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Journal of Visualized Experiments: JoVE

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https://www.readbyqxmd.com/read/28362407/a-high-throughput-compatible-assay-to-evaluate-drug-efficacy-against-macrophage-passaged-mycobacterium-tuberculosis
#1
Kaitlyn Schaaf, Samuel R Smith, Virginia Hayley, Olaf Kutsch, Jim Sun
The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing...
March 24, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362381/vasodilation-of-isolated-vessels-and-the-isolation-of-the-extracellular-matrix-of-tight-skin-mice
#2
Dorothee Weihrauch, John G Krolikowski, Deron W Jones, Tahniyath Zaman, Omoshalewa Bamkole, Janine Struve, Paul S Pagel, Nicole L Lohr, Kirkwood A Pritchard
The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides. IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3...
March 24, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362368/peptide-scanning-assisted-identification-of-a-monoclonal-antibody-recognized-linear-b-cell-epitope
#3
Chien-Wen Chen, Chi-Yao Chang
The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb...
March 24, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362392/fabrication-of-nanopillar-based-split-ring-resonators-for-displacement-current-mediated-resonances-in-terahertz-metamaterials
#4
Chao Liu, Joseph Schauff, Seokhyeong Lee, Jeong-Hyun Cho
Terahertz (THz) split ring resonator (SRR) metamaterials (MMs) has been studied for gas, chemical, and biomolecular sensing applications because the SRR is not affected by environmental characteristics such as the temperature and pressure surrounding the resonator. Electromagnetic radiation in THz frequencies is biocompatible, which is a critical condition especially for the application of the biomolecular sensing. However, the quality factor (Q-factor) and frequency responses of traditional thin-film based split ring resonator (SRR) MMs are very low, which limits their sensitivities and selectivity as sensors...
March 23, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362388/analyzing-dendritic-morphology-in-columns-and-layers
#5
Chun-Yuan Ting, Philip G McQueen, Nishith Pandya, Evan S McCreedy, Matthew McAuliffe, Chi-Hon Lee
In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion...
March 23, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362367/sublimation-of-dan-matrix-for-the-detection-and-visualization-of-gangliosides-in-rat-brain-tissue-for-maldi-imaging-mass-spectrometry
#6
Sarah Caughlin, Dae Hee Park, Ken K-C Yeung, David F Cechetto, Shawn N Whitehead
Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique...
March 23, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362422/in-vivo-imaging-of-transgenic-gene-expression-in-individual-retinal-progenitors-in-chimeric-zebrafish-embryos-to-study-cell-nonautonomous-influences
#7
Stefanie Dudczig, Peter D Currie, Lucia Poggi, Patricia R Jusuf
The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment...
March 22, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362413/assessing-cardiomyocyte-subtypes-following-transcription-factor-mediated-reprogramming-of-mouse-embryonic-fibroblasts
#8
Antonio Fernandez-Perez, Nikhil V Munshi
Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies...
March 22, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362412/conditional-reprogramming-of-pediatric-human-esophageal-epithelial-cells-for-use-in-tissue-engineering-and-disease-investigation
#9
Todd J Jensen, Christopher Foster, Wael Sayej, Christine M Finck
Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype...
March 22, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362408/in-vitro-imaging-and-quantification-of-the-drug-targeting-efficiency-of-fluorescently-labeled-gnrh-analogues
#10
József Murányi, Attila Varga, Bianka Gurbi, Pál Gyulavári, Gábor Mező, Tibor Vántus
GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH receptors. However, the quantitative analysis of GnRH analogues' cellular uptake and the investigated cell types in GnRH-based drug delivery systems are currently limited. Previously introduced, selectively labeled fluorescent GnRH I, -II and -III derivatives provide great detectability, and they have suitable chemical properties for reproducible and robust experiments...
March 21, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362391/absolute-quantification-of-a%C3%AE-1-42-in-csf-using-a-mass-spectrometric-reference-measurement-procedure
#11
Josef Pannee, Kaj Blennow, Henrik Zetterberg, Erik Portelius
Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the diagnosis of AD is based on cognitive tests and mental state exams, but the peptide amyloid-beta (Aβ) in cerebrospinal fluid (CSF) is increasingly used in clinical trials and settings. As for most protein and peptide biomarkers, quantification is performed using antibody-based techniques, such as enzyme-linked immunosorbent assay (ELISA). However, intra- and inter-laboratory variability in these assays hamper its use as a diagnostic marker in clinical routine...
March 21, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362378/combining-intravital-fluorescent-microscopy-ivfm-with-genetic-models-to-study-engraftment-dynamics-of-hematopoietic-cells-to-bone-marrow-niches
#12
Lin Wang, Malgorzata M Kamocka, Amy Zollman, Nadia Carlesso
Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions(1)(,)(2). Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny(3)(,)(4)(,)(5). New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function(6)(,)(7)(,)(8)...
March 21, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362394/techniques-of-sleeve-gastrectomy-and-modified-roux-en-y-gastric-bypass-in-mice
#13
Audrey Ayer, Frederic Borel, Francois Moreau, Xavier Prieur, Michel Neunlist, Bertrand Cariou, Claire Blanchard, Cedric Le May
Obesity is a major public health issue, with a prevalence of 4 to 28% for men and 6.2 to 36.5% for women in Europe (from 2003 to 2008). Morbid obesity is frequently associated with metabolic complications, such as type 2 diabetes, hypertension, and dyslipidemia, reducing life expectancy and quality. In the absence of any effective noninvasive treatments, bariatric surgery is a valuable therapeutic option for patients with morbid obesity (body mass index (BMI) >40 kg/m(2)), leading to long-term, sustained weight loss and improvements in metabolic complications...
March 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362389/accessing-valuable-ligand-supports-for-transition-metals-a-modified-intermediate-scale-preparation-of-1-2-3-4-5-pentamethylcyclopentadiene
#14
Zachary Call, Meagan Suchewski, Christopher A Bradley
A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that derive from initial 2-bromo-2-butene lithiation followed by acid mediated dienol cyclization. The revised synthesis and purification of the ligand avoids the use of mechanical stirring while still permitting access to significant quantities (39 g) of Cp*H in good yield (58%). The procedure offers other additional benefits, including a more controlled quench of excess lithium during the production of the intermediate heptadienols and a simplified isolation of Cp*H of sufficient purity for metallation with transition metals...
March 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362370/transmission-of-multiple-signals-through-an-optical-fiber-using-wavefront-shaping
#15
Daniel Haufe, Nektarios Koukourakis, Lars Büttner, Jürgen W Czarske
The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the light distortion during the propagation within the fiber. Our methodology is based on digital optical phase conjugation employing only a single spatial light modulator, where the optical wavefront is individually modulated at different regions of the modulator, one region per light signal. Digital optical phase conjugation approaches are considered to be faster than other wavefront shaping approaches, where (for example) a complete determination of the wave propagation behavior of the fiber is performed...
March 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362362/combined-intravital-microscopy-and-contrast-enhanced-ultrasonography-of-the-mouse-hindlimb-to-study-insulin-induced-vasodilation-and-muscle-perfusion
#16
Alexander H Turaihi, Erik M van Poelgeest, Victor W M van Hinsbergh, Erik H Serné, Yvo M Smulders, Etto C Eringa
It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion...
March 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362424/dissection-microct-scanning-and-morphometric-analyses-of-the-baculum
#17
Nicholas G Schultz, Erik Otárola-Castillo, Matthew D Dean
Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone)...
March 19, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362390/blue-hazard-free-candlelight-oled
#18
Jwo-Huei Jou, Meenu Singh, Yu-Ting Su, Shih-Hao Liu, Zhe-Kai He
A candlelight-style organic light emitting diode (OLED) is a human-friendly type of lighting because it is blue-hazard-free and has a low correlated color temperature (CCT) illumination. The low CCT lighting is deprived of high-energy blue radiation, and it can be used for a longer duration before causing retinal damage. This work presents the comprehensive protocols for the fabrication of blue-hazard-free candlelight OLEDs. The emission spectrum of the OLED was characterized by the maximum exposure time limit of the retina and the melatonin suppression sensitivity...
March 19, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362369/monovalent-cation-doping-of-ch3nh3pbi3-for-efficient-perovskite-solar-cells
#19
Mojtaba Abdi-Jalebi, M Ibrahim Dar, Aditya Sadhanala, Satyaprasad P Senanayak, Michael Grätzel, Richard H Friend
Here, we demonstrate the incorporation of monovalent cation additives into CH3NH3PbI3 perovskite in order to adjust the optical, excitonic, and electrical properties. The possibility of doping was investigated by adding monovalent cation halides with similar ionic radii to Pb(2+), including Cu(+), Na(+), and Ag(+). A shift in the Fermi level and a remarkable decrease of sub-bandgap optical absorption, along with a lower energetic disorder in the perovskite, was achieved. An order-of-magnitude enhancement in the bulk hole mobility and a significant reduction of transport activation energy within an additive-based perovskite device was attained...
March 19, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28362380/production-and-administration-of-therapeutic-mesenchymal-stem-stromal-cell-msc-spheroids-primed-in-3-d-cultures-under-xeno-free-conditions
#20
Joni H Ylostalo, Nikolay Bazhanov, Arezoo Mohammadipoor, Thomas J Bartosh
Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable...
March 18, 2017: Journal of Visualized Experiments: JoVE
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