Methods in molecular biology
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Antisense-mediated exon skipping and exon inclusion have proven to be powerful tools for treating neuromuscular diseases. The approval of Exondys 51 (eteplirsen) and Spinraza (nusinersen) for the treatment of patients with Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) was the most noteworthy accomplishment in 2016. Exon skipping uses short DNA-like molecules called antisense oligonucleotides (AONs) to correct the disrupted reading frame, allowing the production of functional quasi-dystrophin proteins, and ameliorate the progression of the disease. ⋯ A major challenge in exon skipping and exon inclusion is the difficulty in designing effective AONs. The mechanism of mRNA splicing is highly complex, and the efficacy of AONs is often unpredictable. We will discuss the design of effective AONs for exon skipping and exon inclusion in this chapter.
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Aberrations of the DNA methylome contribute to onset and progression of diseases. Whole genome bisulfite sequencing (WGBS) is the only analytical method covering the complete methylome. ⋯ In tagmentation-based WGBS (TWGBS), several DNA and time-consuming steps of the conventional WGBS library preparation are circumvented by the use of a hyperactive transposase, which simultaneously fragments DNA and appends sequencing adapters. TWGBS requires only nanogram amounts of DNA and, thus, is well suited to study precious biological specimens such as sorted cells or micro-dissected tissue samples.
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Whole genome bisulfite sequencing (WGBS) enables the detection of DNA methylation at single base-pair resolution. The treatment of DNA with sodium bisulfite allows the discrimination of methylated and unmethylated cytosines, but the power of this technology can be limited by the input amounts of DNA and the length of DNA fragments due to DNA damage caused by the desulfonation process. ⋯ Briefly, genomic DNA is sheared, end-repaired, 3'-adenylated, and ligated to adaptors with fewer cleanup steps in between, minimizing DNA loss. The adapter-ligated DNA is then treated with sodium bisulfite and amplified with few PCR cycles to reach the yield needed for sequencing.
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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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Staging is one of the most important factors that determine the prognosis of the patients with esophageal adenocarcinoma. The current staging protocol includes parameters-extent of tumor (T), lymph node status (N), presence of distant metastasis (M), and grade of the adenocarcinoma (G). In addition, different criteria are used in patients with and without neoadjuvant therapy. The silent aspects of the use of new staging protocol are discussed.