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The Precision Medicine Approach in Oncology
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
The DNA sequencing methods used in the 1970s and 1980s such as Maxam-Gilbert and Sanger sequencing were manual techniques requiring a very large amount of manpower, time, and effort. The shift to more rapid automated sequencing methods in the 1990s finally allowed the sequence of whole genomes to be carried out within a reasonable time period. The first bacterial genomes, including that of Haemophilus influenzae, were sequenced by “Shotgun Sequencing” of shorter DNA fragments. The first eukaryote genomes were also sequenced by this method, but involved larger DNA clones from DNA libraries such as Bacterial Artificial Chromosomes (BACs) and Yeast Artificial Chromosomes (YACs). One of the first automated DNA sequencers was the capillary-based ABI PRISM 3100 Genetic Analyzer which allowed a much more rapid and extensive output of data.
Preimplantation Genetic Testing for Aneuploidies: Where We Are and Where We're Going
Published in Darren K. Griffin, Gary L. Harton, Preimplantation Genetic Testing, 2020
Andrea Victor, Cagri Ogur, Alan Thornhill, Darren K. Griffin
Array CGH (aCGH) is similar to CGH in that it involves the fluorescent labeling of a sample to a known reference and assessment of copy number by comparative analysis [68]. Instead of being hybridized to metaphase chromosomes, however, the two-colored DNA sample/reference cocktail is hybridized to bacterial artificial chromosomes (BAC) or synthetic oligonucleotides, bound to glass slides in microarray format. Fluorescence ratios at each arrayed DNA element are analyzed and can provide a locus-by-locus measure of DNA copy number variation at an increased mapping resolution to standard CGH.
Comparative Genomic Hybridization and Copy Number Abnormalities in Breast Cancer
Published in Brian Leyland-Jones, Pharmacogenetics of Breast Cancer, 2020
One of the most important factors when considering a clinical CGH tool is the quality and amount of DNA available for analysis. Depending on which array platform is chosen, the amounts needed for successful hybridization can range from 50 ng to 3 μg. In addition, some protocols have been developed that allow analysis of material from formalin-fixed, paraffin-embedded (FFPE) samples (39,49). The largest amounts of starting material generally are used in protocols in which entire genomes are labeled and hybridized to the array, as is usual for array CGH using BAC or Agilent oligonucleotide arrays. In these experiments, the starting DNA is typically labeled with fluorescent nucleotides using random priming. For BAC arrays, unlabeled repeat-rich DNA is included to suppress hybridization of repeated sequences to repetitive sequences that are present in the BAC probes. Larger amounts of DNA are needed to “drive” these whole-genome hybridization reactions. The amount of starting material needed for analysis is reduced if the DNA is amplified or reduced in complexity—for example, through a PCR amplification step preceded by restriction digestion that reduces the complexity of the hybridized DNA (50). This complexity reduction results in an increase of template sequences, which can utilized in downstream hybridization steps. Both academically and commercially developed platforms result in data sets that are able to detect gains and losses with the caveat that variations and noise may differ between platforms (Fig.4).
From leptin to lasers: the past and present of mouse models of obesity
Published in Expert Opinion on Drug Discovery, 2021
Joshua R. Barton, Adam E. Snook, Scott A. Waldman
Monogenic mouse models have been invaluable for elucidating the molecular mechanisms underlying obesity and metabolic disease. By 2015, mouse models of 221 distinct genes had been studied for obesity-related phenotypes [144]. This list is continually expanding, as are the tools that allow researchers to interrogate the mechanisms behind obesity. Large-scale projects like The GENSAT Project at Rockefeller University have developed hundreds of Bacterial Artificial Chromosome (BAC)-driven GFP and Cre mouse lines that have been used in over 1000 publications [145]. Multi-institutional, international projects have developed suites of neuron specific Cre-drivers and toolboxes of transgenic mice with Cre-dependent optogenetic channels for dissecting the neural circuits that underlie mammalian behavior [146,147]. These herculean efforts provide obesity researchers with thousands of combinations of transgenic mice that can study the interactions between appetitive pathways. While individual circuits are being rigorously mapped and stimulated, discovery of how these pathways work in concert to evoke feeding responses is a crucial next step to precisely modulating satiety in human obesity.
Corneal damage and its recovery after instillation of preservative-free versus preserved latanoprost eye drops
Published in Cutaneous and Ocular Toxicology, 2020
Daisuke Inoue, Yasser Helmy Mohamed, Masafumi Uematsu, Takashi Kitaoka
Among the current ocular hypotensive medications employed in the treatment of open-angle glaucoma and ocular hypertension, the first-line choice is topical application of PG analogues. This choice is based upon previous studies that have shown their efficacy in lowering IOP with few systemic side effects and the ease of the once daily dosing14,15. However, other clinical studies have shown that the topical application of PG analogues can cause ocular surface toxicity16,17. Furthermore, the toxicity of commercial PG analogues has often been associated with the use of BAC. BAC is one of the most commonly used preservatives because of its higher antimicrobial efficiency, stability, and low cost2,18. A huge number of animal and in vitro studies have been conducted for more than two decades using a considerable variety of models, cells, and tissues. These studies have demonstrated that BAC may cause or enhance harmful consequences on the eye structures of the anterior segment, including the tear film, cornea, conjunctiva, and even the trabecular meshwork19. Despite the consistent and solid data, along with the warnings from the results of observational surveys and individual case series, BAC is still used as the main preservative in eye drops, with very few alternatives having been developed19.
Topical hyaluronan alone promotes corneal epithelial cell migration whereas combination with benzalkonium chloride impairs epithelial wound healing
Published in Cutaneous and Ocular Toxicology, 2020
Satoshi Seino, Ryosuke Matsuoka, Yasunobu Masuda, Masaaki Kunou, Yuka Okada, Shizuya Saika
Wounding of the corneal epithelium has several causes, including drug-induced corneal disorder. The impairment is reportedly caused by benzalkonium chloride (BAC). BAC is an antibacterial compound with a quaternary ammonium cation structure and is widely used as a preservative in eye drops owing to its superior antibacterial and physicochemical characteristics. However, it can also impair the cornea depending on the concentration used24,25. HA eye drops were reported to ameliorate the corneal epithelial disorder induced by BAC-preserved eye drops26,27. On the other hand, one study reported that a thickening agent combined with BAC delays wound healing more than BAC alone28. Since HA has aspects of thickening agents, the combined effect of HA and BAC in an ophthalmic solution on corneal epithelial wound healing needs to be examined in more detail. This study investigated the effects of HA on corneal epithelial wound healing in vitro, in situ, and in vivo. Then, based on the same techniques, we examined the effects of HA pre-treatment on the impairment of corneal epithelial healing caused by topical BAC.