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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/28538739/multiplex-pcr-method-for-minion-and-illumina-sequencing-of-zika-and-other-virus-genomes-directly-from-clinical-samples
#2
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
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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
#9
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
#10
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
#11
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
#12
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
https://www.readbyqxmd.com/read/28448484/tissue-engineered-3d-human-lymphatic-microvascular-network-for-in-vitro-studies-of-lymphangiogenesis
#13
Laure Gibot, Todd Galbraith, Jennifer Bourland, Anita Rogic, Mihaela Skobe, François A Auger
This protocol describes a unique in vitro method for the generation of a 3D human lymphatic network within native connective tissue devoid of any exogenous material such as scaffolds or growth factors. In this five-stage protocol, human lymphatic endothelial cells (LECs) cocultured with dermal fibroblasts spontaneously organize into a stable 3D lymphatic capillary network. Stage 1 involves the isolation of primary fibroblasts and LECs from human skin. Fibroblasts are then cultured to produce connective tissue rich in extracellular matrix (stage 2), onto which LECs are seeded to form a network (stage 3)...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28426026/generation-of-multipotent-induced-cardiac-progenitor-cells-from-mouse-fibroblasts-and-potency-testing-in-ex-vivo-mouse-embryos
#14
Pratik A Lalit, Adriana M Rodriguez, Karen M Downs, Timothy J Kamp
Here we describe a protocol to generate expandable and multipotent induced cardiac progenitor cells (iCPCs) from mouse adult fibroblasts using forced expression of Mesp1, Tbx5, Gata4, Nkx2.5 and Baf60c (MTGNB) along with activation of Wnt and JAK/STAT signaling. This method does not use iPS cell factors and thus differs from cell activation and signaling-directed (CASD) reprogramming to cardiac progenitors. Our method is specific to direct CPC reprogramming, whereas CASD reprogramming can generate various cell types depending on culture conditions and raises the possibility of transitioning through a pluripotent cell state...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28426025/modular-low-light-microscope-for-imaging-cellular-bioluminescence-and-radioluminescence
#15
Tae Jin Kim, Silvan Türkcan, Guillem Pratx
Low-light microscopy methods are receiving increased attention as new applications have emerged. One such application is to allow longitudinal imaging of light-sensitive cells with no phototoxicity and no photobleaching of fluorescent biomarkers. Another application is for imaging signals that are inherently dim and undetectable using standard microscopy techniques, such as bioluminescence, chemiluminescence or radioluminescence. In this protocol, we provide instructions on how to build a modular low-light microscope (1-4 d) by coupling two microscope objective lenses, back to back from each other, using standard optomechanical components...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28406496/strategic-and-practical-guidelines-for-successful-structured-illumination-microscopy
#16
REVIEW
Justin Demmerle, Cassandravictoria Innocent, Alison J North, Graeme Ball, Marcel Müller, Ezequiel Miron, Atsushi Matsuda, Ian M Dobbie, Yolanda Markaki, Lothar Schermelleh
Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28406495/quantitative-3d-structured-illumination-microscopy-of-nuclear-structures
#17
REVIEW
Felix Kraus, Ezequiel Miron, Justin Demmerle, Tsotne Chitiashvili, Alexei Budco, Quentin Alle, Atsushi Matsuda, Heinrich Leonhardt, Lothar Schermelleh, Yolanda Markaki
3D structured illumination microscopy (3D-SIM) is the super-resolution technique of choice for multicolor volumetric imaging. Here we provide a validated sample preparation protocol for labeling nuclei of cultured mammalian cells, image acquisition and registration practices, and downstream image analysis of nuclear structures and epigenetic marks. Using immunostaining and replication labeling combined with image segmentation, centroid mapping and nearest-neighbor analyses in open-source environments, 3D maps of nuclear structures are analyzed in individual cells and normalized to fluorescence standards on the nanometer scale...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28384139/antifungal-drug-testing-by-combining-minimal-inhibitory-concentration-testing-with-target-identification-by-gas-chromatography-mass-spectrometry
#18
Christoph Müller, Ulrike Binder, Franz Bracher, Martin Giera
Fungal infections and their increasing resistance to antibiotics are an emerging threat to public health. Novel antifungal drugs, as well technologies that can help us bolster the antimicrobial pipeline and understand resistance mechanisms, are needed. The ergosterol biosynthetic pathway is one potential target for antifungal drugs. Here we describe how antifungal susceptibility testing can be combined with target identification in distal ergosterol biosynthesis by means of gas chromatography-mass spectrometry...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28384138/diverse-protocols-for-correlative-super-resolution-fluorescence-imaging-and-electron-microscopy-of-chemically-fixed-samples
#19
Benjamin G Kopek, Maria G Paez-Segala, Gleb Shtengel, Kem A Sochacki, Mei G Sun, Yalin Wang, C Shan Xu, Schuyler B van Engelenburg, Justin W Taraska, Loren L Looger, Harald F Hess
Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28384137/preparation-of-molecularly-imprinted-polymers-specific-to-glycoproteins-glycans-and-monosaccharides-via-boronate-affinity-controllable-oriented-surface-imprinting
#20
Rongrong Xing, Shuangshou Wang, Zijun Bie, Hui He, Zhen Liu
Molecularly imprinted polymers (MIPs) are materials that are designed to be receptors for a template molecule (e.g., a protein). They are made by polymerizing the polymerizable reagents in the presence of the template; when the template is removed, the material can be used for many applications that would traditionally use antibodies. Thus, MIPs are biomimetic of antibodies and in this capacity have found wide applications, such as sensing, separation and diagnosis. However, many imprinting approaches are uncontrollable, and facile imprinting approaches widely applicable to a large variety of templates remain limited...
May 2017: Nature Protocols
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