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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/28617451/biological-and-chemical-strategies-for-exploring-inter-and-intra-kingdom-communication-mediated-via-bacterial-volatile-signals
#2
Mohamed A Farag, Geun Cheol Song, Yong-Soon Park, Bianca Audrain, Soohyun Lee, Jean-Marc Ghigo, Joseph W Kloepper, Choong-Min Ryu
Airborne chemical signals emitted by bacteria influence the behavior of other bacteria and plants. We present an overview of in vitro methods for evaluating bacterial and plant responses to bacterial volatile compounds (BVCs). Three types of equipment have been used to physically separate the bacterial test strains from either other bacterial strains or plants (in our laboratory we use either Arabidopsis or tobacco plant seedlings): a Petri dish containing two compartments (BI Petri dish); two Petri dishes connected with tubing; and a microtiter-based assay...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28617450/a-human-intestinal-m-cell-like-model-for-investigating-particle-antigen-and-microorganism-translocation
#3
Ana Beloqui, David J Brayden, Per Artursson, Véronique Préat, Anne des Rieux
The specialized microfold cells (M cells) in the follicle-associated epithelium (FAE) of intestinal Peyer's patches serve as antigen-sampling cells of the intestinal innate immune system. Unlike 'classical' enterocytes, they are able to translocate diverse particulates without digesting them. They act as pathways for microorganism invasion and mediate food tolerance by transcellular transport of intestinal microbiota and antigens. Their ability to transcytose intact particles can be used to develop oral drug delivery and oral immunization strategies...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28617449/assessment-of-social-transmission-of-threats-in-humans-using-observational-fear-conditioning
#4
Jan Haaker, Armita Golkar, Ida Selbing, Andreas Olsson
Across the human life span, fear is often acquired indirectly by observation of the emotional expressions of others. The observational fear conditioning protocol was previously developed as a laboratory model for investigating socially acquired threat responses. This protocol serves as a suitable alternative to the widely used Pavlovian fear conditioning, in which threat responses are acquired through direct experiences. In the observational fear conditioning protocol, the participant (observer) watches a demonstrator being presented with a conditioned stimulus (CS) paired with an aversive unconditioned stimulus (US)...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28594816/mrna-quantification-using-single-molecule-fish-in-drosophila-embryos
#5
Tatjana Trcek, Timothée Lionnet, Hari Shroff, Ruth Lehmann
Spatial information is critical to the interrogation of developmental and tissue-level regulation of gene expression. However, this information is usually lost when global mRNA levels from tissues are measured using reverse transcriptase PCR, microarray analysis or high-throughput sequencing. By contrast, single-molecule fluorescence in situ hybridization (smFISH) preserves the spatial information of the cellular mRNA content with subcellular resolution within tissues. Here we describe an smFISH protocol that allows for the quantification of single mRNAs in Drosophila embryos, using commercially available smFISH probes (e...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28594815/preparation-of-biomimetic-hierarchically-helical-fiber-actuators-from-carbon-nanotubes
#6
Jue Deng, Yifan Xu, Sisi He, Peining Chen, Luke Bao, Yajie Hu, Bingjie Wang, Xuemei Sun, Huisheng Peng
Mechanically responsive materials that are able to sense and respond to external stimuli have important applications in soft robotics and the formation of artificial muscles, such as intelligent electronics, prosthetic limbs, comfort-adjusting textiles and miniature actuators for microfluidics. However, previous artificial muscles based on polymer materials are insufficient in generating large actuations, fast responses, diverse deformation modes and high cycle performances. To this end, carbon nanotubes (CNTs) are proposed as promising candidates to be assembled into artificial muscles, as they are lightweight, robust and have high surface-to-volume ratios...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28569763/integrating-macromolecular-x-ray-diffraction-data-with-the-graphical-user-interface-imosflm
#7
Harold R Powell, T Geoff G Battye, Luke Kontogiannis, Owen Johnson, Andrew G W Leslie
X-ray crystallography is the predominant source of structural information for biological macromolecules, providing fundamental insights into biological function. The availability of robust and user-friendly software to process the collected X-ray diffraction images makes the technique accessible to a wider range of scientists. iMosflm/MOSFLM (http://www.mrc-lmb.cam.ac.uk/harry/imosflm) is a software package designed to achieve this goal. The graphical user interface (GUI) version of MOSFLM (called iMosflm) is designed to guide inexperienced users through the steps of data integration, while retaining powerful features for more experienced users...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28569762/quantitative-proteomics-challenges-and-opportunities-in-basic-and-applied-research
#8
Olga T Schubert, Hannes L Röst, Ben C Collins, George Rosenberger, Ruedi Aebersold
In this Perspective, we discuss developments in mass-spectrometry-based proteomic technology over the past decade from the viewpoint of our laboratory. We also reflect on existing challenges and limitations, and explore the current and future roles of quantitative proteomics in molecular systems biology, clinical research and personalized medicine.
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28569761/chemically-induced-mouse-models-of-acute-and-chronic-intestinal-inflammation
#9
Stefan Wirtz, Vanessa Popp, Markus Kindermann, Katharina Gerlach, Benno Weigmann, Stefan Fichtner-Feigl, Markus F Neurath
Inflammatory bowel diseases (IBDs) result in diarrhea and abdominal pain with further potential complications such as tissue fibrosis and stenosis. Animal models help in understanding the immunopathogenesis of IBDs and in the design of novel therapeutic concepts. Here we present an updated version of a protocol we published in 2007 for key models of acute and chronic forms of colitis induced by 2,4,6-trinitro-benzene sulfonic acid (TNBS), oxazolone and dextran sulfate sodium (DSS). This protocol update describes an adaptation of the existing protocol that modifies the technique...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28538739/multiplex-pcr-method-for-minion-and-illumina-sequencing-of-zika-and-other-virus-genomes-directly-from-clinical-samples
#10
Joshua Quick, Nathan D Grubaugh, Steven T Pullan, Ingra M Claro, Andrew D Smith, Karthik Gangavarapu, Glenn Oliveira, Refugio Robles-Sikisaka, Thomas F Rogers, Nathan A Beutler, Dennis R Burton, Lia Laura Lewis-Ximenez, Jaqueline Goes de Jesus, Marta Giovanetti, Sarah C Hill, Allison Black, Trevor Bedford, Miles W Carroll, Marcio Nunes, Luiz Carlos Alcantara, Ester C Sabino, Sally A Baylis, Nuno R Faria, Matthew Loose, Jared T Simpson, Oliver G Pybus, Kristian G Andersen, Nicholas J Loman
Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples (i.e., without isolation and culture) remains challenging for viruses such as Zika, for which metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence-complete genomes, comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimized library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences...
June 2017: Nature Protocols
https://www.readbyqxmd.com/read/28538738/solid-phase-synthesis-cyclization-and-site-specific-functionalization-of-aziridine-containing-tetrapeptides
#11
Benjamin K W Chung, Christopher J White, Andrei K Yudin
Cyclic tetrapeptides comprise a potent and selective class of molecules with a wide range of biological activities, including the phytotoxic activity of tentoxin and the histone deacetylase (HDAC) inhibitory effects of chlamydocin. The incorporation of a functional aziridine group within cyclic peptides enables their conformational control and allows for late-stage and site-selective functionalization of these molecules, thereby creating the potential for covalent protein labeling. This protocol describes the solid-phase synthesis, cyclization, and site-specific structural modification of aziridine-containing tetrapeptides...
June 2017: Nature Protocols
https://www.readbyqxmd.com/read/28518173/simultaneous-quantification-of-n-and-o-glycans-using-a-solid-phase-method
#12
Shuang Yang, Yingwei Hu, Lori Sokoll, Hui Zhang
Glycosylation has a pivotal role in a diverse range of biological activities, modulating the structure and function of proteins. Glycogens coupled to the nitrogen atom (N-linked) of asparagine side chains or to the oxygen atom (O-linked) of serine and threonine side chains represent the two major protein glycosylation forms. N-glycans can be released by glycosidases, whereas O-glycans are often cleaved by chemical reaction. However, it is challenging to combine these enzymatic and chemical reactions in order to analyze both N- and O-glycans...
June 2017: Nature Protocols
https://www.readbyqxmd.com/read/28518172/super-resolution-microscopy-with-dna-paint
#13
Joerg Schnitzbauer, Maximilian T Strauss, Thomas Schlichthaerle, Florian Schueder, Ralf Jungmann
Super-resolution techniques have begun to transform biological and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resolution microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resolution microscopy...
June 2017: Nature Protocols
https://www.readbyqxmd.com/read/28518171/fish-flow-a-protocol-for-the-concurrent-detection-of-mrna-and-protein-in-single-cells-using-fluorescence-in-situ-hybridization-and-flow-cytometry
#14
Riccardo Arrigucci, Yuri Bushkin, Felix Radford, Karim Lakehal, Pooja Vir, Richard Pine, December Martin, Jeffrey Sugarman, Yanlin Zhao, George S Yap, Alfred A Lardizabal, Sanjay Tyagi, Maria Laura Gennaro
We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement...
June 2017: Nature Protocols
https://www.readbyqxmd.com/read/28492527/single-cell-template-strand-sequencing-by-strand-seq-enables-the-characterization-of-individual-homologs
#15
Ashley D Sanders, Ester Falconer, Mark Hills, Diana C J Spierings, Peter M Lansdorp
The ability to distinguish between genome sequences of homologous chromosomes in single cells is important for studies of copy-neutral genomic rearrangements (such as inversions and translocations), building chromosome-length haplotypes, refining genome assemblies, mapping sister chromatid exchange events and exploring cellular heterogeneity. Strand-seq is a single-cell sequencing technology that resolves the individual homologs within a cell by restricting sequence analysis to the DNA template strands used during DNA replication...
June 2017: Nature Protocols
https://www.readbyqxmd.com/read/28492526/differentiation-of-cardiomyocytes-and-generation-of-human-engineered-heart-tissue
#16
Kaja Breckwoldt, David Letuffe-Brenière, Ingra Mannhardt, Thomas Schulze, Bärbel Ulmer, Tessa Werner, Anika Benzin, Birgit Klampe, Marina C Reinsch, Sandra Laufer, Aya Shibamiya, Maksymilian Prondzynski, Giulia Mearini, Dennis Schade, Sigrid Fuchs, Christiane Neuber, Elisabeth Krämer, Umber Saleem, Mirja L Schulze, Marita L Rodriguez, Thomas Eschenhagen, Arne Hansen
Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations...
June 2017: Nature Protocols
https://www.readbyqxmd.com/read/28471460/using-hyperlopit-to-perform-high-resolution-mapping-of-the-spatial-proteome
#17
Claire M Mulvey, Lisa M Breckels, Aikaterini Geladaki, Nina Kočevar Britovšek, Daniel J H Nightingale, Andy Christoforou, Mohamed Elzek, Michael J Deery, Laurent Gatto, Kathryn S Lilley
The organization of eukaryotic cells into distinct subcompartments is vital for all functional processes, and aberrant protein localization is a hallmark of many diseases. Microscopy methods, although powerful, are usually low-throughput and dependent on the availability of fluorescent fusion proteins or highly specific and sensitive antibodies. One method that provides a global picture of the cell is localization of organelle proteins by isotope tagging (LOPIT), which combines biochemical cell fractionation using density gradient ultracentrifugation with multiplexed quantitative proteomics mass spectrometry, allowing simultaneous determination of the steady-state distribution of hundreds of proteins within organelles...
June 2017: Nature Protocols
https://www.readbyqxmd.com/read/28471459/improving-your-four-dimensional-image-traveling-through-a-decade-of-light-sheet-based-fluorescence-microscopy-research
#18
Frederic Strobl, Alexander Schmitz, Ernst H K Stelzer
Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.
June 2017: Nature Protocols
https://www.readbyqxmd.com/read/28471458/comprehensive-analysis-of-mouse-retinal-mononuclear-phagocytes
#19
Anika Lückoff, Rebecca Scholz, Florian Sennlaub, Heping Xu, Thomas Langmann
The innate immune system is activated in a number of degenerative and inflammatory retinal disorders such as age-related macular degeneration (AMD). Retinal microglia, choroidal macrophages, and recruited monocytes, collectively termed 'retinal mononuclear phagocytes', are critical determinants of ocular disease outcome. Many publications have described the presence of these cells in mouse models for retinal disease; however, only limited aspects of their behavior have been uncovered, and these have only been uncovered using a single detection method...
June 2017: Nature Protocols
https://www.readbyqxmd.com/read/28448485/assessment-of-engineered-cells-using-cellnet-and-rna-seq
#20
Arthur H Radley, Remy M Schwab, Yuqi Tan, Jeesoo Kim, Emily K W Lo, Patrick Cahan
CellNet is a computational platform designed to assess cell populations engineered by either directed differentiation of pluripotent stem cells (PSCs) or direct conversion, and to suggest specific hypotheses to improve cell fate engineering protocols. CellNet takes as input gene expression data and compares them with large data sets of normal expression profiles compiled from public sources, in regard to the extent to which cell- and tissue-specific gene regulatory networks are established. CellNet was originally designed to work with human or mouse microarray expression data for 21 cell or tissue (C/T) types...
May 2017: Nature Protocols
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