Methods in molecular biology
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Irreversible electroporation has recently been applied to tissue and tumor ablation. This animal model was developed to evaluate optimal parameters for subcutaneous tumor ablation. ⋯ Under general anesthesia with complete muscle relaxation, electroporation was performed with the NanoKnife device. Post-procedure tumors were recovered and studied at specified time intervals to assess the efficacy.
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Genetically encoded indicators are valuable tools to study intracellular signaling cascades in real time using fluorescent or bioluminescent imaging techniques. Imaging of Ca(2+) indicators is widely used to record transient intracellular Ca(2+) increases associated with bioelectrical activity. The natural bioluminescent Ca(2+) sensor aequorin has been historically the first Ca(2+) indicator used to address biological questions. ⋯ Genetically encoded sensors such as aequorin are commonly used in dissociated cultured cells; however it becomes more challenging to express them in differentiated intact specimen such as brain tissue. Here we describe a method to express a GFP-aequorin (GA) fusion protein in pyramidal cells of neocortical acute slices using recombinant Sindbis virus. This technique allows expressing GA in several hundreds of neurons on the same slice and to perform the bioluminescence recording of Ca(2+) transients in single neurons or multiple neurons simultaneously.
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Composite tissue transplantation is an emerging new era in transplant medicine and has become a viable reconstructive option for patients with large and devastating tissue defects. Advances in microsurgical techniques, transplant immunology and the development of potent immunosuppressive agents have enabled the realization of such types of transplants. ⋯ However, despite the fact that surgical, immunological and functional results are highly encouraging, the need for long-term and high-dose immunosuppression to enable graft survival and to treat/reverse acute skin rejection episodes remains a pace-limiting obstacle towards wide spread application. In this chapter we review the history and development of this novel field, the functional and immunological outcomes based on the world experience, unique biological features of such transplants, mechanisms and treatment protocols for acute skin rejection, as well as novel concepts for immune modulation and tolerance induction.
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The isolation of embryonic stem cells (ESCs) has furthered our understanding of normal embryonic development and fueled the progression of stem cell derived therapies. However, the generation of ESCs requires the destruction of an embryo, making the use of these cells ethically controversial. In 2006 the Yamanaka group overcame this ethical controversy when they described a protocol whereby somatic cells could be dedifferentiated into a pluripotent state following the transduction of a four transcription factor cocktail. ⋯ The fast paced field of cellular reprogramming has recently produced protocols to generate iPSCs using non integrative techniques with an ever improving efficiency. These recent developments have brought us one step closer to developing a safe and efficient method to reprogram cells for clinical use. However, a lot of work is still needed before iPSCs can be implemented in a clinical setting.
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This chapter will outline strategies and ideas for the commercialization a promising wound healing technology discovered in an academic setting. This would include, but not limited to addressing topics such as intellectual property protection, funding, technology development, and regulatory aspects (i.e., navigating through the FDA).