Artificial Intelligence-Generated Draft Replies to Patient Inbox Messages.

Importance: The emergence and promise of generative artificial intelligence (AI) represent a turning point for health care. Rigorous evaluation of generative AI deployment in clinical practice is needed to inform strategic decision-making. Objective: To evaluate the implementation of a large language model used to draft responses to patient messages in the electronic inbox. Design, Setting, and Participants: A 5-week, prospective, single-group quality improvement study was conducted from July 10 through August 13, 2023, at a single academic medical center (Stanford Health Care). All attending physicians, advanced practice practitioners, clinic nurses, and clinical pharmacists from the Divisions of Primary Care and Gastroenterology and Hepatology were enrolled in the pilot. Intervention: Draft replies to patient portal messages generated by a Health Insurance Portability and Accountability Act-compliant electronic health record-integrated large language model. Main Outcomes and Measures: The primary outcome was AI-generated draft reply utilization as a percentage of total patient message replies. Secondary outcomes included changes in time measures and clinician experience as assessed by survey. Results: A total of 197 clinicians were enrolled in the pilot; 35 clinicians who were prepilot beta users, out of office, or not tied to a specific ambulatory clinic were excluded, leaving 162 clinicians included in the analysis. The survey analysis cohort consisted of 73 participants (45.1%) who completed both the presurvey and postsurvey. In gastroenterology and hepatology, there were 58 physicians and APPs and 10 nurses. In primary care, there were 83 physicians and APPs, 4 nurses, and 8 clinical pharmacists. The mean AI-generated draft response utilization rate across clinicians was 20%. There was no change in reply action time, write time, or read time between the prepilot and pilot periods. There were statistically significant reductions in the 4-item physician task load score derivative (mean [SD], 61.31 [17.23] presurvey vs 47.26 [17.11] postsurvey; paired difference, -13.87; 95% CI, -17.38 to -9.50; P < .001) and work exhaustion scores (mean [SD], 1.95 [0.79] presurvey vs 1.62 [0.68] postsurvey; paired difference, -0.33; 95% CI, -0.50 to -0.17; P < .001). Conclusions and Relevance: In this quality improvement study of an early implementation of generative AI, there was notable adoption, usability, and improvement in assessments of burden and burnout. There was no improvement in time. Further code-to-bedside testing is needed to guide future development and organizational strategy.

Data on location and retail price of a standard food basket in supermarkets across New York City.

Previous work has suggested that the price of food sold at supermarkets may vary according to the socioeconomic characteristics of a neighborhood. Given the importance of food prices in securing access to food, understanding how food prices vary across neighborhoods is crucial to assessing affordability. To study food pricing in New York City (NYC) a defined standard food basket (SFB) was collected in supermarkets across NYC neighborhoods. A dataset was created that includes pricing data collected in-person for ten pre-determined food items from 163 supermarkets across 71 of the 181 NYC neighborhoods during March through August of 2019. Included in these data are raw and processed pricing data files that illustrate the complexity of standardizing pricing across items. An additional dataset includes neighborhood-level variables of selected socioeconomic and demographic characteristics from the 2014-2018 American Community Survey that is publicly available via the Census API. The pricing data and the data on neighborhood-level characteristics were merged. Basic statistical measures suggest some distributional differences in the price of a SFB by socioeconomic differences between neighborhoods. This database can be used to describe spatial patterns in food pricing in a dense urban setting, while exploring pricing inequities across neighborhoods. In addition, by working with these data, researchers, policy analysts and educators will gain an understanding of the methodologies used to generate pricing data for an SFB.

Targeting repetitive laboratory testing with electronic health records-embedded predictive decision support: A pre-implementation study.

INTRODUCTION: Unnecessary laboratory testing contributes to patient morbidity and healthcare waste. Despite prior attempts at curbing such overutilization, there remains opportunity for improvement using novel data-driven approaches. This study presents the development and early evaluation of a clinical decision support tool that uses a predictive model to help providers reduce low-yield, repetitive laboratory testing in hospitalized patients. METHODS: We developed an EHR-embedded SMART on FHIR application that utilizes a laboratory test result prediction model based on historical laboratory data. A combination of semi-structured physician interviews, usability testing, and quantitative analysis on retrospective laboratory data were used to inform the tool's development and evaluate its acceptability and potential clinical impact. KEY RESULTS: Physicians identified culture and lack of awareness of repeat orders as key drivers for overuse of inpatient blood testing. Users expressed an openness to a lab prediction model and 13/15 physicians believed the tool would alter their ordering practices. The application received a median System Usability Scale score of 75, corresponding to the 75th percentile of software tools. On average, physicians desired a prediction certainty of 85% before discontinuing a routine recurring laboratory order and a higher certainty of 90% before being alerted. Simulation on historical lab data indicates that filtering based on accepted thresholds could have reduced âˆ¼22% of repeat chemistry panels. CONCLUSIONS: The use of a predictive algorithm as a means to calculate the utility of a diagnostic test is a promising paradigm for curbing laboratory test overutilization. An EHR-embedded clinical decision support tool employing such a model is a novel and acceptable intervention with the potential to reduce low-yield, repetitive laboratory testing.

Using Case Mix Index within Diagnosis-Related Groups to Evaluate Variation in Hospitalization Costs at a Large Academic Medical Center.

In analyzing direct hospitalization cost and clinical data from an academic medical center, commonly used metrics such as diagnosis-related group (DRG) weight explain approximately 37% of cost variability, but a substantial amount of variation remains unaccounted for by case mix index (CMI) alone. Using CMI as a benchmark, we isolate and target individual DRGs with higher than expected average costs for specific quality improvement efforts. While DRGs summarize hospitalization care after discharge, a predictive model using only information known before admission explained up to 60% of cost variability for two DRGs with a high excess cost burden. This level of variability likely reflects underlying patient factors that are not modifiable (e.g., age and prior comorbidities) and therefore less useful for health systems to target for intervention. However, the remaining unexplained variation can be inspected in further studies to discover operational factors that health systems can target to improve quality and value for their patients. Since DRG weights represent the expected resource consumption for a specific hospitalization type relative to the average hospitalization, the data-driven approach we demonstrate can be utilized by any health institution to quantify excess costs and potential savings among DRGs.

Bioengineered optogenetic model of human neuromuscular junction.

Functional human tissues engineered from patient-specific induced pluripotent stem cells (hiPSCs) hold great promise for investigating the progression, mechanisms, and treatment of musculoskeletal diseases in a controlled and systematic manner. For example, bioengineered models of innervated human skeletal muscle could be used to identify novel therapeutic targets and treatments for patients with complex central and peripheral nervous system disorders. There is a need to develop standardized and objective quantitative methods for engineering and using these complex tissues, in order increase their robustness, reproducibility, and predictiveness across users. Here we describe a standardized method for engineering an isogenic, patient specific human neuromuscular junction (NMJ) that allows for automated quantification of NMJ function to diagnose disease using a small sample of blood serum and evaluate new therapeutic modalities. By combining tissue engineering, optogenetics, microfabrication, optoelectronics and video processing, we created a novel platform for the precise investigation of the development and degeneration of human NMJ. We demonstrate the utility of this platform for the detection and diagnosis of myasthenia gravis, an antibody-mediated autoimmune disease that disrupts the NMJ function.

Antenatal course of referred monochorionic diamniotic twins complicated by selective intrauterine growth restriction (SIUGR) type III.

OBJECTIVE: To describe the antenatal course of selective intrauterine growth restriction (SIUGR) type III patients. STUDY DESIGN: Retrospective study of monochorionic diamniotic twins with SIUGR type III. Patients were divided into those who did and did not progress to SIUGR type II or twin-twin transfusion syndrome (TTTS) (Groups A and B, respectively). Patient characteristics and perinatal survival were compared by Group, and continuous data are reported as median (range). RESULTS: Forty-eight patients were studied; Group A [26 (54.2%)] and Group B [22 (45.8%)]. The difference in 30-day survivorship for the appropriate for gestational age twin (88.5 vs. 100%, p = .2394) and for the SIUGR twin (73.1 vs. 95.5%, p = .0551) was not statistically significant. However, dual survivorship was lower in Group A compared to Group B (69.2 vs. 95.4%, p = .0276). CONCLUSIONS: Approximately half of the SIUGR type III patients had antenatal progression. Lack of antenatal progression was associated with 95% dual survivorship. RATIONALE: The antenatal course of monochorionic diamniotic twins complicated by SIUGR type III is not well-understood and antenatal management remains a clinical dilemma. We provide pregnancy outcomes in a referred group of SIUGR type III patients, including the rate of progression to SIUGR type II and TTTS.

Evaluation of cardiac function in the recipient twin in successfully treated twin-to-twin transfusion syndrome using a novel fetal speckle-tracking analysis.

INTRODUCTION: This study was designed to evaluate ventricular size, shape, and function in recipient twins following laser therapy for twin-twin transfusion syndrome (TTTS), using novel speckle-tracking techniques. METHODS: This retrospective study enrolled patients that underwent fetal laser surgery for TTTS and had fetal echocardiograms (FE) performed pre- and post-operatively (op), with adequate resolution in the 4-chamber view for analysis, using a speckle-tracking software, to compute the size, shape, and function of both the right (RV) and left (LV) ventricles. Values were indexed to published normal values. Pre- and post-laser Z-score values for each of the measurements were compared using the Student's t-test, with significance defined as P < 0.05. RESULTS: Fifteen TTTS candidate pregnancies that underwent laser therapy between 2010 and 2017, with adequate pre- and post-op FE, were selected for the analysis. Post-op FE at 28.5 ± 8.3 days showed a significant decrease in RV base dimension, increased LV base dimension, and improvements in many functional measurements: LV global and free wall strain, LV fractional area change, LV basal-apical fractional change, and LV and RV 24-segment fractional shortening (FS) of the basal segments. CONCLUSIONS: Cardiac remodeling, following laser surgery in TTTS recipient twins, was demonstrated in the basal portion of both the RV and LV with improved biventricular function.

Surface Chemical Functionalization of Wrinkled Thiol-ene Elastomers for Promoting Cellular Alignment.

Wrinkled polymer surfaces find broad applicability; however, the polymer substrates are often limited to poly(dimethylsiloxane) (PDMS), which limits spatial control over wrinkle features and surface chemistry. An approach to surface functionalization of wrinkled elastomer substrates is demonstrated through versatile, multistep thiol-ene click chemistry. The elastomer is formed using a thiol-Michael reaction of tetrathiol with excess diacrylates while wrinkle formation is induced through a second free radical UV polymerization of the acrylates on the surface of the elastomer. Due to oxygen inhibition of the free radical polymerization, pendant acrylates at the surface remain unreacted and are subsequently functionalized with a multi-functional thiol, which can be further reacted through a number of thiol-X 'click' reactions. As a demonstration, these thiol surfaces are further modified to either promote cell adhesion of human mesenchymal stem cells (hMSCs) through coupling with RGDS-containing peptides or surface passivation through reaction with hydrophilic hydroxyl ethyl acrylate moieties. Through engineering a combination of surface chemistry and surface topography, hMSCs exhibited increased spreading and cell density on RGDS-functionalized surfaces and a two-fold increase in cell alignment when cultured on wrinkled substrates. Gradient functionalized surfaces created by tuning the wrinkle wavelength with UV irradiation enabled rapid screening of the effect of topography on the hMSCs. Further, this novel application of click chemistry enables simultaneous tuning of wrinkle topology and surface chemistry towards targeted material applications.

Engineering of human cardiac muscle electromechanically matured to an adult-like phenotype.

The application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues' functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force-frequency and force-length relationships, calcium handling, and responses to ß-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs.

Author Correction: Advanced maturation of human cardiac tissue grown from pluripotent stem cells.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Quantification of human neuromuscular function through optogenetics.

The study of human neuromuscular diseases has traditionally been performed in animal models, due to the difficulty of performing studies in human subjects. Despite the unquestioned value of animal models, inter-species differences hamper the translation of these findings to clinical trials. Tissue-engineered models of the neuromuscular junction (NMJ) allow for the recapitulation of the human physiology in tightly controlled in vitro settings. Methods: Here we report the first human patient-specific tissue-engineered model of the neuromuscular junction (NMJ) that combines stem cell technology with tissue engineering, optogenetics, microfabrication and image processing. The combination of custom-made hardware and software allows for repeated, quantitative measurements of NMJ function in a user-independent manner. Results: We demonstrate the utility of this model for basic and translational research by characterizing in real time the functional changes during physiological and pathological processes. Principal Conclusions: This system holds great potential for the study of neuromuscular diseases and drug screening, allowing for the extraction of quantitative functional data from a human, patient-specific system.

The potential role of circulating tumor DNA (ctDNA) in the further investigation of colorectal cancer patients with nonspecific findings on standard investigations.

Early detection of metastatic colorectal cancer, at initial diagnosis or during routine surveillance, can improve survival outcomes. Current routine investigations, including CEA and CT, have limited sensitivity and specificity. Recent studies of colorectal cancer cohorts under post surgery surveillance indicate circulating tumor DNA (ctDNA) evidence of recurrence can occur many months before clinical detection. Another possible role for ctDNA is in the further assessment of indeterminate findings on standard CEA or CT investigations. To further explore this potential, we undertook a prospective study. Further investigation, including FDG-PET imaging, was at clinician discretion, blinded to ctDNA analysis. Forty-nine patients were enrolled. Analyzed here are the 45 patients with an evaluable blood sample of whom 6 had an isolated elevated CEA, 30 had indeterminate CT findings, and 9 had both. FDG-PET scans were performed in 30 patients. Fourteen of 45 patients (31%) had detectable ctDNA. At completion of the planned 2 year follow-up, recurrence has occurred in 21 (47%) patients. Detectable ctDNA at study entry was associated with inferior relapse free survival (HR 4.85, p < 0.0001). Where FDG-PET scan was normal/equivocal (n = 15, 50%) 1 of 1 with detectable ctDNA versus 3 of 14 with undetectable ctDNA ultimately had recurrence confirmed. In summary, for colorectal cancer patients with indeterminate findings on routine investigations, ctDNA detection increases the probability that the findings indicate metastatic disease, including in a nonpredefined subset that also underwent FDG-PET imaging. Further studies of the value of ctDNA analysis during patient surveillance are warranted.

Dual IFN-γ/hypoxia priming enhances immunosuppression of mesenchymal stromal cells through regulatory proteins and metabolic mechanisms.

The immunosuppressive capacity of human mesenchymal stromal cells (MSCs) renders them promising candidates for treating diverse immune disorders. However, after hundreds of clinical trials, there are still no MSC therapies approved in the United States. MSCs require specific cues to adopt their immunosuppressive phenotype, and yet most clinical trials use cells expanded in basic culture medium and growth conditions. We propose that priming MSCs prior to administration will improve their therapeutic efficacy. Interferon-gamma (IFN-γ) priming are cues common to situations of immune escape that have individually shown promise as MSC priming cues but have not been systematically compared. Using mixed lymphocyte reactions, we show that priming MSCs with either cue alone improves T-cell inhibition. However, combining the two cues results in additive effects and markedly enhances the immunosuppressive phenotype of MSCs. We demonstrate that IFN-γ induces expression of numerous immunosuppressive proteins (IDO, PD-L1, HLA-E, HLA-G), whereas hypoxia switches MSCs to glycolysis, causing rapid glucose consumption and production of T-cell inhibitory lactate levels. Dual IFN-γ/hypoxia primed MSCs display both attributes and have even higher induction of immunosuppressive proteins over IFN-γ priming alone (IDO and HLA-G), which may reflect another benefit of metabolic reconfiguration.

Advanced maturation of human cardiac tissue grown from pluripotent stem cells.

Cardiac tissues generated from human induced pluripotent stem cells (iPSCs) can serve as platforms for patient-specific studies of physiology and disease1-6. However, the predictive power of these models is presently limited by the immature state of the cells1, 2, 5, 6. Here we show that this fundamental limitation can be overcome if cardiac tissues are formed from early-stage iPSC-derived cardiomyocytes soon after the initiation of spontaneous contractions and are subjected to physical conditioning with increasing intensity over time. After only four weeks of culture, for all iPSC lines studied, such tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiological sarcomere length (2.2 µm) and density of mitochondria (30%), the presence of transverse tubules, oxidative metabolism, a positive force-frequency relationship and functional calcium handling. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiological responses to isoproterenol and recapitulating pathological hypertrophy, supporting the utility of this tissue model for studies of cardiac development and disease.

BAK α6 permits activation by BH3-only proteins and homooligomerization via the canonical hydrophobic groove.

BAK and BAX are the essential effectors of apoptosis because without them a cell is resistant to most apoptotic stimuli. BAK and BAX undergo conformation changes to homooligomerize then permeabilize the mitochondrial outer membrane during apoptosis. How BCL-2 homology 3 (BH3)-only proteins bind to activate BAK and BAX is unclear. We report that BH3-only proteins bind inactive full-length BAK at mitochondria and then dissociate following exposure of the BAK BH3 and BH4 domains before BAK homodimerization. Using a functional obstructive labeling approach, we show that activation of BAK involves important interactions of BH3-only proteins with both the canonical hydrophobic binding groove (α2-5) and α6 at the rear of BAK, with interaction at α6 promoting an open groove to receive a BH3-only protein. Once activated, how BAK homodimers multimerize to form the putative apoptotic pore is unknown. Obstructive labeling of BAK beyond the BH3 domain and hydrophobic groove did not inhibit multimerization and mitochondrial damage, indicating that critical protein-protein interfaces in BAK self-association are limited to the α2-5 homodimerization domain.

Cisplatin Increases Sensitivity to FGFR Inhibition in Patient-Derived Xenograft Models of Lung Squamous Cell Carcinoma.

Lung squamous cell carcinoma (SqCC) is a molecularly complex and genomically unstable disease. No targeted therapy is currently approved for lung SqCC, although potential oncogenic drivers of SqCC have been identified, including amplification of the fibroblast growth factor receptor 1 (FGFR1). Reports from a recently completed clinical trial indicate low response rates in patients treated with FGFR tyrosine kinase inhibitors, suggesting inadequacy of FGFR1 amplification as a biomarker of response, or the need for combination treatment. We aimed to develop accurate models of lung SqCC and determine improved targeted therapies for these tumors. We show that detection of FGFR1 mRNA by RNA in situ hybridization is a better predictor of response to FGFR inhibition than FGFR1 gene amplification using clinically relevant patient-derived xenograft (PDX) models of lung SqCC. FGFR1-overexpressing tumors were observed in all histologic subtypes of non-small cell lung cancers (NSCLC) as assessed on a tissue microarray, indicating a broader range of tumors that may respond to FGFR inhibitors. In FGFR1-overexpressing PDX tumors, we observed increased differentiation and reduced proliferation following FGFR inhibition. Combination therapy with cisplatin was able to increase tumor cell death, and dramatically prolonged animal survival compared to single-agent treatment. Our data suggest that FGFR tyrosine kinase inhibitors can benefit NSCLC patients with FGFR1-overexpressing tumors and provides a rationale for clinical trials combining cisplatin with FGFR inhibitors. Mol Cancer Ther; 16(8); 1610-22. ©2017 AACR.

Lung Basal Stem Cells Rapidly Repair DNA Damage Using the Error-Prone Nonhomologous End-Joining Pathway.

Lung squamous cell carcinoma (SqCC), the second most common subtype of lung cancer, is strongly associated with tobacco smoking and exhibits genomic instability. The cellular origins and molecular processes that contribute to SqCC formation are largely unexplored. Here we show that human basal stem cells (BSCs) isolated from heavy smokers proliferate extensively, whereas their alveolar progenitor cell counterparts have limited colony-forming capacity. We demonstrate that this difference arises in part because of the ability of BSCs to repair their DNA more efficiently than alveolar cells following ionizing radiation or chemical-induced DNA damage. Analysis of mice harbouring a mutation in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key enzyme in DNA damage repair by nonhomologous end joining (NHEJ), indicated that BSCs preferentially repair their DNA by this error-prone process. Interestingly, polyploidy, a phenomenon associated with genetically unstable cells, was only observed in the human BSC subset. Expression signature analysis indicated that BSCs are the likely cells of origin of human SqCC and that high levels of NHEJ genes in SqCC are correlated with increasing genomic instability. Hence, our results favour a model in which heavy smoking promotes proliferation of BSCs, and their predilection for error-prone NHEJ could lead to the high mutagenic burden that culminates in SqCC. Targeting DNA repair processes may therefore have a role in the prevention and therapy of SqCC.

Real-Time Bioluminescence Imaging of Cell Distribution, Growth, and Differentiation in a Three-Dimensional Scaffold Under Interstitial Perfusion for Tissue Engineering.

Bioreactor systems allow safe and reproducible production of tissue constructs and functional analysis of cell behavior in biomaterials. However, current procedures for the analysis of tissue generated in biomaterials are destructive. We describe a transparent perfusion system that allows real-time bioluminescence imaging of luciferase expressing cells seeded in scaffolds for the study of cell-biomaterial interactions and bioreactor performance. A prototype provided with a poly(lactic) acid scaffold was used for "proof of principle" studies to monitor cell survival in the scaffold (up to 22 days). Moreover, using cells expressing a luciferase reporter under the control of inducible tissue-specific promoters, it was possible to monitor changes in gene expression resulting from hypoxic state and endothelial cell differentiation. This system should be useful in numerous tissue engineering applications, the optimization of bioreactor operation conditions, and the analysis of cell behavior in three-dimensional scaffolds.

Tissue-Engineering for the Study of Cardiac Biomechanics.

The notion that both adaptive and maladaptive cardiac remodeling occurs in response to mechanical loading has informed recent progress in cardiac tissue engineering. Today, human cardiac tissues engineered in vitro offer complementary knowledge to that currently provided by animal models, with profound implications to personalized medicine. We review here recent advances in the understanding of the roles of mechanical signals in normal and pathological cardiac function, and their application in clinical translation of tissue engineering strategies to regenerative medicine and in vitro study of disease.

Assembly of the Bak apoptotic pore: a critical role for the Bak protein α6 helix in the multimerization of homodimers during apoptosis.

Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins.