual or panels of biomarkers, may revolutionize disease diagnostics and enable minimally invasive therapy monitoring. However, selection of the most relevant biomarkers from liquid biosources remains an immense challenge. We recently presented the thromboSeq pipeline, which enables RNA sequencing and cancer classification via self-learning and swarm intelligence-enhanced bioinformatics algorithms using blood platelet RNA. Read More

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http://dx.doi.org/10.1038/s41596-019-0139-5DOI Listing
April 2019
2 Reads

A non-enzymatic method for dissection of mouse bladder urothelial tissue.

Nat Protoc 2019 04 20;14(4):1280-1292. Epub 2019 Mar 20.

Department of Urology, Yale University School of Medicine, New Haven, CT, USA.

Urothelial cells contribute to bladder functions, including urine storage, urine emptying, and innate immune response. Functional studies of urothelial cells usually use either freshly isolated cells or cultured cells. Most methods of isolating urothelial cells require enzymes; however, these techniques remove proteins that connect the cells and disrupt the orientation of the cells within the multilayered urothelium. Read More

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http://dx.doi.org/10.1038/s41596-019-0142-xDOI Listing

INFOGEST static in vitro simulation of gastrointestinal food digestion.

Nat Protoc 2019 04 18;14(4):991-1014. Epub 2019 Mar 18.

Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM), Madrid, Spain.

Developing a mechanistic understanding of the impact of food structure and composition on human health has increasingly involved simulating digestion in the upper gastrointestinal tract. These simulations have used a wide range of different conditions that often have very little physiological relevance, and this impedes the meaningful comparison of results. The standardized protocol presented here is based on an international consensus developed by the COST INFOGEST network. Read More

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http://dx.doi.org/10.1038/s41596-018-0119-1DOI Listing
April 2019
1 Read

Isolating the human cochlea to generate bone powder for ancient DNA analysis.

Nat Protoc 2019 04 6;14(4):1194-1205. Epub 2019 Mar 6.

Department of Evolutionary Anthropology, University of Vienna, Vienna, Austria.

The cortical bone that forms the structure of the cochlea, part of the osseous labyrinth of the inner ear, is now one of the most frequently used skeletal elements in analyses of human ancient DNA. However, there is currently no published, standardized method for its sampling. This protocol describes the preparation of bone powder from the cochlea of fragmented skulls in which the petrous pyramid of the temporal bone is accessible. Read More

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http://www.nature.com/articles/s41596-019-0137-7
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http://dx.doi.org/10.1038/s41596-019-0137-7DOI Listing
April 2019
12 Reads

Measuring nanoscale diffusion dynamics in cellular membranes with super-resolution STED-FCS.

Nat Protoc 2019 04 6;14(4):1054-1083. Epub 2019 Mar 6.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

Super-resolution microscopy techniques enable optical imaging in live cells with unprecedented spatial resolution. They unfortunately lack the temporal resolution required to directly investigate cellular dynamics at scales sufficient to measure molecular diffusion. These fast time scales are, on the other hand, routinely accessible by spectroscopic techniques such as fluorescence correlation spectroscopy (FCS). Read More

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http://www.nature.com/articles/s41596-019-0127-9
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http://dx.doi.org/10.1038/s41596-019-0127-9DOI Listing
April 2019
5 Reads

Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization.

Nat Protoc 2019 04 4;14(4):1027-1053. Epub 2019 Mar 4.

Children's Cancer and Blood Foundation Laboratories, Department of Pediatrics and Department of Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA.

We describe the protocol development and optimization of asymmetric-flow field-flow fractionation (AF4) technology for separating and characterizing extracellular nanoparticles (ENPs), particularly small extracellular vesicles (sEVs), known as exosomes, and even smaller novel nanoparticles, known as exomeres. This technique fractionates ENPs on the basis of hydrodynamic size and demonstrates a unique capability to separate nanoparticles with sizes ranging from a few nanometers to an undefined level of micrometers. ENPs are resolved by two perpendicular flows-channel flow and cross-flow-in a thin, flat channel with a semi-permissive bottom wall membrane. Read More

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http://dx.doi.org/10.1038/s41596-019-0126-xDOI Listing
April 2019
4 Reads

Microfluidic reprogramming to pluripotency of human somatic cells.

Nat Protoc 2019 03 26;14(3):722-737. Epub 2019 Feb 26.

Shanghai Institute for Advanced Immunochemical Studies (SIAIS), ShanghaiTech University, Shanghai, China.

Human induced pluripotent stem cells (hiPSCs) have a number of potential applications in stem cell biology and regenerative medicine, including precision medicine. However, their potential clinical application is hampered by the low efficiency, high costs, and heavy workload of the reprogramming process. Here we describe a protocol to reprogram human somatic cells to hiPSCs with high efficiency in 15 d using microfluidics. Read More

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http://www.nature.com/articles/s41596-018-0108-4
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http://dx.doi.org/10.1038/s41596-018-0108-4DOI Listing
March 2019
9 Reads

Holographic two-photon activation for synthetic optogenetics.

Nat Protoc 2019 03 25;14(3):864-900. Epub 2019 Feb 25.

Department of Neuroscience, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.

Optogenetic tools provide users the ability to photocontrol the activity of cells. Commonly, activation is achieved by expression of proteins from photosynthetic organisms, for example, microbial opsins (e.g. Read More

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http://dx.doi.org/10.1038/s41596-018-0118-2DOI Listing
March 2019
1 Read

Protocol Update for large-scale genome and gene function analysis with the PANTHER classification system (v.14.0).

Nat Protoc 2019 03 25;14(3):703-721. Epub 2019 Feb 25.

Division of Bioinformatics, Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.

The PANTHER classification system ( http://www.pantherdb.org ) is a comprehensive system that combines genomes, gene function classifications, pathways and statistical analysis tools to enable biologists to analyze large-scale genome-wide experimental data. Read More

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http://dx.doi.org/10.1038/s41596-019-0128-8DOI Listing
March 2019
3 Reads

Nonviral ultrasound-mediated gene delivery in small and large animal models.

Nat Protoc 2019 04 25;14(4):1015-1026. Epub 2019 Feb 25.

Skeletal Biotech Laboratory, The Hebrew University-Hadassah Faculty of Dental Medicine, Ein Kerem, Jerusalem, Israel.

Ultrasound-mediated gene delivery (sonoporation) is a minimally invasive, nonviral and clinically translatable method of gene therapy. This method offers a favorable safety profile over that of viral vectors and is less invasive as compared with other physical gene delivery approaches (e.g. Read More

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http://dx.doi.org/10.1038/s41596-019-0125-yDOI Listing

Creation and analysis of biochemical constraint-based models using the COBRA Toolbox v.3.0.

Nat Protoc 2019 03;14(3):639-702

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Belvaux, Luxembourg.

Constraint-based reconstruction and analysis (COBRA) provides a molecular mechanistic framework for integrative analysis of experimental molecular systems biology data and quantitative prediction of physicochemically and biochemically feasible phenotypic states. The COBRA Toolbox is a comprehensive desktop software suite of interoperable COBRA methods. It has found widespread application in biology, biomedicine, and biotechnology because its functions can be flexibly combined to implement tailored COBRA protocols for any biochemical network. Read More

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http://dx.doi.org/10.1038/s41596-018-0098-2DOI Listing
March 2019
2 Reads

Fractionation of lignocellulosic biomass to produce uncondensed aldehyde-stabilized lignin.

Nat Protoc 2019 03 18;14(3):921-954. Epub 2019 Feb 18.

Laboratory of Sustainable and Catalytic Processing, Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Lignin is one of the most promising sources of renewable aromatic hydrocarbons. Current methods for its extraction from lignocellulosic biomass-which include the kraft, sulfite, and organosolv processes-result in the rapid formation of carbon-carbon bonds, leading to a condensed lignin that cannot be effectively depolymerized into its constituent monomers. Treatment of lignocellulosic biomass with aldehydes during lignin extraction generates an aldehyde-stabilized lignin that is uncondensed and can be converted into its monomers at near-theoretical yields. Read More

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http://dx.doi.org/10.1038/s41596-018-0121-7DOI Listing

A practical guide to optimization in X10 expansion microscopy.

Nat Protoc 2019 03;14(3):832-863

Institute of Science and Technology Austria, Klosterneuburg, Austria.

Expansion microscopy is a relatively new approach to super-resolution imaging that uses expandable hydrogels to isotropically increase the physical distance between fluorophores in biological samples such as cell cultures or tissue slices. The classic gel recipe results in an expansion factor of ~4×, with a resolution of 60-80 nm. We have recently developed X10 microscopy, which uses a gel that achieves an expansion factor of ~10×, with a resolution of ~25 nm. Read More

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http://www.nature.com/articles/s41596-018-0117-3
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http://dx.doi.org/10.1038/s41596-018-0117-3DOI Listing
March 2019
5 Reads

Publisher Correction: Live imaging of stem cells in the germarium of the Drosophila ovary using a reusable gas-permeable imaging chamber.

Nat Protoc 2019 Feb 15. Epub 2019 Feb 15.

Department of Biomedical Engineering, Columbia University, New York, NY, USA.

The version of this paper originally published contained an incorrect unit abbreviation in Step 21: "0.20 g/mL" should have been "0.20 mg/mL. Read More

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http://www.nature.com/articles/s41596-019-0134-x
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http://dx.doi.org/10.1038/s41596-019-0134-xDOI Listing
February 2019
7 Reads

Using the tube test to measure social hierarchy in mice.

Nat Protoc 2019 03 15;14(3):819-831. Epub 2019 Feb 15.

Center for Neuroscience and Department of Psychiatry of First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

Investigation of the neural mechanisms underlying social hierarchy requires a reliable and effective behavioral test. The tube test is a simple and robust behavioral assay that we recently validated as a reliable measure of social hierarchy in mice. The test was demonstrated to produce results largely consistent with the results seen when using other dominance measures, including the warm spot test, territory urine marking or the courtship ultrasound vocalization test. Read More

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http://dx.doi.org/10.1038/s41596-018-0116-4DOI Listing

Using CAPTURE to detect spacer acquisition in native CRISPR arrays.

Nat Protoc 2019 03 11;14(3):976-990. Epub 2019 Feb 11.

Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands.

CRISPR-Cas systems are able to acquire immunological memories (spacers) from bacteriophages and plasmids in order to survive infection; however, this often occurs at low frequency within a population, which can make it difficult to detect. Here we describe CAPTURE (CRISPR adaptation PCR technique using reamplification and electrophoresis), a versatile and adaptable protocol to detect spacer-acquisition events by electrophoresis imaging with high-enough sensitivity to identify spacer acquisition in 1 in 10 cells. Our method harnesses two simple PCR steps, separated by automated electrophoresis and extraction of size-selected DNA amplicons, thus allowing the removal of unexpanded arrays from the sample pool and enabling 1,000-times more sensitive detection of new spacers than alternative PCR protocols. Read More

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http://dx.doi.org/10.1038/s41596-018-0123-5DOI Listing

Design, execution, and analysis of CRISPR-Cas9-based deletions and genetic interaction networks in the fungal pathogen Candida albicans.

Nat Protoc 2019 03 8;14(3):955-975. Epub 2019 Feb 8.

Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.

The study of fungal pathogens is of immediate importance, yet progress is hindered by the technical challenges of genetic manipulation. For Candida species, their inability to maintain plasmids, unusual codon usage, and inefficient homologous recombination are among the obstacles limiting efficient genetic manipulation. New advances in genomic biotechnologies-particularly CRISPR-based tools-have revolutionized genome editing for many fungal species. Read More

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http://dx.doi.org/10.1038/s41596-018-0122-6DOI Listing

Multiplexed profiling of RNA and protein expression signatures in individual cells using flow or mass cytometry.

Nat Protoc 2019 03 6;14(3):901-920. Epub 2019 Feb 6.

Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, UK.

Advances in single-cell analysis technologies are providing novel insights into phenotypic and functional heterogeneity within seemingly identical cell populations. RNA within single cells can be analyzed using unbiased sequencing protocols or through more targeted approaches using in situ hybridization (ISH). The proximity ligation assay for RNA (PLAYR) approach is a sensitive and high-throughput technique that relies on in situ and proximal ligation to measure at least 27 specific RNAs by flow or mass cytometry. Read More

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http://dx.doi.org/10.1038/s41596-018-0120-8DOI Listing
March 2019
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Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute.

Nat Protoc 2019 03 1;14(3):756-780. Epub 2019 Feb 1.

Shanghai Key Laboratory of Tuberculosis, Clinical Translational Research Center, Shanghai Pulmonary Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China.

Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR-Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. Read More

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http://www.nature.com/articles/s41596-018-0113-7
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http://dx.doi.org/10.1038/s41596-018-0113-7DOI Listing
March 2019
26 Reads

Pipelines for cross-species and genome-wide prediction of long noncoding RNA binding.

Nat Protoc 2019 03;14(3):795-818

Bioinformatics Section, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

Abundant long, noncoding RNAs (lncRNAs) in mammals can bind to DNA sequences and recruit histone- and DNA-modifying enzymes to binding sites to epigenetically regulate target genes. However, most lncRNAs' binding motifs and target sites are unknown. The large numbers of lncRNAs and target sites in the whole genome make it infeasible to examine lncRNA binding to DNA purely experimentally. Read More

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http://dx.doi.org/10.1038/s41596-018-0115-5DOI Listing
March 2019
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Micropipette force sensors for in vivo force measurements on single cells and multicellular microorganisms.

Nat Protoc 2019 02;14(2):594-615

Max Planck Institute for Dynamics and Self-Organization (MPIDS), Göttingen, Germany.

Measuring forces from the piconewton to millinewton range is of great importance for the study of living systems from a biophysical perspective. The use of flexible micropipettes as highly sensitive force probes has become established in the biophysical community, advancing our understanding of cellular processes and microbial behavior. The micropipette force sensor (MFS) technique relies on measurement of the forces acting on a force-calibrated, hollow glass micropipette by optically detecting its deflections. Read More

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http://www.nature.com/articles/s41596-018-0110-x
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http://dx.doi.org/10.1038/s41596-018-0110-xDOI Listing
February 2019
9 Reads

Publisher Correction: Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum).

Nat Protoc 2019 Jan 29. Epub 2019 Jan 29.

The Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.

In the version of this protocol originally published, the recipe for CAS9 buffer was incorrectly identified as a recipe for sodium acetate solution, and vice versa. These errors have been corrected in the PDF and HTML versions of the paper. Read More

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http://dx.doi.org/10.1038/s41596-019-0138-6DOI Listing
January 2019
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A procedure for identifying possible products in the assembly-disassembly-organization-reassembly (ADOR) synthesis of zeolites.

Nat Protoc 2019 03 25;14(3):781-794. Epub 2019 Jan 25.

School of Chemistry and EaStCHEM, University of St. Andrews, St. Andrews, UK.

High-silica zeolites, some of the most important and widely used catalysts in industry, have potential for application across a wide range of traditional and emerging technologies. The many structural topologies of zeolites have a variety of potential uses, so a strong drive to create new zeolites exists. Here, we present a protocol, the assembly-disassembly-organization-reassembly (ADOR) process, for a relatively new method of preparing these important solids. Read More

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http://dx.doi.org/10.1038/s41596-018-0114-6DOI Listing

A surgical orthotopic approach for studying the invasive progression of human bladder cancer.

Nat Protoc 2019 03;14(3):738-755

Division of Urologic Oncology, Department of Urology, University of Michigan, Ann Arbor, MI, USA.

The invasion of bladder cancer into the sub-urothelial muscle and vasculature are key determinants leading to lethal metastatic progression. However, the molecular basis is poorly understood, partly because of the lack of uncomplicated and reliable models that recapitulate the biology of locally invasive disease. We developed a surgical grafting technique, characterized by a simple, rapid, reproducible and high-efficiency approach, to recapitulate the pathobiological events of human bladder cancer invasion in mice. Read More

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http://dx.doi.org/10.1038/s41596-018-0112-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463286PMC
March 2019
2 Reads

Ex vivo and in vivo stable isotope labelling of central carbon metabolism and related pathways with analysis by LC-MS/MS.

Nat Protoc 2019 02;14(2):313-330

Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, USA.

Targeted tandem mass spectrometry (LC-MS/MS) has been extremely useful for profiling small molecules extracted from biological sources, such as cells, bodily fluids and tissues. Here, we present a protocol for analysing incorporation of the non-radioactive stable isotopes carbon-13 (C) and nitrogen-15 (N) into polar metabolites in central carbon metabolism and related pathways. Our platform utilizes selected reaction monitoring (SRM) with polarity switching and amide hydrophilic interaction liquid chromatography (HILIC) to capture transitions for carbon and nitrogen incorporation into selected metabolites using a hybrid triple quadrupole (QQQ) mass spectrometer. Read More

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http://www.nature.com/articles/s41596-018-0102-x
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http://dx.doi.org/10.1038/s41596-018-0102-xDOI Listing
February 2019
6 Reads

Using enhanced number and brightness to measure protein oligomerization dynamics in live cells.

Nat Protoc 2019 02;14(2):616-638

Translational Imaging Center, Molecular and Computational Biology, University of Southern California, Los Angeles, CA, USA.

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. Read More

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http://www.nature.com/articles/s41596-018-0111-9
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http://dx.doi.org/10.1038/s41596-018-0111-9DOI Listing
February 2019
2 Reads

Generation of lung organoids from human pluripotent stem cells in vitro.

Nat Protoc 2019 02;14(2):518-540

Program in Cell and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI, USA.

The lung epithelium is derived from the endodermal germ layer, which undergoes a complex series of endoderm-mesoderm-mediated signaling events to generate the final arborized network of conducting airways (bronchi, bronchioles) and gas-exchanging units (alveoli). These stages include endoderm induction, anterior-posterior and dorsal-ventral patterning, lung specification, lung budding, branching morphogenesis, and, finally, maturation. Here we describe a protocol that recapitulates several of these milestones in order to differentiate human pluripotent stem cells (hPSCs) into ventral-anterior foregut spheroids and further into two distinct types of organoids: human lung organoids and bud tip progenitor organoids. Read More

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http://dx.doi.org/10.1038/s41596-018-0104-8DOI Listing
February 2019
2 Reads

Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap.

Nat Protoc 2019 02;14(2):482-517

The Donnelly Centre, University of Toronto, Toronto, ON, Canada.

Pathway enrichment analysis helps researchers gain mechanistic insight into gene lists generated from genome-scale (omics) experiments. This method identifies biological pathways that are enriched in a gene list more than would be expected by chance. We explain the procedures of pathway enrichment analysis and present a practical step-by-step guide to help interpret gene lists resulting from RNA-seq and genome-sequencing experiments. Read More

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http://dx.doi.org/10.1038/s41596-018-0103-9DOI Listing
February 2019
1 Read