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Nature Protocols

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https://www.readbyqxmd.com/read/28079880/nonradioactive-quantification-of-autophagic-protein-degradation-with-l-azidohomoalanine-labeling
#1
Jigang Wang, Jianbin Zhang, Yew Mun Lee, Shukie Ng, Yin Shi, Zi-Chun Hua, Qingsong Lin, Han-Ming Shen
At present, several assays that use radioisotope labeling to quantify the degradation of long-lived proteins have been developed to measure autophagic flux. Here, we describe a nonradioactive pulse-chase protocol using L-azidohomoalanine (AHA) labeling to quantify long-lived protein degradation during autophagy. AHA is used as a surrogate for L-methionine, and, when added to cultured cells grown in methionine-free medium, AHA is incorporated into proteins during de novo protein synthesis. After a chase period to remove short-lived proteins, autophagy is induced by starvation or other stimuli...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/28102837/precise-and-efficient-scarless-genome-editing-in-stem-cells-using-correct
#2
Dylan Kwart, Dominik Paquet, Shaun Teo, Marc Tessier-Lavigne
CRISPR/Cas9 is a promising tool for genome-editing DNA in cells with single-base-pair precision, which allows novel in vitro models of human disease to be generated-e.g., in pluripotent stem cells. However, the accuracy of intended sequence changes can be severely diminished by CRISPR/Cas9's propensity to re-edit previously modified loci, causing unwanted mutations (indels) alongside intended changes. Here we describe a genome-editing framework termed consecutive re-guide or re-Cas steps to erase CRISPR/Cas-blocked targets (CORRECT), which, by exploiting the use of highly efficacious CRISPR/Cas-blocking mutations in two rounds of genome editing, enables accurate, efficient and scarless introduction of specific base changes-for example, in human induced pluripotent (iPS) stem cells...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28102836/preparation-characterization-and-in-vitro-dosimetry-of-dispersed-engineered-nanomaterials
#3
Glen M DeLoid, Joel M Cohen, Georgios Pyrgiotakis, Philip Demokritou
Evidence continues to grow of the importance of in vitro and in vivo dosimetry in the hazard assessment and ranking of engineered nanomaterials (ENMs). Accurate dose metrics are particularly important for in vitro cellular screening to assess the potential health risks or bioactivity of ENMs. To ensure meaningful and reproducible quantification of in vitro dose, with consistent measurement and reporting between laboratories, it is necessary to adopt standardized and integrated methodologies for (i) generation of stable ENM suspensions in cell culture media; (ii) colloidal characterization of suspended ENMs, particularly of properties that determine particle kinetics in an in vitro system (size distribution and formed agglomerate effective density); and (iii) robust numerical fate and transport modeling for accurate determination of the ENM dose delivered to cells over the course of the in vitro exposure...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28102835/spinal-cord-regeneration-in-xenopus-laevis
#4
Gabriela Edwards-Faret, Rosana Muñoz, Emilio E Méndez-Olivos, Dasfne Lee-Liu, Victor S Tapia, Juan Larraín
Here we present a protocol for the husbandry of Xenopus laevis tadpoles and froglets, and procedures to study spinal cord regeneration. This includes methods to induce spinal cord injury (SCI); DNA and morpholino electroporation for genetic studies; in vivo imaging for cell analysis; a swimming test to measure functional recovery; and a convenient model for screening for new compounds that promote neural regeneration. These protocols establish X. laevis as a unique model organism for understanding spinal cord regeneration by comparing regenerative and nonregenerative stages...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28079879/the-cluspro-web-server-for-protein-protein-docking
#5
Dima Kozakov, David R Hall, Bing Xia, Kathryn A Porter, Dzmitry Padhorny, Christine Yueh, Dmitri Beglov, Sandor Vajda
The ClusPro server (https://cluspro.org) is a widely used tool for protein-protein docking. The server provides a simple home page for basic use, requiring only two files in Protein Data Bank (PDB) format. However, ClusPro also offers a number of advanced options to modify the search; these include the removal of unstructured protein regions, application of attraction or repulsion, accounting for pairwise distance restraints, construction of homo-multimers, consideration of small-angle X-ray scattering (SAXS) data, and location of heparin-binding sites...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28079877/genome-wide-transposon-screening-and-quantitative-insertion-site-sequencing-for-cancer-gene-discovery-in-mice
#6
Mathias J Friedrich, Lena Rad, Iraad F Bronner, Alexander Strong, Wei Wang, Julia Weber, Matthew Mayho, Hannes Ponstingl, Thomas Engleitner, Carolyn Grove, Anja Pfaus, Dieter Saur, Juan Cadiñanos, Michael A Quail, George S Vassiliou, Pentao Liu, Allan Bradley, Roland Rad
Transposon-mediated forward genetics screening in mice has emerged as a powerful tool for cancer gene discovery. It pinpoints cancer drivers that are difficult to find with other approaches, thus complementing the sequencing-based census of human cancer genes. We describe here a large series of mouse lines for insertional mutagenesis that are compatible with two transposon systems, PiggyBac and Sleeping Beauty, and give guidance on the use of different engineered transposon variants for constitutive or tissue-specific cancer gene discovery screening...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28055037/advances-in-the-field-of-single-particle-cryo-electron-microscopy-over-the-last-decade
#7
Joachim Frank
In single-particle cryo-electron microscopy (cryo-EM), molecules suspended in a thin aqueous layer are rapidly frozen and imaged at cryogenic temperature in the transmission electron microscope. From the random projection views, a three-dimensional image is reconstructed, enabling the structure of the molecule to be obtained. In this article I discuss technological progress over the past decade, which has, in my own field of study, culminated in the determination of ribosome structure at 2.5-Å resolution. I also discuss likely future improvements in methodology...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28055036/preparation-and-implementation-of-optofluidic-neural-probes-for-in-vivo-wireless-pharmacology-and-optogenetics
#8
Jordan G McCall, Raza Qazi, Gunchul Shin, Shuo Li, Muhammad Hamza Ikram, Kyung-In Jang, Yuhao Liu, Ream Al-Hasani, Michael R Bruchas, Jae-Woong Jeong, John A Rogers
This Protocol Extension describes the fabrication and technical procedures for implementing ultrathin, flexible optofluidic neural probe systems that provide targeted, wireless delivery of fluids and light into the brains of awake, freely behaving animals. As a Protocol Extension article, this article describes an adaptation of an existing Protocol that offers additional applications. This protocol serves as an extension of an existing Nature Protocol describing optoelectronic devices for studying intact neural systems...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28055035/dna-sequencing-technologies-2006-2016
#9
Elaine R Mardis
Recent advances in the field of genomics have largely been due to the ability to sequence DNA at increasing throughput and decreasing cost. DNA sequencing was first introduced in 1977, and next-generation sequencing technologies have been available only during the past decade, but the diverse experiments and corresponding analyses facilitated by these techniques have transformed biological and biomedical research. Here, I review developments in DNA sequencing technologies over the past 10 years and look to the future for further applications...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28055034/applying-the-intact-method-to-purify-endosperm-nuclei-and-to-generate-parental-specific-epigenome-profiles
#10
Jordi Moreno-Romero, Juan Santos-González, Lars Hennig, Claudia Köhler
The early endosperm tissue of dicot species is very difficult to isolate by manual dissection. This protocol details how to apply the INTACT (isolation of nuclei tagged in specific cell types) system for isolating early endosperm nuclei of Arabidopsis at high purity and how to generate parental-specific epigenome profiles. As a Protocol Extension, this article describes an adaptation of an existing Nature Protocol that details the use of the INTACT method for purification of root nuclei. We address how to obtain the INTACT lines, generate the starting material and purify the nuclei...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28005069/depletion-of-mitochondria-in-mammalian-cells-through-enforced-mitophagy
#11
Clara Correia-Melo, Gabriel Ichim, Stephen W G Tait, João F Passos
Mitochondria are not only the 'powerhouse' of the cell; they are also involved in a multitude of processes that include calcium storage, the cell cycle and cell death. Traditional means of investigating mitochondrial importance in a given cellular process have centered upon depletion of mtDNA through chemical or genetic means. Although these methods severely disrupt the mitochondrial electron transport chain, mtDNA-depleted cells still maintain mitochondria and many mitochondrial functions. Here we describe a straightforward protocol to generate mammalian cell populations with low to nondetectable levels of mitochondria...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/28005068/immuno-engineered-organoids-for-regulating-the-kinetics-of-b-cell-development-and-antibody-production
#12
Alberto Purwada, Ankur Singh
Induction of B-cell immunity against infection depends on the initiation of the germinal center (GC) reaction in secondary lymphoid organs. Ex vivo recapitulation of the GC reaction in 2D cultures results in transient cell growth, with poor yield and short-term survival. Furthermore, no reported 2D ex vivo system can modulate the kinetics of a GC-like phenotype or the rate of antibody class switching. This protocol describes a methodology for developing immune organoids that partially mimic the B-cell zone of a lymphoid tissue, for efficient and rapid generation of B cells with a GC-like phenotype from naive murine B cells...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/28005067/generation-of-nephron-progenitor-cells-and-kidney-organoids-from-human-pluripotent-stem-cells
#13
Ryuji Morizane, Joseph V Bonventre
A variety of protocols have been developed that demonstrate the capability to differentiate human pluripotent stem cells (hPSCs) into kidney structures. Our goal was to develop a high-efficiency protocol to generate nephron progenitor cells (NPCs) and kidney organoids to facilitate applications for tissue engineering, disease modeling and chemical screening. Here, we describe a detailed protocol resulting in high-efficiency production (80-90%) of NPCs from hPSCs within 9 d of differentiation. Kidney organoids were generated from NPCs within 12 d with high reproducibility using 96-well plates suitable for chemical screening...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27977023/direct-long-term-intrathecal-application-of-therapeutics-to-the-rodent-cns
#14
Benjamin V Ineichen, Lisa Schnell, Miriam Gullo, Julia Kaiser, Marc P Schneider, Alice C Mosberger, Nicolas Good, Michael Linnebank, Martin E Schwab
Systemic application of therapeutics to the CNS tissue often results in subtherapeutic drug levels, because of restricted and selective penetration through the blood-brain barrier (BBB). Here, we give a detailed description of a standardized technique for intrathecal drug delivery in rodents, analogous to the technique used in humans. The intrathecal drug delivery method bypasses the BBB and thereby offers key advantages over oral or intravenous administration, such as maximized local drug doses with minimal systemic side effects...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27977022/capture-and-sequencing-of-nad-capped-rna-sequences-with-nad-captureseq
#15
Marie-Luise Winz, Hana Cahová, Gabriele Nübel, Jens Frindert, Katharina Höfer, Andres Jäschke
Here we describe a protocol for NAD captureSeq that allows for the identification of nicotinamide-adenine dinucleotide (NAD)-capped RNA sequences in total RNA samples from different organisms. NAD-capped RNA is first chemo-enzymatically biotinylated with high efficiency, permitting selective capture on streptavidin beads. Then, a highly efficient library preparation protocol tailored to immobilized, 5'-modified RNA is applied, with adaptor ligation to the RNA's 3' terminus and reverse transcription (RT) performed on-bead...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27977021/correlated-fluorescence-microscopy-and-cryo-electron-tomography-of-virus-infected-or-transfected-mammalian-cells
#16
Cheri M Hampton, Joshua D Strauss, Zunlong Ke, Rebecca S Dillard, Jason E Hammonds, Eric Alonas, Tanay M Desai, Mariana Marin, Rachel E Storms, Fredrick Leon, Gregory B Melikyan, Philip J Santangelo, Paul W Spearman, Elizabeth R Wright
Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27929523/single-cell-barcoding-and-sequencing-using-droplet-microfluidics
#17
Rapolas Zilionis, Juozas Nainys, Adrian Veres, Virginia Savova, David Zemmour, Allon M Klein, Linas Mazutis
Single-cell RNA sequencing has recently emerged as a powerful tool for mapping cellular heterogeneity in diseased and healthy tissues, yet high-throughput methods are needed for capturing the unbiased diversity of cells. Droplet microfluidics is among the most promising candidates for capturing and processing thousands of individual cells for whole-transcriptome or genomic analysis in a massively parallel manner with minimal reagent use. We recently established a method called inDrops, which has the capability to index >15,000 cells in an hour...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27929522/merging-allylic-c-h-bond-activation-and-c-c-bond-cleavage-en-route-to-the-formation-of-a-quaternary-carbon-stereocenter-in-acyclic-systems
#18
Alexandre Vasseur, Ilan Marek
This protocol describes a diastereoselective approach for the synthesis of complex molecular architectures containing two stereogenic centers in a 1,4 relationship, one of which being an all-carbon quaternary stereogenic center. Such molecules could be intermediates in the synthesis of steroids, for example. Conceived as a single-flask synthetic sequence from ω-ene cyclopropanes, the protocol involves a concerted allylic C-H and C-C bond activation promoted by the Negishi reagent (Cp2Zr(η(2)-butene)). This zirconium-promenade-based procedure affords bifunctionalized products in high diastereomeric ratios after reaction of ω-ene cyclopropanes with the Negishi complex, followed by a thermal treatment and sequential addition of two different electrophiles...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27929521/efficient-footprint-free-human-ipsc-genome-editing-by-consolidation-of-cas9-crispr-and-piggybac-technologies
#19
Gang Wang, Luhan Yang, Dennis Grishin, Xavier Rios, Lillian Y Ye, Yong Hu, Kai Li, Donghui Zhang, George M Church, William T Pu
Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27906170/generating-high-purity-cardiac-and-endothelial-derivatives-from-patterned-mesoderm-using-human-pluripotent-stem-cells
#20
Nathan J Palpant, Lil Pabon, Clayton E Friedman, Meredith Roberts, Brandon Hadland, Rebecca J Zaunbrecher, Irwin Bernstein, Ying Zheng, Charles E Murry
Human pluripotent stem cells (hPSCs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and requires experience in hPSC culture and differentiation techniques. Building on lessons taken from early development, this monolayer-directed differentiation protocol uses different concentrations of activin A and bone morphogenetic protein 4 (BMP4) to polarize cells into mesodermal subtypes that reflect mid-primitive-streak cardiogenic mesoderm and posterior-primitive-streak hemogenic mesoderm...
January 2017: Nature Protocols
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