High-Resolution Multi-Photon Imaging of Morphological Structures of Caenorhabditis elegans.
Authors: Bixel GM, Fretham SJ, Aschner M
Abstract
In this protocol, we combine two-photon excitation fluorescence with nonlinear optical measurements to reconstruct the three-dimensional architecture of the pharyngeal region and the muscular system of the anterior and mid-body region of Caenorhabditis elegans (C. elegans). Femto-second laser pulses excite second-harmonic generation (SHG) and third-harmonic generation (THG) signals, which show detailed structural information regarding the organization of myofibrils that are arranged around the central pharynx region. The combination of two-photon excitation...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Growth and Quantification of MERS-CoV Infection.
Authors: Coleman CM, Frieman MB
Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging highly pathogenic respiratory virus. Although MERS-CoV only emerged in 2012, we and others have developed assays to grow and quantify infectious MERS-CoV and RNA products of replication in vitro. MERS-CoV is able to infect a range of cell types, but replicates to high titers in Vero E6 cells. Protocols for the propagation and quantification of MERS-CoV are presented. © 2015 by John Wiley & Sons, Inc.
PMID: 26344222 [PubMed - in process] (Source: Current Protocols in Toxicology)
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Detection of Bulky Endogenous Oxidative DNA Lesions Derived from 8,5'-Cyclo-2'-deoxyadenosine by 32P-Postlabeling Assay.
Authors: Zhou GD, Moorthy B
Abstract
8,5'-Cyclopurine-2'-deoxynucleotides represent a class of oxidative DNA lesions that are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. The 32P-postlabeling assay is an ultrasensitive method that has been extensively used for the detection of carcinogen-DNA adducts in laboratory animal and epidemiological studies. This assay under modified chromatographic conditions is also a suitable and sensitive method for the detection of 8,5'-cyclo-2'-deoxyadenosine (cA). After enzymatic digestion of DNA, and enric...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
microRNA Profiling as Tool for Developmental Neurotoxicity Testing (DNT).
Authors: Smirnova L, Seiler AE, Luch A
Abstract
microRNAs (miRNAs) are small non-coding RNA molecules functioning as post-transcriptional regulators of gene expression. miRNAs play a significant role in organism development, regulating developmental timing, cell differentiation, and specification. In the developing brain, miRNAs regulate neural stem cell differentiation, lineage specification, synaptogenesis, and brain morphogenesis. Temporal and spatial specificity of miRNA expression make them an attractive marker to study cellular responses to toxicant exposure. Neural differentiation of murine embryoni...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Immune Cell Phenotyping Using Flow Cytometry.
Authors: Pockley AG, Foulds GA, Oughton JA, Kerkvliet NI, Multhoff G
Abstract
Fluorescent immunophenotyping uses fluorescently-conjugated antibodies to identify, characterize and quantify distinct subpopulations of cells within heterogeneous single-cell populations, either in the context of tissue (using fluorescence and imaging microscopy) or in a single-cell suspension (using multiparameter imaging microscopy, imaging cytometry, and/or flow cytometry). Flow cytometry is an optical, laser-based technology which analyzes the physical and fluorescent properties of cells in suspension in real-time as they fl...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Caenorhabditis elegans as a Model for Toxic Effects of Nanoparticles: Lethality, Growth, and Reproduction.
Authors: Maurer LL, Ryde IT, Yang X, Meyer JN
Abstract
The nematode Caenorhabditis elegans is extensively utilized in toxicity studies. C. elegans offers a high degree of homology with higher organisms, and its ease of use and relatively inexpensive maintenance have made it an attractive complement to mammalian and ecotoxicological models. C. elegans provides multiple benefits, including the opportunity to perform relatively high-throughput assays on whole organisms, a wide range of genetic tools permitting investigation of mechanisms and genetic sensitivity, and transparent bodies that facilitate toxicoki...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Measuring p66Shc Signaling Pathway Activation and Mitochondrial Translocation in Cultured Cells.
Authors: Wieckowski MR, Deus CM, Couto R, Oparka M, Lebiedzińska-Arciszewska M, Duszyński J, Oliveira PJ
Abstract
The adaptor protein p66Shc links membrane receptors to intracellular signaling pathways, with downstream consequences on mitochondrial metabolism and reactive oxygen species production. Moreover, p66Shc has also been implicated in cancer development, progression, and metastasis. Increased phosphorylation of serine 36 residue of p66Shc very often correlates with oxidative stress-associated pathologies. The pro-oxidative role of p66Shc also appears to be involved in chemical toxicity, being an ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Seahorse Xf(e) 24 Extracellular Flux Analyzer-Based Analysis of Cellular Respiration in Caenorhabditis elegans.
Authors: Luz AL, Smith LL, Rooney JP, Meyer JN
Abstract
Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and intercellular as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XF(e) 24 Extracellular Flux Analyzer and the pharmacological inhibitors dic...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Use of Ciliogenesis to Detect Aneugens: The Role of Primary Cilia.
Authors: Divi KV, Ward Y, Poirier MC, Olivero OA
Abstract
Primary cilia arise from the centrosomes of quiescent or post-mitotic cells, and serve as sensory organelles that communicate mechanical and chemical stimuli from the environment to the interior of the cell. Cilium formation may, therefore, become a useful end point signaling exposure to genotoxins or aneugens. Here we have used the aneugen, zidovudine (AZT), an antiretroviral drug that induces DNA replication arrest and centrosomal amplification (>2 centrosomes per quiescent cell), to evaluate cilia formation in retinal epithelial (pigmented) ce...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
