Many Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications.

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Brain death, also commonly referred to as death by neurologic criteria, has been considered a legal definition of death for decades. Its determination involves many considerations and subtleties. In this review, we discuss the philosophy and history of brain death, its clinical determination, and special considerations. We discuss performance of the main clinical components of the brain death exam: assessment of coma, cranial nerves, motor testing, and apnea testing. We also discuss common ancillary tests, including advantages and pitfalls. Special discussion is given to extracorporeal membrane oxygenation, target temperature management, and determination of brain death in pediatric populations. Lastly, we discuss existing controversies and future directions in the field.

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