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Journal of Visualized Experiments: JoVE

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https://www.readbyqxmd.com/read/28745647/utilization-of-capsules-for-negative-staining-of-viral-samples-within-biocontainment
#1
Candace D Blancett, Mitchell K Monninger, Chrystal A Nguessan, Kathleen A Kuehl, Cynthia A Rossi, Scott P Olschner, Priscilla L Williams, Steven L Goodman, Mei G Sun
Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents...
July 19, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745646/reducing-state-anxiety-using-working-memory-maintenance
#2
Nicholas L Balderston, Abigail Hsiung, Jeffrey Liu, Monique Ernst, Christian Grillon
The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working Memory (WM) and the threat of shock paradigms. In the Sternberg WM paradigm, subjects are required to maintain a series of letters in the WM for a brief interval and respond by identifying whether the position of a given letter in the series matches a numerical prompt. In the threat of shock paradigm, subjects are exposed to alternating blocks where they are either at risk of receiving unpredictable presentations of a mild electric shock or are safe from the shock...
July 19, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745645/a-simple-neuronal-mechanical-injury-methodology-to-study-drosophila-motor-neuron-degeneration
#3
Erika B Danella, Lani C Keller
The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases...
July 19, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745619/quantification-of-drosophila-grooming-behavior
#4
Francesca Barradale, Kairav Sinha, Tim Lebestky
Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye...
July 19, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745649/high-resolution-melting-pcr-for-complement-receptor-1-length-polymorphism-genotyping-an-innovative-tool-for-alzheimer-s-disease-gene-susceptibility-assessment
#5
Aymric Kisserli, Thierry Tabary, Jacques Henri Max Cohen, Valérie Duret, Rachid Mahmoudi
Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (Aβ) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene...
July 18, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745637/isolation-of-circulating-tumor-cells-in-an-orthotopic-mouse-model-of-colorectal-cancer
#6
Susan Kochall, May-Linn Thepkaysone, Sebastián A García, Alexander M Betzler, Jürgen Weitz, Christoph Reissfelder, Sebastian Schölch
Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical...
July 18, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745634/synthesis-and-testing-of-supported-pt-cu-solid-solution-nanoparticle-catalysts-for-propane-dehydrogenation
#7
Zixue Ma, Zhenwei Wu, Jeffrey T Miller
A convenient method for the synthesis of bimetallic Pt-Cu catalysts and performance tests for propane dehydrogenation and characterization are demonstrated here. The catalyst forms a substitutional solid solution structure, with a small and uniform particle size around 2 nm. This is realized by careful control over the impregnation, calcination, and reduction steps during catalyst preparation and is identified by advanced in situ synchrotron techniques. The catalyst propane dehydrogenation performance continuously improves with increasing Cu:Pt atomic ratio...
July 18, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745632/differentiating-chondrocytes-from-peripheral-blood-derived-human-induced-pluripotent-stem-cells
#8
Yueying Li, Yong Hai, Jiayu Chen, Tie Liu
In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers...
July 18, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745629/intrarenal-injection-of-escherichia-coli-in-a-rat-model-of-pyelonephritis
#9
Karishma Gupta, Shannon B Donnola, Zhina Sadeghi, Lan Lu, Bernadette O Erokwu, Michael Kavran, Adonis Hijaz, Chris A Flask
Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney...
July 18, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745642/assessing-spatial-memory-impairment-in-a-mouse-model-of-traumatic-brain-injury-using-a-radial-water-tread-maze
#10
Marcella M Cline, Megan A Ostlie, Chloe G Cross, Gregory G Garwin, Satoshi Minoshima, Donna J Cross
Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and validated for rat use. The Radial Water Tread (RWT) maze test of spatial memory (designed specifically for mice and requiring no swimming) has been shown previously to successfully distinguish between controlled cortical impact-induced TBI mice and sham controls. Here, a detailed protocol for this task is presented. The RWT maze capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the sides of an apparatus (thigmotaxis)...
July 17, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745633/determination-of-the-glycogen-content-in-cyanobacteria
#11
Alice De Porcellinis, Niels-Ulrik Frigaard, Yumiko Sakuragi
Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities...
July 17, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745628/a-method-to-test-the-effect-of-environmental-cues-on-mating-behavior-in-drosophila-melanogaster
#12
Jenke A Gorter, Jean-Christophe Billeter
An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior...
July 17, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745620/identification-of-erythromyeloid-progenitors-and-their-progeny-in-the-mouse-embryo-by-flow-cytometry
#13
Lorea Iturri, Javier Saenz Coronilla, Yvan Lallemand, Elisa Gomez Perdiguero
Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others...
July 17, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745639/a-lipid-extraction-and-analysis-method-for-characterizing-soil-microbes-in-experiments-with-many-samples
#14
Lawrence G Oates, Harry W Read, Jessica L M Gutknecht, David S Duncan, Teri B Balser, Randall D Jackson
Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes...
July 16, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745621/investigating-protein-sequence-structure-dynamics-relationships-with-bio3d-web
#15
Shashank Jariwala, Lars Skjærven, Xin-Qiu Yao, Barry J Grant
We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality for: (1) The identification of related protein structure sets to user specified thresholds of similarity; (2) Their multiple alignment and structure superposition; (3) Sequence and structure conservation analysis; (4) Inter-conformer relationship mapping with principal component analysis, and (5) comparison of predicted internal dynamics via ensemble normal mode analysis...
July 16, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745648/fizzy-extraction-of-volatile-organic-compounds-combined-with-atmospheric-pressure-chemical-ionization-quadrupole-mass-spectrometry
#16
Hao-Chun Yang, Cheng-Hao Chang, Pawel L Urban
Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to the gas phase, and efficiently transferred to a detection system. Fizzy extraction takes advantage of the effervescence phenomenon. First, a carrier gas (here, carbon dioxide) is dissolved in the sample by applying overpressure and stirring the sample. Second, the sample chamber is decompressed abruptly. Decompression leads to the formation of numerous carrier gas bubbles in the sample liquid...
July 14, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745643/isolation-and-culture-of-primary-mouse-keratinocytes-from-neonatal-and-adult-mouse-skin
#17
Fengwu Li, Christopher A Adase, Ling-Juan Zhang
The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS)...
July 14, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745640/determination-of-sialic-acids-in-liver-and-milk-samples-of-wild-type-and-cmah-knock-out-mice
#18
Cui Cao, Wen J Wang, Ying Y Huang, Hong L Yao, Louis P Conway, Li Liu, Josef Voglmeir
CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice...
July 14, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745631/integration-of-miniaturized-solid-phase-extraction-and-lc-ms-ms-detection-of-3-nitrotyrosine-in-human-urine-for-clinical-applications
#19
Xiaoguang Sunny Li, Shu Li, Melissa Ahrens, Gottfried Kellermann
Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions...
July 14, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745644/profiling-anti-neu5gc-igg-in-human-sera-with-a-sialoglycan-microarray-assay
#20
Shani Leviatan Ben-Arye, Hai Yu, Xi Chen, Vered Padler-Karavani
Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5'monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH)...
July 13, 2017: Journal of Visualized Experiments: JoVE
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