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Journal of Visualized Experiments: JoVE

Priyadip Das, Tal Duanias-Assaf, Meital Reches
The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications...
March 6, 2017: Journal of Visualized Experiments: JoVE
Charlotte Leidinger, Felix Herrmann, Christa Thöne-Reineke, Nadine Baumgart, Jan Baumgart
Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward...
March 6, 2017: Journal of Visualized Experiments: JoVE
Joanna A Guse, Timothy W Jones, Andrew Danos, Dane R McCamey
A method for investigating recombination dynamics of photo-induced charge carriers in thin film semiconductors, specifically in photovoltaic materials such as organo-lead halide perovskites is presented. The perovskite film thickness and absorption coefficient are initially characterized by profilometry and UV-VIS absorption spectroscopy. Calibration of both laser power and cavity sensitivity is described in detail. A protocol for performing Flash-photolysis Time Resolved Microwave Conductivity (TRMC) experiments, a non-contact method of determining the conductivity of a material, is presented...
March 6, 2017: Journal of Visualized Experiments: JoVE
Kaitlyn D Sniffen, Christopher M Sales, Mira S Olson
An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ammonia removal, nitrogen removal and algal growth are compared over an 8-week period in paired sets of small (100 L) and large (1,000 L) reactors designed for algal remediation of landfill wastewater. Contents of the small and large scale reactors were mixed before the beginning of each weekly testing interval to maintain equivalent initial conditions across the two scales...
March 6, 2017: Journal of Visualized Experiments: JoVE
Zhong-Dong Shi, Chew-Li Soh, Zengrong Zhu, Danwei Huangfu
Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus...
March 5, 2017: Journal of Visualized Experiments: JoVE
Robyn M Johnston, Anne W Sylvester, Michael J Scanlon
Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments...
March 5, 2017: Journal of Visualized Experiments: JoVE
Ramakrishnan B Kumar, Lin Zhu, Hans Hebert, Caroline Jegerschöld
Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc...
March 5, 2017: Journal of Visualized Experiments: JoVE
Jun Tang, Carlos Pérez-Medina, Yiming Zhao, Ahmad Sadique, Willem J M Mulder, Thomas Reiner
Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy...
March 4, 2017: Journal of Visualized Experiments: JoVE
Sara Gonia, Judith Berman, Cheryl A Gale
Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively...
March 4, 2017: Journal of Visualized Experiments: JoVE
Ibrahim B Durowoju, Kamaljit S Bhandal, Jian Hu, Bruce Carpick, Marina Kirkitadze
Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural "fingerprint" that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; ∆H) of proteins...
March 4, 2017: Journal of Visualized Experiments: JoVE
Anne Mayer-Scholl, Edoardo Pozio, Jennifer Gayda, Nora Thaben, Peter Bahn, Karsten Nöckler
Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses. There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard...
March 3, 2017: Journal of Visualized Experiments: JoVE
Paolo Benigni, Rebecca Marin, Kathia Sandoval, Piero Gardinali, Francisco Fernandez-Lima
Multiple chemical processes control how crude oil is incorporated into seawater and also the chemical reactions that occur overtime. Studying this system requires the careful preparation of the sample in order to accurately replicate the natural formation of the water-accommodated fraction that occurs in nature. Low-energy water-accommodated fractions (LEWAF) are carefully prepared by mixing crude oil and water at a set ratio. Aspirator bottles are then irradiated, and at set time points, the water is sampled and extracted using standard techniques...
March 3, 2017: Journal of Visualized Experiments: JoVE
Matthew M Scase, Kyle A Baldwin, Richard J A Hill
Classical techniques for investigating the Rayleigh-Taylor instability include using compressed gasses(1), rocketry(2) or linear electric motors(3) to reverse the effective direction of gravity, and accelerate the lighter fluid toward the denser fluid. Other authors(e.g.)(4)(,)(5)(,)(6) have separated a gravitationally unstable stratification with a barrier that is removed to initiate the flow. However, the parabolic initial interface in the case of a rotating stratification imposes significant technical difficulties experimentally...
March 3, 2017: Journal of Visualized Experiments: JoVE
Collins Cheruiyot, Zemplen Pataki, Robert Williams, Bharat Ramratnam, Ming Li
Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies...
March 3, 2017: Journal of Visualized Experiments: JoVE
Yu Wang, Sharmin Alhaque, Kate Cameron, Jose Meseguer-Ripolles, Baltasar Lucendo-Villarin, Hassan Rashidi, David C Hay
Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes...
March 2, 2017: Journal of Visualized Experiments: JoVE
Hung-Chang Shen, Tsai-Chi Hsu, Pei-Chi Chung, Hung-Hsiang Yu
Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis...
March 2, 2017: Journal of Visualized Experiments: JoVE
Katrien De Clercq, Aurélie Hennes, Joris Vriens
Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization...
March 2, 2017: Journal of Visualized Experiments: JoVE
Mitchel C Schiewe, Shane Zozula, Nancy Nugent, Kelley Waggoner, Jessica Borba, Lisa Gamboa, John B Whitney
Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21(st) century and with the misconception that ultra-rapid cooling in an "open" system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety)...
March 2, 2017: Journal of Visualized Experiments: JoVE
Ke Ma, Dominique P Lippelt, Bernhard Hommel
Studies investigating how people represent themselves and their own body often use variants of "ownership illusions", such as the traditional rubber-hand illusion or the more recently discovered enfacement illusion. However, these examples require rather artificial experimental setups, in which the artificial effector needs to be stroked in synchrony with the participants' real hand or face-a situation in which participants have no control over the stroking or the movements of their real or artificial effector...
March 1, 2017: Journal of Visualized Experiments: JoVE
Xue Liu, Guy K German
Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both cleansing agents and daily variations in ambient moisture; both of which can alter the water content of the tissue. Reductions in water content from severe barrier dysfunction or low humidity environments can alter SC stiffness and cause a build-up of drying stresses. In extreme conditions, these factors can cause mechanical rupture of the tissue. We have established a high throughput method of quantifying dynamic changes in the mechanical properties of SC upon drying...
March 1, 2017: Journal of Visualized Experiments: JoVE
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