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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Introduction: Time-limited dual antiplatelet therapy (DAPT) with aspirin and clopidogrel has emerged as an important treatment modality for secondary stroke prevention in patients with symptomatic intracranial atherosclerotic disease, minor stroke, or high-risk transient ischemic attack. However, continuation beyond the indicated treatment period may expose patients to bleeding risk without additional benefit. Methods: We conducted a retrospective study of a prospectively maintained registry (the Get-With-The-Guidelines database at our hospital) evaluating patients discharged on time-limited DAPT for secondary stroke prevention between January 1st, 2019 and July 31st, 2020. Intended and actual duration of therapy were extracted from clinical notes and prescription records. We assessed the frequency of inappropriate continuation within the entire cohort and performed univariate analyses to assess for differences in outcome with respect to clinical and demographic characteristics, including social determinants of health (such as race and primary spoken language). Results: Of the 127 patients included, 17 (13%) had inappropriately prolonged treatment with DAPT. Black patients had a significantly higher incidence of inappropriate continuation than non-Black patients and individuals speaking languages other than English or Spanish exhibited a trend toward more frequent occurrence. Patients intended to transition to monotherapy with clopidogrel and those initially presenting with stroke (rather than transient ischemic attack) had similar trends toward more frequent occurrence. Conclusions: Our study represents the first attempt to approximate the incidence of inappropriate prolongation of time-limited DAPT for secondary stroke prevention at a hospital caring for a large underserved population. Our results suggest that social determinants of health may contribute to this phenomenon, but further studies are required to replicate our observations in larger multicenter cohorts. Systems-based interventions should be aimed at ensuring proper duration of therapy to minimize bleeding risk and other side effects.

Aneurysmal subarachnoid hemorrhage can be a devastating disease, with an in-hospital mortality rate of up to 20%. The American Heart Association/American Stroke Association 2023 Aneurysmal Subarachnoid Hemorrhage Guidelines provide a comprehensive update to the 2012 Guidelines based on a systematic review of the intervening evidence. The guidelines are broad in scope, covering prehospital care, aneurysm treatment modality, medical complications, detection and treatment of delayed cerebral ischemia, and recovery. Here, we comment on salient aspects of aneurysmal subarachnoid hemorrhage care, compare these guidelines with the 2023 Neurocritical Care aneurysmal subarachnoid hemorrhage guidelines, and review relevant updates.

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Introduction: The FOUR (Full Outline of UnResponsiveness) score was developed as a more effective alternative to the Glasgow Coma Scale for prognostication of critically-ill neurology patients. Current research on the FOUR score in cardiac arrest is limited, but suggests that it may be useful. Given that whether or not a patient over-breathes the ventilator can often be confounded by the set respiratory rate, hypocarbia, and other factors, we evaluated the FOUR score with and without the respiratory component (FOUR-). Methods: We retrospectively studied 83 cardiac arrest patients treated at an urban hospital from 2011-2018 from the Multimodal outcome characterization in comatose cardiac arrest (MOCHA) registry. FOUR and FOUR- score within first 24 hours (day 1) and 72-96 hours (day 4) after arrest were evaluated for ability to predict in-hospital mortality and survival to discharge. Results: Day 1 FOUR score < 4 had 78% (67%-87%) sensitivity and 57% (29%-82%) specificity, while FOUR- score < 4 had 84% (73%-92%) sensitivity and 50% (23%-77%) specificity for predicting in-hospital mortality. Day 4 FOUR and FOUR- scores < 4 had higher specificities (both 94% [71%-100%]) but lower sensitivities (63% [45%-79%] and 69% [51%-83%], respectively) for mortality than day 1. With outcome changed to survival to discharge, day 1 FOUR score > 8 had 29% (8%-58%) sensitivity and 97% (90%-100%) specificity, while FOUR- > 8 had 21% (5%-51%) sensitivity and 100% (95%-100%) specificity. Day 4 FOUR and FOUR- scores > 8 had lower specificities (89% [73%-97%] and 91% [77%-98%], respectively), but higher sensitivities (53% [28%-77%]) and 47% [23%-72%], respectively) than day 1. There were no differences in mortality between FOUR and FOUR- < 4 on day 1 (p=0.89) or 4 (p=0.95), and no differences in survival between FOUR and FOUR- > 8 on day 1 (p=0.26) or 4 (p=0.85). Conclusions: Both the FOUR and FOUR- scores had high specificity for mortality and survival, which is important given that incorrectly predicting a bad outcome could lead to premature withdrawal of life support. The absence of a significant difference between the FOUR and FOUR- and the stronger prognostic ability of the FOUR- suggest that the respiratory component may not provide additional prognostic utility in cardiac arrest.