Methods in molecular biology
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Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder due to the lack of dystrophin production. The disease is characterized by muscle wasting, with the most common causes of death being respiratory failure or heart failure. ⋯ Researchers have previously relied on high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS) methods for detecting PPMO uptake, but an enzyme-linked immunosorbent assay (ELISA) has been shown to have greater sensitivity. Here, we present methodologies to determine the uptake efficiency of a PPMO into the heart and efficacy of exon 51 skipping by a PPMO injected retro-orbitally into a humanized DMD mouse model via ELISA and RT-PCR, respectively.
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Exon-skipping therapy is an emerging approach that uses synthetic DNA-like molecules called antisense oligonucleotides (ASOs) to splice out frame-disrupting parts of mRNA, restore the reading frame, and produce truncated yet functional proteins. Phosphorodiamidate morpholino oligomer (PMO) is one of the safest among therapeutic ASOs for patients and has recently been approved under the accelerated approval program by the US Food and Drug Administration (FDA) as the first ASO-based drug for Duchenne muscular dystrophy (DMD). Multi-exon skipping utilizing ASOs can theoretically treat 80-90% of patients with DMD. Here, we describe the systemic delivery of a cocktail of ASOs to skip exon 51 and exons 45-55 in the mdx52 mouse, an exon 52 deletion model of DMD produced by gene targeting, and the evaluation of their efficacies in vivo.
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Liquid biopsy of cancers is an area of increasing interest in medical practice for the surveillance, management, and potential detection of malignant cells, using minimally invasive collection of body fluids. A liquid biopsy is particularly useful for metastatic cancers, which may be difficult to be sampled by core biopsy, due to difficulty of access or an occult location. Access to DNA shed from esophageal adenocarcinoma can enable the detection of mutations confirming the presence of malignant cells or the evolution of clonal lines with altered treatment response profiles. In this chapter, we detail a method for the isolation of cell-free DNA from blood plasma and DNA associated with exosomes in blood from patients with esophageal adenocarcinoma.
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Esophagogastrectomy for esophageal adenocarcinoma remains a procedure of significant morbidity and mortality rates. Management should be within the context of a multidisciplinary team. ⋯ Enhanced recovery after surgery (ERAS) protocols are evidence based and improve outcome. Standard protocol for open transthoracic esophagectomy is described.
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Quorum sensing (QS) systems play global regulatory roles in bacterial virulence. They synchronize the expression of multiple virulence factors and they control and modulate bacterial antibiotic tolerance systems and host defense mechanisms. ⋯ This chapter describes methods to study bacterial pathogenesis in murine acute and persistent/relapsing infection models, using the Gram-negative bacterial pathogen Pseudomonas aeruginosa as an example. These infection models can be used to probe bacterial virulence functions and in mechanistic studies, as well as for the assessment of the therapeutic potential of antibacterials, including anti-virulence agents.