Methods in molecular biology
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Spinal muscular atrophy (SMA), the most common gentic cause of infantile death caused by mutations in the SMN1 gene, presents a unique case in the field of splice modulation therapy, where a gene (or lack of) is responsible for causing the disease phenotype but treatment is not focused around it. Antisense therapy targeting SMN2 which leads to SMN protein expression has been at the forefront of research when it comes to developing a feasible therapy for treating SMA. ⋯ This propelled the research community to investigate new chemistries of antisense oligonucleotides (ASOs) that may be better in both treatment and cost efficiency. Here we describe two types of ASOs, phosphorodiamidate morpholino oligomers (PMOs) and locked nucleic acids (LNA)-DNA mixmers, being investigated as potential treatments for SMA, and methods used to test their efficacy, including quantitative RT-PCR, Western blotting, and immunofluorescence staining to detect SMN in nuclear gems/Cajal bodies, in type I SMA patient fibroblast cell lines.
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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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Pathological assessment of tissue is the gold standard for diagnosis and staging of neoplasia and provides key prognostic information for clinical management. Proper macroscopic assessment and cut-up technique is essential to ensure that the overall assessment is correct and reproducible. Endoscopic mucosal resection is a technique used for removing early neoplastic glandular lesions of the esophagus at the level of submucosa. Here, we describe the macroscopic assessment and dissection techniques used for the routine handling of endoscopic mucosal resection specimens in the clinical laboratory.
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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.
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Tissue microarray technology could allow immunohistochemical staining or in situ hybridization on hundreds of different tissue samples simultaneously. It allows faster analysis and considerably reducing costs incurred in staining. ⋯ In the literature, many researches of esophageal adenocarcinoma use tissue microarray to enhance the output. In this chapter, we have a brief overview of tissue microarray technologies, the advantages and disadvantages of tissue microarray, and related troubleshootings.