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Arnaoutova I.,Trevigen, Inc. | Kleinman H.K.,U.S. National Institutes of Health
Nature Protocols | Year: 2010

A protocol is presented here for a rapid, quantitative and reliable in vitro angiogenesis assay that can be adapted for high throughput use. Endothelial cells are plated on a gelled basement matrix, their natural substrate, and form capillary-like structures with a lumen. The assay can be used to identify inhibitors or stimulators of angiogenesis, as well as genes and signaling pathways involved in angiogenesis. It has also been used to identify endothelial progenitor cells. This assay involves endothelial cell adhesion, migration, protease activity and tubule formation. This tube formation assay is preferred, as other in vitro assays for angiogenesis, such as cell adhesion, migration and invasion, measure limited steps in the angiogenesis process. The tube formation assay on basement membrane can be completed in a day because transformed endothelial cells form tubes within 3 h, whereas non-transformed endothelial cells form tubes within 6 h. © 2010 Nature Publlshing Group. Source

Reha-Krantz L.J.,University of Alberta | Woodgate S.,Trevigen, Inc. | Goodman M.F.,University of Southern California
Frontiers in Microbiology | Year: 2014

DNA polymerases need to be engineered to achieve optimal performance for biotechnological applications, which often require high fidelity replication when using modified nucleotides and when replicating difficult DNA sequences. These tasks are achieved for the bacteriophage T4 DNA polymerase by replacing leucine with methionine in the highly conserved Motif A sequence (L412M). The costs are minimal. Although base substitution errors increase moderately, accuracy is maintained for templates with mono- and dinucleotide repeats while replication efficiency is enhanced. The L412M substitution increases intrinsic processivity and addition of phage T4 clamp and single-stranded DNA binding proteins further enhance the ability of the phage T4 L412M-DNA polymerase to replicate all types of difficult DNA sequences. Increased pyrophosphorolysis is a drawback of increased processivity, but pyrophosphorolysis is curbed by adding an inorganic pyrophosphatase or divalent metal cations, Mn2+ or Ca2+. In the absence of pyrophosphorolysis inhibitors, the T4 L412M-DNA polymerase catalyzed sequence-dependent pyrophosphorolysis under DNA sequencing conditions. The sequence specificity of the pyrophosphorolysis reaction provides insights into how the T4 DNA polymerase switches between nucleotide incorporation, pyrophosphorolysis and proofreading pathways. The L-to-M substitution was also tested in the yeast DNA polymerases delta and alpha. Because the mutant DNA polymerases displayed similar characteristics, we propose that amino acid substitutions in Motif A have the potential to increase processivity and to enhance performance in biotechnological applications. An underlying theme in this chapter is the use of genetic methods to identify mutant DNA polymerases with potential for use in current and future biotechnological applications. © 2014 Reha-Krantz, Woodgate and Goodman. Source

Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 153.00K | Year: 2016

DESCRIPTION provided by applicant The objective of this Phase STTR research is to quantify genotoxicity in basal cell keratinocytes from organotypic cultures Epiderm in response to commonly used chemical agents The proposed product is a Comet Chip assay that measures DNA damage in basal cells derived from a reconstructed human epidermis Technical questions that will be addressed are Can we modify our currently used Comet Chip assay to incorporate extracellular matrix proteins or antibodies Can we isolate individual basal keratinocytes from a D organotypic skin culture on the basis of their preferential adhesion to these matrix proteins including collagen I and IV or by using immobilized antibodies to integrin Can we confirm their identity by quantum dot coupled antibodies specific for or collagen ligand integrins Can we use the isolated antibody labeled epidermal basal cells to detect and quantify levels of DNA damage in response to known environmental genotoxic agents Can we use our Immuno CometChip assay to screen large numbers of agents currently or proposed to be marketed The impact of the proposed research will be to reduce animal model use for toxic agent screening since human organotypic culture has been shown to be almost identical to human skin with respect to its cytokine profile in response to corrosive or irritating agents The market for screening skin genotoxic agents is immense since the current screening procedures cannot keep pace with the number of new agents currently being introduced Aim will be to develop a new method for the isolation of basal keratinocytes and an immunostaining method for simultaneous visualization of specific antigens including integrin and DNA damage Aim will be to validate the Immuno CometChip assay using known DNA damaging agents including H O This adds three new parameters to the Comet assay The first is obtaining treatment groups of single basal epidermal keratinocytes from an organotypic culture on a single chip the second is verifying their identity with surface markers and the third is the simultaneous reproducible assay for DNA damage PUBLIC HEALTH RELEVANCE The Comet assay has appeal for many reasons The assay is rapid simple sensitive reliable and fairly inexpensive Its use for testing for skin toxicity hs been hampered by a number of factors including background DNA damage incurred during the normal process of differentiation in organotypic skin models which mimic human skin We therefore propose to develop a Comet Chip assay a high throughput reproducible and quantitative method to isolate basal keratinocytes and measure genotoxicity in response to commonly used chemical agents

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 318.78K | Year: 2011

DESCRIPTION (provided by applicant): Human exposure to dangerous genotoxins is unavoidable, as DNA damaging agents are ubiquitous both in our environment and within our cells. DNA damaging agents and other genotoxins that arise from cellular metabolism, environmental sources or disease-related cellular defects contribute to cell death (e.g., neurodegeneration), gene mutations, gene rearrangements and in many cases, the onset of cancer, disease and aging phenotypes. In addition, many exogenous exposures suchas chemotherapy and radiation treatment rely on the induction of tumor cell genotoxicity to mediate therapeutic response. Further, the ability to effectively and accurately repair spontaneous or induced DNA damage depends on the cellular DNA repair capacity. Therefore, the ability to quantify DNA damage and the rate of repair of the damage to the nuclear genome directly in human cells is critical in applications ranging from epidemiology to drug development. To address this technological need in the research community, to be better positioned to characterize the genotoxicity of newly developed pharmaceuticals, and to quantify DNA repair capacity without the need to identify specific DNA Repair gene defects, we propose the development of the next generationin DNA damage detection and quantification technology. This proposal, to develop the 'DNA Repair on a Chip' technology, combines the use of agarose-based Microwell arrays, spatially- encoded cellular recognition, human tumor cell lines with genetically-defined DNA repair status and extra-cellular matrix proteins to optimize, validate and commercialize a series of Spatially Encoded Microwell Arrays that will function as a tool to quantify DNA damage and measure cellular DNA Repair capacity at baseline and following genotoxin exposure on a single array or chip (DNA Repair on a Chip). The studies described in Aim 1 involve the development of a series of 24-well Spatially Encoded Microwell Arrays, with Microwells ranging from 10-50 5M in diameter and 20-50 5M indepth, suitable for gravity capture of a single cell of various sizes. Efficacy of the Microwell Arrays will be validated using radiation and small molecule inhibitors. Further, the sensitivity of the Microwell Arrays for analysis of cellular DNA Repair capacity will be evaluated using an isogenic panel of human tumor cell lines with defined defects in DNA Repair gene expression and following genotoxic stress. Iterative analysis and Microwell characterization will inform to finalize a set of 24-well Microwell Arrays for production and distribution. The studies described in Aim 2 involve additives to the Microwell Arrays that will enhance cell growth and attachment, providing optimal analysis of baseline DNA damage and most importantly, critical data on cellular capacity for in vivo repair post-damage. This technological advance opens the door to new strategies for drug discovery, genotoxicity testing, and environmental health research through objective, quantitative analyses. Phase II of the project will beexpanded to offer 96-well capability, end-user software for spatial recognition and quantitation plus micro-well additive options for specialized cell growth and attachment. PUBLIC HEALTH RELEVANCE: We describe a new methodology that provides for robust, high-throughput DNA damage and repair analysis by exploiting gravity capture of single cells into a Microwell array. DNA damage levels are revealed morphologically by single-cell gel electrophoresis. The Microwell array enables fully automated DNA damage and DNA repair measurement of multiple experimental conditions simultaneously. This technological advance opens the door to new strategies for drug discovery, genotoxicity testing, and environmental health research through objective, quantitative analyses.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 202.05K | Year: 2014

Project Summary / Abstract DNA repair pathways maintain the integrity of the genome and thereby help prevent the onset of cancer, disease and aging phenotypes. Further, many cancer treatments function by inducing genomic DNA damage. As such, the critical requirement for DNA repair proteins and pathways in response to radiation and genotoxic chemotherapeutics implicates DNA repair proteins as prime targets for improving response to currently available anti-cancer regimens. Essential to the development of specific DNA repair inhibitors is the availability of robust, highly sensitive assays to measure DNA repair capacity. In addition, defects in critical DNA repair pathways or proteins can predispose to cancer onset and may also provide an option for therapeutic selectivity. Many of these defects in the 150 or more DNA repair proteins can be detected using current omics technologies. However, there are many defects that can only be detected using functional assays such as those described herein. To effectivel

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