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Journal of Visualized Experiments: JoVE

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https://www.readbyqxmd.com/read/29286491/microprobe-capillary-electrophoresis-mass-spectrometry-for-single-cell-metabolomics-in-live-frog-xenopus-laevis-embryos
#1
Rosemary M Onjiko, Erika P Portero, Sally A Moody, Peter Nemes
The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology...
December 22, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286428/stenosis-of-the-inferior-vena-cava-a-murine-model-of-deep-vein-thrombosis
#2
Holly Payne, Alexander Brill
Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created...
December 22, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286420/comprehensive-workflow-for-the-genome-wide-identification-and-expression-meta-analysis-of-the-atl-e3-ubiquitin-ligase-gene-family-in-grapevine
#3
Pietro Ariani, Elodie Vandelle, Darren Wong, Alejandro Giorgetti, Andrea Porceddu, Salvatore Camiolo, Annalisa Polverari
Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis Tóxicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine...
December 22, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286419/generation-and-culturing-of-primary-human-keratinocytes-from-adult-skin
#4
Claus Johansen
The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin...
December 22, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286485/characterization-of-tumor-cells-using-a-medical-wire-for-capturing-circulating-tumor-cells-a-3d-approach-based-on-immunofluorescence-and-dna-fish
#5
Giulia Gallerani, Claudia Cocchi, Martine Bocchini, Filippo Piccinini, Francesco Fabbri
Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire...
December 21, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286479/method-for-labeling-transcripts-in-individual-escherichia-coli-cells-for-single-molecule-fluorescence-in-situ-hybridization-experiments
#6
Rinat Arbel-Goren, Yonatan Shapira, Joel Stavans
A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes...
December 21, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286414/genome-wide-quantification-of-translation-in-budding-yeast-by-ribosome-profiling
#7
Carine Beaupere, Rosalyn B Chen, William Pelosi, Vyacheslav M Labunskyy
Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast...
December 21, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286411/monitoring-the-effects-of-illumination-on-the-structure-of-conjugated-polymer-gels-using-neutron-scattering
#8
Brian Morgan, Samantha J Rinehart, Mark D Dadmun
We demonstrate a protocol to effectively monitor the gelation process of a high concentration solution of conjugated polymer both in the presence and absence of white light exposure. By instituting a controlled temperature ramp, the gelation of these materials can be precisely monitored as they proceed through this structural evolution, which effectively mirrors the conditions experienced during the solution deposition phase of organic electronic device fabrication. Using small angle neutron scattering (SANS) and ultra-small angle neutron scattering (USANS) along with appropriate fitting protocols we quantify the evolution of select structural parameters throughout this process...
December 21, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286477/biotinylated-cell-penetrating-peptides-to-study-intracellular-protein-protein-interactions
#9
Myriam Jaraíz-Rodríguez, Ana González-Sánchez, Laura García-Vicente, Jose M Medina, Arantxa Tabernero
Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas...
December 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286471/in-vitro-enzyme-measurement-to-test-pharmacological-chaperone-responsiveness-in-fabry-and-pompe-disease
#10
Jan Lukas, Anne-Marie Knospe, Susanne Seemann, Valentina Citro, Maria V Cubellis, Arndt Rolfs
The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, high costs, and low patient numbers. Hundreds of mutant alleles are implicated in most of the LSDs. The diseases are typically classified into 2 to 3 different clinical types according to severity. Moreover, molecular characterization of the genotype can help predict clinical outcomes and inform patient care. Therefore, we developed a simple cell culture assay based on HEK293H cells heterologously over-expressing the mutations identified in Fabry and Pompe disease...
December 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286409/the-ingestion-of-fluorescent-magnetic-nanoparticles-for-determining-fluid-uptake-abilities-in-insects
#11
Matthew S Lehnert, Kristen E Reiter, Andrew Bennett, Patrick D Gerard, Qi-Huo Wei, Miranda Byler, Huan Yan, Wah-Keat Lee
Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unaccompanied with experimental evidence. Here, we report a novel method for assessing fluid-uptake abilities with butterflies (Lepidoptera) and flies (Diptera) using small amounts of liquids...
December 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286392/overlapping-peptide-library-to-map-qa-1-epitopes-in-a-protein
#12
Yi Xu, Samiksha Wasnik, David J Baylink, Edmundo Carreon Berumen, Xiaolei Tang
Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8+ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes...
December 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286486/whole-body-pet-mri-of-pediatric-patients-the-details-that-matter
#13
Anuj Pareek, Anne M Muehe, Ashok J Theruvath, Praveen K Gulaka, Sheri L Spunt, Heike E Daldrup-Link
Integrated PET/MRI is a hybrid imaging technique enabling clinicians to acquire diagnostic images for tumor assessment and treatment monitoring with both high soft tissue contrast and added metabolic information. Integrated PET/MRI has shown to be valuable in the clinical setting and has many promising future applications. The protocol presented here will provide step-by-step instructions for the acquisition of whole-body 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) PET/MRI data in children with cancer. It also provides instructions on how to combine a whole-body staging scan with a local tumor scan for evaluation of the primary tumor...
December 19, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286480/3d-mitochondrial-ultrastructure-of-drosophila-indirect-flight-muscle-revealed-by-serial-section-electron-tomography
#14
Yi-Fan Jiang, Hsiang-Ling Lin, Chi-Yu Fu
Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique cristae-rich double membrane ultrastructure of this organelle is elegantly arranged to carry out multiple functions by partitioning biomolecules. Mitochondrial ultrastructure is intimately linked with various functions; however, the fine details of these structure-function relationships are only beginning to be described. Here, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle...
December 19, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286422/methodology-for-biomimetic-chemical-neuromodulation-of-rat-retinas-with-the-neurotransmitter-glutamate-in-vitro
#15
Corey M Rountree, John B Troy, Laxman Saggere
Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients...
December 19, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286400/experimental-column-setup-for-studying-anaerobic-biogeochemical-interactions-between-iron-oxy-hydroxides-trace-elements-and-bacteria
#16
Jennifer Hellal, André Burnol, Aude Locatelli, Fabienne Battaglia-Brunet
Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg°...
December 19, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286482/real-time-measurement-of-epithelial-barrier-permeability-in-human-intestinal-organoids
#17
David R Hill, Sha Huang, Yu-Hwai Tsai, Jason R Spence, Vincent B Young
Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters...
December 18, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286478/a-murine-model-of-vertical-sleeve-gastrectomy
#18
Darline Garibay, Bethany P Cummings
Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. Bariatric surgery is currently the most effective long-term treatment for obesity. In addition to weight loss, bariatric surgery produces additional health benefits such as remission of type 2 diabetes, remission of hypertension, and decreased risk of developing certain types of cancer. The mechanisms beyond weight loss for these benefits remain incompletely defined...
December 18, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286410/an-engineered-split-tet2-enzyme-for-chemical-inducible-dna-hydroxymethylation-and-epigenetic-remodeling
#19
Minjung Lee, Yubin Zhou, Yun Huang
DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution...
December 18, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29286406/high-throughput-parallel-sequencing-to-measure-fitness-of-leptospira-interrogans-transposon-insertion-mutants-during-golden-syrian-hamster-infection
#20
Kristel Lourdault, James Matsunaga, Karen V Evangelista, David A Haake
In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant...
December 18, 2017: Journal of Visualized Experiments: JoVE
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