Network-based approach elucidates critical genes in BRCA subtypes and chemotherapy response in triple negative breast cancer.

Breast cancers (BRCA) exhibit substantial transcriptional heterogeneity, posing a significant clinical challenge. The global transcriptional changes in a disease context, however, are likely mediated by few key genes which reflect disease etiology better than the differentially expressed genes (DEGs). We apply our network-based tool PathExt to 1,059 BRCA tumors across 4 subtypes to identify key mediator genes in each subtype. Compared to conventional differential expression analysis, PathExt-identified genes exhibit greater concordance across tumors, revealing shared and subtype-specific biological processes; better recapitulate BRCA-associated genes in multiple benchmarks, and are more essential in BRCA subtype-specific cell lines. Single-cell transcriptomic analysis reveals a subtype-specific distribution of PathExt-identified genes in multiple cell types from the tumor microenvironment. Application of PathExt to a TNBC chemotherapy response dataset identified subtype-specific key genes and biological processes associated with resistance. We described putative drugs that target key genes potentially mediating drug resistance.

Draft genome sequence of 'Candidatus Phytoplasma asteris,' strain SW86 associated with sandal spike disease (SSD).

The sandal spike disease (SSD), related to 'Ca. Phytoplasma asteris' (Aster Yellows group), poses a significant threat to Indian sandalwood (Santalum album L.), making it the second most expensive wood globally due to declining population density. The epidemiology of SSD and the nature of the pathogen remain poorly understood. The SW86 isolate, collected from the Marayoor Sandalwood Reserve, was chosen for genome sequencing subsequent to confirming its titer and enriching phytoplasma DNA. Genome sequencing, utilizing Illumina and Oxford Nanopore Technology platforms, enabled a targeted hybrid metagenomic assembly resulting in 20 scaffolds totaling 554,025 bp, housing 436 protein-coding genes, 27 tRNA, and 1 rRNA operon. The genome analysis highlighted specific gene distributions, emphasizing translation, ribosomal structure, and biogenesis, with 352 genes assigned to 18 functional categories. Additionally, 322 proteins received functional assignments in the KEGG database, emphasizing 'Genetic Information Processing' and 'Environmental Information Processing'. Key potential pathogenicity factors, including signal peptide proteins and virulence proteins, were identified. Noteworthy findings include homologs of effectors genes like SAP11 and SAP05 and pathogenesis-related proteins, such as hemolysin III and SodA genes, in the SW86 genome. The duplicated cation-transporting P-type ATPase in the SW86 genome suggests a role in enhancing adaptability and contributing to the severity of SSD symptoms. This genome analysis provides crucial insights into the genomic features and potential virulence factors of 'Ca. Phytoplasma asteris' strain SW86, advancing our understanding of pathogenicity mechanisms and offering avenues for future disease management strategies in Indian sandalwood. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03952-5.

A quality improvement initiative to reduce the time to initial maternal visit in the neonatal intensive care unit.

OBJECTIVE: We aimed to reduce the time interval between an infant's admission to the Neonatal Intensive Care Unit (NICU) and first maternal interaction. METHODS: We identified three key drivers: 1. Collaboration with Labor and Delivery, 2. Education of staff and parents, and 3. Improved documentation of maternal presence. We measured the time interval from NICU admission to the initial maternal presence. We followed length of stay as a balancing measure to assay whether use of remote televisitation impeded efficient parental teaching and delayed discharge. RESULTS: We reduced the time interval from an average of 19.7 h in February 2020 to 12.3 h in June 2021. We expanded an already existing televisitation program as a surrogate to in-person interaction during COVID-19 pandemic. Televisitation did not affect in-person parental presence or LOS. CONCLUSION: Our multidisciplinary efforts resulted in a significantly accelerated time to initial maternal presence and did not prolong LOS.

Charting the transcriptomic landscape of primary and metastatic cancers in relation to their origin and target normal tissues.

Metastasis is a leading cause of cancer-related deaths, yet understanding how metastatic tumors adapt from their origin to target tissues is challenging. To address this, we assessed whether primary and metastatic tumors resemble their tissue of origin or target tissue in terms of gene expression. We analyzed gene expression profiles from various cancer types, including single-cell and bulk RNA-seq data, in both paired and unpaired primary and metastatic patient cohorts. We quantified the transcriptomic distances between tumor samples and their normal tissues, revealing that primary tumors are more similar to their tissue of origin, while metastases shift towards the target tissue. Pathway-level analysis highlighted critical transcriptomic changes during metastasis. Notably, primary cancers exhibited higher activity in cancer hallmarks, including Activating Invasion and Metastasis , compared to metastatic cancers. This comprehensive landscape analysis provides insight into how cancer tumors adapt to their metastatic environments, providing a transcriptome-wide view of the processes involved.

Network-based approach elucidates critical genes in BRCA subtypes and chemotherapy response in Triple Negative Breast Cancer.

Breast cancers exhibit substantial transcriptional heterogeneity, posing a significant challenge to the prediction of treatment response and prognostication of outcomes. Especially, translation of TNBC subtypes to the clinic remains a work in progress, in part because of a lack of clear transcriptional signatures distinguishing the subtypes. Our recent network-based approach, PathExt, demonstrates that global transcriptional changes in a disease context are likely mediated by a small number of key genes, and these mediators may better reflect functional or translationally relevant heterogeneity. We apply PathExt to 1059 BRCA tumors and 112 healthy control samples across 4 subtypes to identify frequent, key-mediator genes in each BRCA subtype. Compared to conventional differential expression analysis, PathExt-identified genes (1) exhibit greater concordance across tumors, revealing shared as well as BRCA subtype-specific biological processes, (2) better recapitulate BRCA-associated genes in multiple benchmarks, and (3) exhibit greater dependency scores in BRCA subtype-specific cancer cell lines. Single cell transcriptomes of BRCA subtype tumors reveal a subtype-specific distribution of PathExt-identified genes in multiple cell types from the tumor microenvironment. Application of PathExt to a TNBC chemotherapy response dataset identified TNBC subtype-specific key genes and biological processes associated with resistance. We described putative drugs that target top novel genes potentially mediating drug resistance. Overall, PathExt applied to breast cancer refines previous views of gene expression heterogeneity and identifies potential mediators of TNBC subtypes, including potential therapeutic targets.

Transcriptome analysis provides insights into the stress response in cultivated peanut (Arachis hypogaea L.) subjected to drought-stress.

BACKGROUND: Peanut (Arachis hypogaea L.) is one of the valuable oilseed crops grown in drought-prone areas worldwide. Drought severely limits peanut production and productivity significantly. METHOD AND RESULTS: In order to decipher the drought tolerance mechanism in peanut under drought stress, RNA sequencing was performed in TAG - 24 (drought tolerant genotype) and JL-24 (drought susceptible genotype). Approximately 51 million raw reads were generated from four different libraries of two genotypes subjected to drought stress exerted by 20% PEG 6000 stress and control conditions, of which ~ 41 million (80.87%) filtered reads were mapped to the Arachis hypogaea L. reference genome. The transcriptome analysis detected 1,629 differentially expressed genes (DEGs), 186 genes encoding transcription factors (TFs) and 30,199 SSR among the identified DEGs. Among the differentially expressed TF encoding genes, the highest number of genes were WRKY followed by bZIP, C2H2, and MYB during drought stress. The comparative analysis between the two genotypes revealed that TAG-24 exhibits activation of certain key genes and transcriptional factors that are involved in essential biological processes. Specifically, TAG-24 showed activation of genes involved in the plant hormone signaling pathway such as PYL9, Auxin response receptor gene, and ABA. Additionally, genes related to water deprivation such as LEA protein and those involved in combating oxidative damage such as Glutathione reductase were also found to be activated in TAG-24. CONCLUSION: This genome-wide transcription map, therefore, provides a valuable tool for future transcript profiling under drought stress and enriches the genetic resources available for this important oilseed crop.

Epidemiologic Characteristics of Mpox among People Experiencing Homelessness, Los Angeles County, California, USA, 2022.

In Los Angeles County, California, USA, public health surveillance identified 118 mpox cases among persons experiencing homelessness (PEH) during July-September 2022. Age and sex were similar for mpox case-patients among PEH and in the general population. Seventy-one (60%) PEH mpox case-patients were living with HIV, 35 (49%) of them virally suppressed. Hospitalization was required for 21% of case-patients because of severe disease. Sexual contact was likely the primary mode of transmission; 84% of patients reported sexual contact <3 weeks before symptom onset. PEH case-patients lived in shelters, encampments, cars, or on the street, or stayed briefly with friends or family (couch surfed). Some case-patients stayed at multiple locations during the 3-week incubation period. Public health follow-up and contact tracing detected no secondary mpox cases among PEH in congregate shelters or encampments. Equitable efforts should continue to identify, treat, and prevent mpox among PEH, who often experience severe disease.

Clinically oriented prediction of patient response to targeted and immunotherapies from the tumor transcriptome.

BACKGROUND: Precision oncology is gradually advancing into mainstream clinical practice, demonstrating significant survival benefits. However, eligibility and response rates remain limited in many cases, calling for better predictive biomarkers. METHODS: We present ENLIGHT, a transcriptomics-based computational approach that identifies clinically relevant genetic interactions and uses them to predict a patient's response to a variety of therapies in multiple cancer types without training on previous treatment response data. We study ENLIGHT in two translationally oriented scenarios: personalized oncology (PO), aimed at prioritizing treatments for a single patient, and clinical trial design (CTD), selecting the most likely responders in a patient cohort. FINDINGS: Evaluating ENLIGHT's performance on 21 blinded clinical trial datasets in the PO setting, we show that it can effectively predict a patient's treatment response across multiple therapies and cancer types. Its prediction accuracy is better than previously published transcriptomics-based signatures and is comparable with that of supervised predictors developed for specific indications and drugs. In combination with the interferon-γ signature, ENLIGHT achieves an odds ratio larger than 4 in predicting response to immune checkpoint therapy. In the CTD scenario, ENLIGHT can potentially enhance clinical trial success for immunotherapies and other monoclonal antibodies by excluding non-responders while overall achieving more than 90% of the response rate attainable under an optimal exclusion strategy. CONCLUSIONS: ENLIGHT demonstrably enhances the ability to predict therapeutic response across multiple cancer types from the bulk tumor transcriptome. FUNDING: This research was supported in part by the Intramural Research Program, NIH and by the Israeli Innovation Authority.

Sex Biases in Cancer and Autoimmune Disease Incidence Are Strongly Positively Correlated with Mitochondrial Gene Expression across Human Tissues.

Cancer occurs more frequently in men while autoimmune diseases (AIDs) occur more frequently in women. To explore whether these sex biases have a common basis, we collected 167 AID incidence studies from many countries for tissues that have both a cancer type and an AID that arise from that tissue. Analyzing a total of 182 country-specific, tissue-matched cancer-AID incidence rate sex bias data pairs, we find that, indeed, the sex biases observed in the incidence of AIDs and cancers that occur in the same tissue are positively correlated across human tissues. The common key factor whose levels across human tissues are most strongly associated with these incidence rate sex biases is the sex bias in the expression of the 37 genes encoded in the mitochondrial genome.

Respiratory support strategies in the management of severe, longstanding bronchopulmonary dysplasia.

Despite efforts to minimize ventilator-induced lung injury, some preterm infants require positive pressure support after 36 weeks' post-menstrual age. Infants with severe BPD typically experience progressive mismatch of ventilation and perfusion, which manifests as respiratory distress, hypoxemia in room air, hypercarbia, and growth failure. Lung compliance varies, but lung resistance generally increases with prolonged exposure to positive pressure ventilation and other sources of inflammation. Serial lung radiographs reveal a heterogeneous pattern, with areas of both hyperinflation and atelectasis; in extreme cases, macrocystic changes may be noted. Efforts to wean the respiratory support are often unsuccessful, and trials of high frequency ventilation, exogenous corticosteroids, and diuretics are common. The incidence of pulmonary hypertension increases with the severity of BPD, as does the mortality rate. Therefore, periodic screening and efforts to mitigate the risk of PH is fundamental to the management of longstanding BPD. Failure of conventional, lung-protective strategies (e.g., high rate/low tidal-volume and/or high frequency ventilation) warrants consideration of ventilatory strategies individualized to the disease physiology. Non-invasive modes of respiratory support may be successful in infants with mild to moderate BPD phenotypes. However, infants with moderate to severe BPD phenotypes often require invasive respiratory support, and pressure-limited or volume-targeted conventional ventilation may be better suited to the physiology than high-frequency ventilation. The consistent provision of adequate support is fundamental to the management of longstanding BPD and is best achieved with a stepwise increase in ventilator support until comfortable spontaneous respirations are achieved. Adequately supported infants typically experience improvements in both oxygenation and ventilation, which, if sustained, may arrest and generally reverses the course of a potentially lethal lung disease. Care should be individualized to address the most likely pulmonary mechanics, including variable lung compliance, elevated airway resistance, and variable airway obstruction.

Evaluation of the Homeless Management Information System for COVID-19 Surveillance Among People Experiencing Homelessness.

BACKGROUND: Variable and incomplete reporting of housing status creates challenges in the surveillance of coronavirus disease 2019 (COVID-19) among the homeless population in Los Angeles County (LA County) and nationwide. METHODS: We developed standard investigation procedures to assess the housing status of LA County COVID-19 patients. Using data sharing procedures, we matched COVID-19 patients to Homeless Management Information System (HMIS) client profiles and supplemented with additional data sources for contributory data points and to further housing status ascertainment. RESULTS: We identified 10 586 COVID-19 patients among people experiencing homelessness (PEH) between 30 March 2020 and 30 December 2021; 2801 (26.5%) patients were first identified from HMIS profile matches, 1877 (17.7%) from quarantine/isolation housing intake rosters, 573 (5.4%) from hospital records, 749 (7.1%) from case and contact interviews, 3659 (34.6%) directly from PEH medical and service providers, and 927 (8.8%) had unknown sources. Among COVID-19 patients matched to HMIS profiles, 5351 (42.5%) were confirmed to be PEH at the time of COVID-19 diagnosis. CONCLUSIONS: Interoperability between public health data, HMIS, and external partners have been critical components in evaluating the impact of COVID-19 among the LA County homeless population. No one data source was complete for COVID-19 surveillance in this population.

Rothia santali sp. nov., endophytic bacteria isolated from sandalwood (Santalum album L.) seedling.

A novel, mustard yellow-pigmented aerobic bacterial strain designated AR01T was isolated from hypocotyl tissue of a sandalwood seedling from Bangalore, India. The 16S rRNA gene of strain AR01T had the highest 98.97% sequence similarity with Rothia halotolerans YIM 90716T (KCTC 19172) followed by Rothia kristinae PM 129T (NBRC 15354T) (97.31%) and Rothia koreensis P31T (JCM 15915) (97.11%), respectively. The strain AR01T was coccoid-shaped, non-motile, non-spore forming, oxidase negative and catalase positive. The strain AR01T has a genome size of 3.31 Mb containing 2993 protein-coding genes including 48 tRNA and 10 rRNAs spread across 84 contigs. The genomic DNA G + C content was 71.77 mol%. The calculated dDDH was 31.10% and the OrthoANI value was 85.27% when compared with its closest related type strain Rothia halotolerans YIM 90716T. The predominant cellular fatty acids were C16:0 iso, C15:0 anteiso and C17:0 anteiso. The strain AR01T contains major polar lipids including diphosphatidylglycerol and phosphatidylglycerol. The distinct physiological, biochemical characteristics and genotypic relatedness indicated that AR01T represents a novel species of the genus Rothia, for which the name Rothia santali sp. nov. (Type strain AR01T = MCC 4800T = JCM 35593T) is proposed.

Cross-species identification of cancer resistance-associated genes that may mediate human cancer risk.

Cancer is a predominant disease across animals. We applied a comparative genomics approach to systematically characterize genes whose conservation levels correlate positively (PC) or negatively (NC) with cancer resistance estimates across 193 vertebrates. Pathway analysis reveals that NC genes are enriched for metabolic functions and PC genes in cell cycle regulation, DNA repair, and immune response, pointing to their corresponding roles in mediating cancer risk. We find that PC genes are less tolerant to loss-of-function (LoF) mutations, are enriched in cancer driver genes, and are associated with germline mutations that increase human cancer risk. Their relevance to cancer risk is further supported via the analysis of mouse functional genomics and cancer mortality of zoo mammals' data. In sum, our study describes a cross-species genomic analysis pointing to candidate genes that may mediate human cancer risk.

COVID-19 Mortality Rates in Los Angeles County Among People Experiencing Homelessness, March 2020-February 2021.

OBJECTIVE: Understanding COVID-19-related mortality among the large population of people experiencing homelessness (PEH) in Los Angeles County (LA County) may inform public health policies to protect this vulnerable group. We investigated the impact of COVID-19 on PEH compared with the general population in LA County. METHODS: We calculated crude COVID-19 mortality rates per 100 000 population and mortality rates adjusted for age, race, and sex/gender among PEH and compared them with the general population in LA County from March 1, 2020, through February 28, 2021. RESULTS: Among adults aged ≥18 years, the crude mortality rate per 100 000 population among PEH was 20% higher than among the general LA County population (348.7 vs 287.6). After adjusting for age, the mortality rate among PEH was 570.7 per 100 000 population. PEH had nearly twice the risk of dying from COVID-19 as people in the general LA County population; PEH aged 18-29 years had almost 8 times the risk of dying compared with their peers in the general LA County population. PEH had a higher risk of mortality than the general population after adjusting for race (standardized mortality ratio [SMR] = 1.4; 95% CI, 1.2-1.6) and sex/gender (SMR = 1.3; 95% CI, 1.1-1.5). CONCLUSIONS: A higher risk of COVID-19-related death among PEH compared with the general population indicates the need for public health policies and interventions to protect this vulnerable group.

Pleiotropy-guided transcriptome imputation from normal and tumor tissues identifies candidate susceptibility genes for breast and ovarian cancer.

Familial, sequencing, and genome-wide association studies (GWASs) and genetic correlation analyses have progressively unraveled the shared or pleiotropic germline genetics of breast and ovarian cancer. In this study, we aimed to leverage this shared germline genetics to improve the power of transcriptome-wide association studies (TWASs) to identify candidate breast cancer and ovarian cancer susceptibility genes. We built gene expression prediction models using the PrediXcan method in 681 breast and 295 ovarian tumors from The Cancer Genome Atlas and 211 breast and 99 ovarian normal tissue samples from the Genotype-Tissue Expression project and integrated these with GWAS meta-analysis data from the Breast Cancer Association Consortium (122,977 cases/105,974 controls) and the Ovarian Cancer Association Consortium (22,406 cases/40,941 controls). The integration was achieved through application of a pleiotropy-guided conditional/conjunction false discovery rate (FDR) approach in the setting of a TWASs. This identified 14 candidate breast cancer susceptibility genes spanning 11 genomic regions and 8 candidate ovarian cancer susceptibility genes spanning 5 genomic regions at conjunction FDR < 0.05 that were >1 Mb away from known breast and/or ovarian cancer susceptibility loci. We also identified 38 candidate breast cancer susceptibility genes and 17 candidate ovarian cancer susceptibility genes at conjunction FDR < 0.05 at known breast and/or ovarian susceptibility loci. The 22 genes identified by our cross-cancer analysis represent promising candidates that further elucidate the role of the transcriptome in mediating germline breast and ovarian cancer risk.

Remote Lactation Support in the COVID-19 Era.

Breast milk provides optimal nourishment for all infants and has special advantages in preterm infants. Breast milk is associated with lower rates of necrotizing enterocolitis and bronchopulmonary dysplasia and improved neurodevelopmental outcomes in the preterm population. Mothers in the NICU may experience multiple psychological, physical, and social/cultural barriers that impede successful breastfeeding. Professional lactation support is of crucial importance in this population. With the social distancing requirements of the pandemic, many clinicians have adopted novel methods of education and communication to ensure continued timely support for NICU mothers.

Benzimidazole-1,2,3-triazole hybrid molecules: synthesis and study of their interaction with G-quadruplex DNA.

A series of new benzimidazole-1,2,3-triazole hybrid derivatives have been synthesized via 'click' reaction and evaluated for their in vitro cytotoxicity as well as DNA binding affinity. MTT assay showed that all the six compounds are cytotoxic to PC3 and B16-F10 cancer cell lines. Though all the compounds showed moderate interaction with G4, c-Myc promoter DNA and dsDNA, 4f exhibited selective interaction with G-quadruplex DNA over duplex DNA as demonstrated by spectroscopic experiments like UV-vis spectroscopy, fluorescence spectroscopy, CD spectroscopy, thermal melting and fluorescence lifetime experiments. They also confirm the G-quadruplex DNA stabilizing potential of 4f. Viscosity measurements also confirm that 4f exhibits high G-quadruplex DNA selectivity over duplex DNA. Docking studies supported the spectroscopic observations. Cell cycle analysis showed that 4f induces G2/M phase arrest and induces apoptosis. Hence, from these experimental results it is evident that compound 4f may be a G-quadruplex DNA groove binding molecule with anticancer activity.

Metabolomic Profiling of Drought-Tolerant and Susceptible Peanut (Arachis hypogaea L.) Genotypes in Response to Drought Stress.

Peanut is frequently constrained by extreme environmental conditions such as drought. To reveal the involvement of metabolites, TAG 24 (drought-tolerant) and JL 24 (drought-sensitive) peanut genotypes were investigated under control and 20% PEG 6000-mediated water scarcity conditions at the seedling stage. Samples were analyzed by gas chromatography-mass spectrometry (GC-MS) to identify untargeted metabolites and targeted metabolites, i.e., polyamines and polyphenols by high-performance liquid chromatography (HPLC) and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), respectively. The principal component analysis (PCA), partial least-squares discriminant analysis (PLS-DA), heat map, and cluster analysis were applied to the metabolomics data obtained by the GC-MS technique to determine the important metabolites for drought tolerance. Among 46 resulting metabolites, pentitol, phytol, xylonic acid, d-xylopyranose, stearic acid, and d-ribose were important drought-responsive metabolites. Agmatine and cadaverine were present in TAG 24 leaves and roots, respectively, during water-deficit conditions and believed to be the potential polyamines for drought tolerance. Polyphenols such as syringic acid and vanillic acid were produced more in the leaves of TAG 24, while catechin production was high in JL 24 during stress conditions. Seven metabolic pathways, namely, galactose metabolism, starch and sucrose metabolism, fructose and mannose metabolism, pentose and glucuronate interconversion, propanoate metabolism, amino sugar and nucleotide sugar metabolism, and biosynthesis of unsaturated fatty acids were significantly affected by water-deficit conditions. This study provides valuable information about the metabolic response of peanut to drought stress and metabolites identified, which encourages further study by transcriptome and proteomics to improve drought tolerance in peanut.

PhenomeXcan: Mapping the genome to the phenome through the transcriptome.

Large-scale genomic and transcriptomic initiatives offer unprecedented insight into complex traits, but clinical translation remains limited by variant-level associations without biological context and lack of analytic resources. Our resource, PhenomeXcan, synthesizes 8.87 million variants from genome-wide association study summary statistics on 4091 traits with transcriptomic data from 49 tissues in Genotype-Tissue Expression v8 into a gene-based, queryable platform including 22,515 genes. We developed a novel Bayesian colocalization method, fast enrichment estimation aided colocalization analysis (fastENLOC), to prioritize likely causal gene-trait associations. We successfully replicate associations from the phenome-wide association studies (PheWAS) catalog Online Mendelian Inheritance in Man, and an evidence-based curated gene list. Using PhenomeXcan results, we provide examples of novel and underreported genome-to-phenome associations, complex gene-trait clusters, shared causal genes between common and rare diseases via further integration of PhenomeXcan with ClinVar, and potential therapeutic targets. PhenomeXcan (phenomexcan.org) provides broad, user-friendly access to complex data for translational researchers.

Development and Pilot Testing of a Simulation to Study How Physicians Facilitate Surrogate Decision Making Based on Critically Ill Patients' Values and Preferences.

CONTEXT: There are no evidence-based programs to train physicians to facilitate shared decision making based on incapacitated intensive care unit patients' values and preferences. OBJECTIVES: The objective of this study was to develop a high-fidelity simulation to fill this gap. METHODS: Case development involved six steps: 1) drafting a case about an elderly patient receiving prolonged mechanical ventilation; 2) engaging an expert advisory board to optimize case content; 3) revising the case based on advisory board input; 4) training actors to portray the case patient's daughter; 5) obtaining physician feedback on the simulation; and 6) revising the case based on their feedback. We conducted a cross-sectional pilot study with 50 physicians to assess feasibility and acceptability, defined a priori as an enrollment rate >40 physicians/year, study procedures <75 minutes/participant, >95% actor adherence to standardization rules, and high physician ratings of realism and acceptability. RESULTS: Advisory panel feedback yielded two modifications: 1) refocusing the case on decision making about tracheostomy and percutaneous gastrostomy and 2) making the patient's values more authentic. Physician feedback yielded two additional modifications: 1) reducing how readily the actor divulged the patient's values and 2) making her more emotional. All 50 physicians enrolled in the pilot study over 11 months completed study procedures in <75 minutes. Actor adherence to standardization rules was 95.8%. Physicians' mean ratings of realism and acceptability were 8.4 and 9.1, respectively, on a 10-point scale. CONCLUSION: Simulation is feasible, is acceptable, and can be adequately standardized to study physicians' skills for facilitating surrogate decision making based on an incapacitated intensive care unit patient's values and preferences.