Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis.

In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2-α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other's surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.

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.

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.

Inhibitor of apoptosis proteins limit RIP3 kinase-dependent interleukin-1 activation.

Interleukin-1ß (IL-1ß) is a potent inflammatory cytokine that is usually cleaved and activated by inflammasome-associated caspase-1. To determine whether IL-1ß activation is regulated by inhibitor of apoptosis (IAP) proteins, we treated macrophages with an IAP-antagonist "Smac mimetic" compound or genetically deleted the genes that encode the three IAP family members cIAP1, cIAP2, and XIAP. After Toll-like receptor priming, IAP inhibition triggered cleavage of IL-1ß that was mediated not only by the NLRP3-caspase-1 inflammasome, but also by caspase-8 in a caspase-1-independent manner. In the absence of IAPs, rapid and full generation of active IL-1ß by the NLRP3-caspase-1 inflammasome, or by caspase-8, required the kinase RIP3 and reactive oxygen species production. These results demonstrate that activation of the cell death-inducing ripoptosome platform and RIP3 can generate bioactive IL-1ß and implicate them as additional targets for the treatment of pathological IL-1-driven inflammatory responses.

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.

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.

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.

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.

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.

Electronic Phenotyping of Urinary Tract Infections as a Silver Standard Label for Machine Learning.

This study explored the efficacy of electronic phenotyping in data labeling for machine learning with a focus on urinary tract infections (UTIs). We contrasted labels from electronic phenotyping against previously published labels such as urine culture positivity. In comparison, electronic phenotyping showed the potential to enhance specificity in UTI labeling while maintaining similar sensitivity and was easily scaled for application to a large dataset suitable for machine learning, which we used to train and validate a machine learning model. Electronic phenotyping offers a valuable method for machine learning label generation in healthcare, with potential benefits for patient care and antimicrobial stewardship. Further research will expand its application and optimize techniques for increased performance.

"Covering provider": an effort to streamline clinical communication chaos.

Objective: This report describes a root cause analysis of incorrect provider assignments and a standardized workflow developed to improve the clarity and accuracy of provider assignments. Materials and Methods: A multidisciplinary working group involving housestaff was assembled. Key drivers were identified using value stream mapping and fishbone analysis. A report was developed to allow for the analysis of correct provider assignments. A standardized workflow was created and piloted with a single service line. Pre- and post-pilot surveys were administered to nursing staff and participating housestaff on the unit. Results: Four key drivers were identified. A standardized workflow was created with an exclusive treatment team role in Epic held by a single provider at any given time, with a corresponding patient list column displaying provider information for each patient. Pre- and post-survey responses report decreased confusion, decreased provider identification errors, and increased user satisfaction among RNs and residents with sustained uptake over time. Conclusion: This work demonstrates structured root cause analysis, notably engaging housestaff, to develop a standardized workflow for an understudied and growing problem. The development of tools and strategies to address the widespread burdens resulting from clinical communication failures is needed.

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.

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.

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.

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.

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.

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.