Aligning patient values and code status: Choice of Diction's Effect (CODE) study.

BACKGROUND: Decisions regarding resuscitation after cardiac arrest are critical from ethical, patient satisfaction, outcome, and healthcare cost standpoints. Physician-reported discussion barriers include topic discomfort, fear of time commitment, and difficulty articulating end-of-life concepts. The influence of language used in these discussions has not been tested. This study explored whether utilizing the alternate term "allow (a) natural death" changed code status decisions in hospitalized patients versus "do not resuscitate" (DNR). METHODS: All patients age 65 and over admitted to a general medicine hospital teaching service were screened (English-speaking, not ICU-level care, no active psychiatric illness, no substance misuse, no active DNR). Participants were randomized to resuscitation discussions with either DNR or "allow natural death" as the "no code" phrasing. Outcomes included patient resuscitation decision, satisfaction with and duration of the conversation, and decision correlation with illness severity and predicted resuscitation success. RESULTS: 102 participants were randomized to the "allow natural death" (N = 49) or DNR (N = 53) arms. The overall "no code" rate for our sample of hospitalized general medicine inpatients age >65 was 16.7%, with 13% in the DNR and 20.4% in the "allow natural death" arms (p = 0.35). Discussion length was similar in the DNR and "allow natural death" arms (3.9 + 3.2 vs. 4.9 + 3.9 minutes), and not significantly different (p = 0.53). Over 90% of participants were highly satisfied with their code status decision, without difference between arms (p = 0.49). CONCLUSIONS: Participants' code status discussions did not differ in "no code" rate between "allow natural death" and DNR arms but were short in length and had high patient satisfaction. Previously reported code status discussion barriers were not encountered. It is appropriate to screen code status in all hospitalized patients regardless of phrasing used.

Factors associated with attitudes toward research MRI in older Asian Americans.

INTRODUCTION: South Asian (SA) and East Asian (EA) older adults represent the fastest-growing racial/ethnic groups of Americans at risk for dementia. While recruiting older SA adults into a brain health study, we encountered unexpected hesitancy toward structural brain magnetic resonance imaging (MRI) analysis and stigmatizing attitudes related to internal locus of control (LoC) for future dementia risks. We hypothesized that support for MRI-related research was influenced by these attitudes as well as personal MRI experience, perceived MRI safety, and concerns for personal risk for future dementia/stroke. METHODS: We developed a brief cross-sectional survey to assess older adults' MRI experiences and perceptions, desire to learn of six incidental findings of increasing impact on health, and attitudes related to dementia (including LoC) and research participation. We recruited a convenience sample of 256 respondents (74% reporting as 50+) from the New Jersey/New York City area to complete the survey (offered in English, Chinese, Korean, and Spanish) and modeled the proportional odds (PO) for favorable attitudes toward research activities. RESULTS: Seventy-seven SA and 84 EA respondents were analyzed alongside 95 White, Black, or Hispanic adults. White (PO = 2.54, p = 0.013) and EA (PO = 2.14, p = 0.019) respondents were both more likely than SA respondents to endorse healthy volunteers' participation in research, and the difference between White and SA respondents was mediated by the latter's greater internal LoC for dementia risks. EA respondents had more worries for future dementia/stroke than SA respondents (p = 0.006) but still shared SA respondents' lower wish (measured by proportion of total) to learn of incidental MRI findings. DISCUSSION: SA-and EA compared to SA-older adults had low desire to learn of incidental MRI findings but had different attitudes toward future dementia/stroke risks. A culturally appropriate protocol to disclose incidental MRI findings may improve SA and EA participation in brain health research. Highlights: Older Asian Americans have limited interest in incidental findings on research MRISouth Asians are most likely to attribute dementia to people's own behaviorsSouth Asians' attitudes mediate lower support for healthy volunteers in researchSouth and East Asians differ in dementia worries and research-related attitudes.

Electronic Health Record Prompt to Improve Lung Cancer Screening in Primary Care.

INTRODUCTION: Lung cancer is the leading cause of cancer death in the U.S. Combusted tobacco use, the primary risk factor, accounts for 90% of all lung cancers. Early detection of lung cancer improves survival, yet lung cancer screening rates are much lower than those of other cancer screening tests. Electronic health record (EHR) systems are an underutilized tool that could improve screening rates. METHODS: This study was conducted in the Rutgers Robert Wood Johnson Medical Group, a university-affiliated network in New Brunswick, NJ. Two novel EHR workflow prompts were implemented on July 1, 2018. These prompts included fields to determine tobacco use and lung cancer screening eligibility and facilitated low-dose computed tomography ordering for eligible patients. The prompts were designed to improve tobacco use data entry, allowing for better lung cancer screening eligibility identification. Data were analyzed in 2022 retrospectively for the period July 1, 2017 to June 30, 2019. The analyses represented 48,704 total patient visits. RESULTS: The adjusted odds of patient record completeness to determine eligibility for low-dose computed tomography (AOR=1.19, 95% CI=1.15, 1.23), eligibility for low-dose computed tomography (AOR=1.59, 95% CI=1.38, 1.82), and whether low-dose computed tomography was ordered (AOR=1.04, 95% CI=1.01, 1.07) all significantly increased after the electronic medical record prompts were implemented. CONCLUSIONS: These findings show the utility and benefit of EHR prompts in primary care settings to increase identification for lung cancer screening eligibility as well as increased low-dose computed tomography ordering.

Too much information? Asian Americans' preferences for incidental brain MRI findings.

INTRODUCTION: South Asian (SA) and East Asian (EA) older adults represent the fastest growing group of Americans at risk for dementia, but their participation in aging and dementia research has been limited. While recruiting healthy SA older adults into a brain health study, we encountered unexpected hesitancy towards structural brain MRI analysis along with some stigmatizing attitudes related to internal locus of control (LoC) for future dementia risks. We hypothesized that support for MRI-related research was influenced by these attitudes as well as one's own MRI experience, perceived MRI safety, and concerns for one's own risks for future dementia/stroke. METHODS: We developed a brief cross-sectional survey to assess older adults' MRI experiences and perceptions, desire to learn of six incidental findings of increasing health implications, and attitudes related to dementia as well as research participation. We recruited a convenience sample of 256 respondents (74% reporting as 50+) from the New Jersey/New York City area to complete the survey, and modeled the proportional odds (P.O.) for pro-research attitudes. RESULTS: 77 SA and 84 EA respondents were analyzed with 95 non-Asian adults. White (P.O.=2.54, p=0.013) and EA (P.O.=2.14, p=0.019) respondents were both more likely than SA respondents to endorse healthy volunteers' participation in research, and the difference between White and SA respondents was mediated by the latter's greater internal LoC for dementia risks. EA respondents had more worries for future dementia/stroke than SA respondents (p=0.006), but still shared SA respondents' low desire to learn of incidental MRI findings. DISCUSSION: SA and EA older adults had different attitudes towards future dementia/stroke risks, but shared a low desire to learn of incidental MRI findings. A culturally-appropriate protocol to disclose incidental MRI findings may improve SA and EA participation in brain health research.

Lung Cancer Screening Knowledge and Perceived Barriers Among Physicians in the United States.

Introduction: Lung cancer remains the leading cause of cancer death in the United States and has historically been detected late in its course. Low-dose computed tomography scan (LDCT) reduces lung cancer mortality by 20% and is currently recommended by clinical practice guidelines. However, compared with other cancer screening modalities, LDCT utilization remains low. This study surveyed office-based primary care physicians across the United States to better understand LDCT utilization. Methods: A total of 1500 family and internal medicine physicians selected from the American Medical Association's physician master file were surveyed between April and July 2019 regarding LDCT practices, eligibility, clinical scenarios, and perceived barriers. Results: The American Association for Public Opinion Research response rate 3 was 59% (652 respondents); 599 completed supplemental questions regarding lung cancer screening. A total of 88% of respondents discussed LDCT in the previous year, and 78% had ordered at least one LDCT. Most (59%) knew the tobacco exposure criteria for LDCT and correctly identified appropriate clinical scenarios (49%-86% responded correctly). Less than half of respondents correctly identified the age eligibility criteria (44%-45% responded correctly). In general, male physicians, those who graduated after 1990, and family medicine physicians were more likely to report accurate knowledge regarding LDCT eligibility. The top perceived barriers to LDCT were cost to the patient (48% identified as a major barrier), insurance not covering screening (46% major), and patients being unaware of lung cancer screening (40% major). Conclusion: Knowledge and practices about lung cancer screening are improving, though remain suboptimal. The most common barriers remain cost or insurance-based and suggest the need for a systems-based response to increase awareness and reduce the underutilization of LDCT.

Targeted mutation detection in breast cancer using MammaSeq™.

BACKGROUND: Breast cancer is the most common invasive cancer among women worldwide. Next-generation sequencing (NGS) has revolutionized the study of cancer across research labs around the globe; however, genomic testing in clinical settings remains limited. Advances in sequencing reliability, pipeline analysis, accumulation of relevant data, and the reduction of costs are rapidly increasing the feasibility of NGS-based clinical decision making. METHODS: We report the development of MammaSeq, a breast cancer-specific NGS panel, targeting 79 genes and 1369 mutations, optimized for use in primary and metastatic breast cancer. To validate the panel, 46 solid tumors and 14 plasma circulating tumor DNA (ctDNA) samples were sequenced to a mean depth of 2311× and 1820×, respectively. Variants were called using Ion Torrent Suite 4.0 and annotated with cravat CHASM. CNVKit was used to call copy number variants in the solid tumor cohort. The oncoKB Precision Oncology Database was used to identify clinically actionable variants. Droplet digital PCR was used to validate select ctDNA mutations. RESULTS: In cohorts of 46 solid tumors and 14 ctDNA samples from patients with advanced breast cancer, we identified 592 and 43 protein-coding mutations. Mutations per sample in the solid tumor cohort ranged from 1 to 128 (median 3), and the ctDNA cohort ranged from 0 to 26 (median 2.5). Copy number analysis in the solid tumor cohort identified 46 amplifications and 35 deletions. We identified 26 clinically actionable variants (levels 1-3) annotated by OncoKB, distributed across 20 out of 46 cases (40%), in the solid tumor cohort. Allele frequencies of ESR1 and FOXA1 mutations correlated with CA.27.29 levels in patient-matched blood draws. CONCLUSIONS: In solid tumor biopsies and ctDNA, MammaSeq detects clinically actionable mutations (OncoKB levels 1-3) in 22/46 (48%) solid tumors and in 4/14 (29%) of ctDNA samples. MammaSeq is a targeted panel suitable for clinically actionable mutation detection in breast cancer.

Whole genome amplification of cell-free DNA enables detection of circulating tumor DNA mutations from fingerstick capillary blood.

The ability to measure mutations in plasma cell-free DNA (cfDNA) has the potential to revolutionize cancer surveillance and treatment by enabling longitudinal monitoring not possible with solid tumor biopsies. However, obtaining sufficient quantities of cfDNA remains a challenge for assay development and clinical translation; consequently, large volumes of venous blood are typically required. Here, we test proof-of-concept for using smaller volumes via fingerstick collection. Matched venous and fingerstick blood were obtained from seven patients with metastatic breast cancer. Fingerstick blood was separated at point-of-care using a novel paper-based concept to isolate plasma centrifuge-free. Patient cfDNA was then analyzed with or without a new method for whole genome amplification via rolling-circle amplification (WG-RCA). We identified somatic mutations by targeted sequencing and compared the concordance of mutation detection from venous and amplified capillary samples by droplet-digital PCR. Patient mutations were detected with 100% concordance after WG-RCA, although in some samples, allele frequencies showed greater variation likely due to differential amplification or primer inaccessibility. These pilot findings provide physiological evidence that circulating tumor DNA is accessible by fingerstick and sustains presence/absence of mutation detection after whole-genome amplification. Further refinement may enable simpler and less-invasive methods for longitudinal or theranostic surveillance of metastatic cancer.

Detection of ESR1 mutations in circulating cell-free DNA from patients with metastatic breast cancer treated with palbociclib and letrozole.

ESR1 mutations are frequently acquired in hormone-resistant metastatic breast cancer (MBC). CDK4/6 inhibition along with endocrine therapy is a promising strategy in hormone receptor-positive MBC. However, the incidence and impact of ESR1 mutations on clinical outcome in patients treated with CDK4/6 inhibitors have not been defined. In this study, we evaluated the frequency of ESR1 mutations in cfDNA from 16 patients with MBC undergoing palbociclib and letrozole therapy. Four common ESR1 mutations (D538G, Y537C, Y537N, and Y537S) were analyzed in serial blood draws using ddPCR. Mutation rate was 31.3% (5/16) (n=3; de novo, n=2; acquired). D538G was the most frequent mutation (n=3), followed by Y537N and Y537S (n=2). One patient showed multiple ESR1 mutations. Mutations were enriched during therapy. Progression-free survival (PFS) and overall survival (OS) were similar in patients with and without mutation detected at any given time during treatment. However, PFS was significantly shorter in patients with ESR1 mutation at initial blood draw (3.3 versus 9.0 months, P-value=0.038). In conclusion, ESR1 mutation prevalence is consistent with recent studies in hormone-refractory breast cancer. Further, treatment with palbociclib and letrozole does not prevent selection of ESR1 mutations in later lines of therapy. Larger studies are warranted to validate these findings.

Estradiol as a Targeted, Late-Line Therapy in Metastatic Breast Cancer with Estrogen Receptor Amplification.

Estradiol is a major regulator of growth for the subset of breast cancers that express the estrogen receptor (ER, ESR1). Strategies to block ER action, via reduction of estradiol or direct inhibition of ER, have shown major success in the prevention and treatment of breast cancer. However, most ER-positive (ER+) metastatic breast cancers (MBC) eventually become resistant to these interventions. Interestingly, high dose estrogen can induce apoptosis in breast cancer cell lines, and high-dose estrogen has been used for over 50 years as therapy for ER+ breast cancer. The mechanism for growth control of MBC by high dose estrogen is unclear. We present a patient with metastatic breast cancer whose tumor was found to have amplification of ESR1 by tumor genome sequencing. This patient was treated with high dose estradiol and subsequently experienced a sustained partial response, which was predicted by prior experiments with patient-derived xenograft animal models containing breast cancers with ER amplification.

Sustained, Long-Term Maintenance of Remission with Single-Agent Veliparib in Recurrent Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBC) have poorer outcomes than hormone positive or human epidermal growth factor receptor 2 (HER2)-positive breast cancers, with chemotherapy being the usual standard of care. Veliparib, a poly ADP-ribose polymerase (PARP) inhibitor, has been studied in both breast cancer susceptibility genes 1 and 2 (BRCA)-mutation related and sporadic cancers as a single agent and in combination with chemotherapy. Here, we describe a patient whose metastatic recurrence of TNBC was treated with combination chemotherapy and veliparib followed by maintenance single-therapy veliparib.

mBLAST: Keeping up with the sequencing explosion for (meta)genome analysis.

Recent advances in next-generation sequencing technologies require alignment algorithms and software that can keep pace with the heightened data production. Standard algorithms, especially protein similarity searches, represent significant bottlenecks in analysis pipelines. For metagenomic approaches in particular, it is now often necessary to search hundreds of millions of sequence reads against large databases. Here we describe mBLAST, an accelerated search algorithm for translated and/or protein alignments to large datasets based on the Basic Local Alignment Search Tool (BLAST) and retaining the high sensitivity of BLAST. The mBLAST algorithms achieve substantial speed up over the National Center for Biotechnology Information (NCBI) programs BLASTX, TBLASTX and BLASTP for large datasets, allowing analysis within reasonable timeframes on standard computer architectures. In this article, the impact of mBLAST is demonstrated with sequences originating from the microbiota of healthy humans from the Human Microbiome Project. mBLAST is designed as a plug-in replacement for BLAST for any study that involves short-read sequences and includes high-throughput analysis. The mBLAST software is freely available to academic users at www.multicorewareinc.com.

Exploration of bacterial community classes in major human habitats.

BACKGROUND: Determining bacterial abundance variation is the first step in understanding bacterial similarity between individuals. Categorization of bacterial communities into groups or community classes is the subsequent step in describing microbial distribution based on abundance patterns. Here, we present an analysis of the groupings of bacterial communities in stool, nasal, skin, vaginal and oral habitats in a healthy cohort of 236 subjects from the Human Microbiome Project. RESULTS: We identify distinct community group patterns in the anterior nares, four skin sites, and vagina at the genus level. We also confirm three enterotypes previously identified in stools. We identify two clusters with low silhouette values in most oral sites, in which bacterial communities are more homogeneous. Subjects sharing a community class in one habitat do not necessarily share a community class in another, except in the three vaginal sites and the symmetric habitats of the left and right retroauricular creases. Demographic factors, including gender, age, and ethnicity, significantly influence community composition in several habitats. Community classes in the vagina, retroauricular crease and stool are stable over approximately 200 days. CONCLUSION: The community composition, association of demographic factors with community classes, and demonstration of community stability deepen our understanding of the variability and dynamics of human microbiomes. This also has significant implications for experimental designs that seek microbial correlations with clinical phenotypes.

The prevalence of species and strains in the human microbiome: a resource for experimental efforts.

Experimental efforts to characterize the human microbiota often use bacterial strains that were chosen for historical rather than biological reasons. Here, we report an analysis of 380 whole-genome shotgun samples from 100 subjects from the NIH Human Microbiome Project. By mapping their reads to 1,751 reference genome sequences and analyzing the resulting relative strain abundance in each sample we present metrics and visualizations that can help identify strains of interest for experimentalists. We also show that approximately 14 strains of 10 species account for 80% of the mapped reads from a typical stool sample, indicating that the function of a community may not be irreducibly complex. Some of these strains account for >20% of the sequence reads in a subset of samples but are absent in others, a dichotomy that could underlie biological differences among subjects. These data should serve as an important strain selection resource for the community of researchers who take experimental approaches to studying the human microbiota.

Individuality and temporal stability of the human gut microbiome.

INTRODUCTION: The breakthrough of next generation sequencing-technologies has enabled large-scale studies of natural microbial communities and the 16S rRNA genes have been widely used as a phylogenetic marker to study community structure. However, major limitations of this approach are that neither strain-level resolution nor genomic context of microorganisms can be provided. This information, however, is crucial to answer fundamental questions about the temporal stability and distinctiveness of natural microbial communities. MATERIAL AND METHODS: We developed a methodological framework for metagenomic single nucleotide polymorphism (SNP) variation analysis and applied it to publicly available data from 252 human fecal samples from 207 European and North American individuals. We further analyzed samples from 43 healthy subjects that were sampled at least twice over time intervals of up to one year and measured population similarities of dominant gut species. RESULTS: We detected 10.3 million SNPs in 101 species, which nearly amounts to the number identified in more than 1,000 humans. CONCLUSION: The most striking result was that host-specific strains appear to be retained over long time periods. This indicates that individual-specific strains are not easily exchanged with the environment and furthermore, that an individuals appear to have a unique metagenomic genotype. This, in turn, is linked to implications for human gut physiology, such as the stability of antibiotic resistance potential.

Coverage theories for metagenomic DNA sequencing based on a generalization of Stevens' theorem.

Metagenomic project design has relied variously upon speculation, semi-empirical and ad hoc heuristic models, and elementary extensions of single-sample Lander-Waterman expectation theory, all of which are demonstrably inadequate. Here, we propose an approach based upon a generalization of Stevens' Theorem for randomly covering a domain. We extend this result to account for the presence of multiple species, from which are derived useful probabilities for fully recovering a particular target microbe of interest and for average contig length. These show improved specificities compared to older measures and recommend deeper data generation than the levels chosen by some early studies, supporting the view that poor assemblies were due at least somewhat to insufficient data. We assess predictions empirically by generating roughly 4.5 Gb of sequence from a twelve member bacterial community, comparing coverage for two particular members, Selenomonas artemidis and Enterococcus faecium, which are the least ([Formula: see text]3 %) and most ([Formula: see text]12 %) abundant species, respectively. Agreement is reasonable, with differences likely attributable to coverage biases. We show that, in some cases, bias is simple in the sense that a small reduction in read length to simulate less efficient covering brings data and theory into essentially complete accord. Finally, we describe two applications of the theory. One plots coverage probability over the relevant parameter space, constructing essentially a "metagenomic design map" to enable straightforward analysis and design of future projects. The other gives an overview of the data requirements for various types of sequencing milestones, including a desired number of contact reads and contig length, for detection of a rare viral species.

Genomic variation landscape of the human gut microbiome.

Whereas large-scale efforts have rapidly advanced the understanding and practical impact of human genomic variation, the practical impact of variation is largely unexplored in the human microbiome. We therefore developed a framework for metagenomic variation analysis and applied it to 252 faecal metagenomes of 207 individuals from Europe and North America. Using 7.4 billion reads aligned to 101 reference species, we detected 10.3 million single nucleotide polymorphisms (SNPs), 107,991 short insertions/deletions, and 1,051 structural variants. The average ratio of non-synonymous to synonymous polymorphism rates of 0.11 was more variable between gut microbial species than across human hosts. Subjects sampled at varying time intervals exhibited individuality and temporal stability of SNP variation patterns, despite considerable composition changes of their gut microbiota. This indicates that individual-specific strains are not easily replaced and that an individual might have a unique metagenomic genotype, which may be exploitable for personalized diet or drug intake.

Optimizing read mapping to reference genomes to determine composition and species prevalence in microbial communities.

The Human Microbiome Project (HMP) aims to characterize the microbial communities of 18 body sites from healthy individuals. To accomplish this, the HMP generated two types of shotgun data: reference shotgun sequences isolated from different anatomical sites on the human body and shotgun metagenomic sequences from the microbial communities of each site. The alignment strategy for characterizing these metagenomic communities using available reference sequence is important to the success of HMP data analysis. Six next-generation aligners were used to align a community of known composition against a database comprising reference organisms known to be present in that community. All aligners report nearly complete genome coverage (>97%) for strains with over 6X depth of coverage, however they differ in speed, memory requirement and ease of use issues such as database size limitations and supported mapping strategies. The selected aligner was tested across a range of parameters to maximize sensitivity while maintaining a low false positive rate. We found that constraining alignment length had more impact on sensitivity than does constraining similarity in all cases tested. However, when reference species were replaced with phylogenetic neighbors, similarity begins to play a larger role in detection. We also show that choosing the top hit randomly when multiple, equally strong mappings are available increases overall sensitivity at the expense of taxonomic resolution. The results of this study identified a strategy that was used to map over 3 tera-bases of microbial sequence against a database of more than 5,000 reference genomes in just over a month.

The metalloprotease of Listeria monocytogenes is regulated by pH.

Listeria monocytogenes is an intracytosolic bacterial pathogen. Among the factors contributing to escape from vacuoles are a phosphatidylcholine phospholipase C (PC-PLC) and a metalloprotease (Mpl). Both enzymes are translocated across the bacterial membrane as inactive proproteins, whose propeptides serve in part to maintain them in association with the bacterium. We have shown that PC-PLC maturation is regulated by Mpl and pH and that Mpl maturation occurs by autocatalysis. In this study, we tested the hypothesis that Mpl activity is pH regulated. To synchronize the effect of pH on bacteria, the cytosolic pH of infected cells was manipulated immediately after radiolabeling de novo-synthesized bacterial proteins. Immunoprecipitation of secreted Mpl from host cell lysates revealed the presence of the propeptide and catalytic domain in samples treated at pH 6.5 but not at pH 7.3. The zymogen was present in small amounts under all conditions. Since proteases often remain associated with their respective propeptide following autocatalysis, we aimed at determining whether pH regulates autocatalysis or secretion of the processed enzyme. For this purpose, we used an Mpl construct that contains a Flag tag at the N terminus of its catalytic domain and antibodies that can distinguish N-terminal and non-N-terminal Flag. By fluorescence microscopy, we observed the Mpl zymogen associated with the bacterium at physiological pH but not following acidification. Mature Mpl was not detected in association with the bacterium at either pH. Using purified proteins, we determined that processing of the PC-PLC propeptide by mature Mpl is also pH sensitive. These results indicate that pH regulates the activity of Mpl on itself and on PC-PLC.

Reuse of pacemakers and defibrillators in developing countries: logistical, legal, and ethical barriers and solutions.

In the wealthy nations of the world, access to implantable cardiac rhythm management devices is widespread. In many underserved low- and middle-income countries (LMIC), where cardiovascular disease is fast becoming a major public health problem, access is often limited. Reuse of pulse generators was practiced regularly in some European nations in the 1990s with good results. It is performed in LMIC, although the rates of device reuse are unknown. The available literature suggests there is no increased risk of morbidity or mortality with the reuse of devices. Donations of pacemaker and defibrillator pulse generators from developed nations constitute an important source of devices for the poor in LMIC. There are opportunities to increase this supply, but logistical barriers and legal and ethical concerns must be addressed. With proper sterilization, meticulous chains of custody, and advance directives for device handling (pacemaker/defibrillator living wills), patients in LMIC who would otherwise lack access to these devices could benefit from their reuse.

Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, an ancient family of proteins found in all phyla. In nearly all cases, ABC proteins are transporters that couple the hydrolysis of ATP to the transmembrane movement of substrate via an alternating access mechanism. In contrast, CFTR is best known for its activity as an ATP-dependent chloride channel. We asked why CFTR, which shares the domain architecture of ABC proteins that function as transporters, exhibits functional divergence. We compared CFTR protein sequences to those of other ABC transporters, which identified the ABCC4 proteins as the closest mammalian paralogs, and used statistical analysis of the CFTR-ABCC4 multiple sequence alignment to identify the specific domains and residues most likely to be involved in the evolutionary transition from transporter to channel activity. Among the residues identified as being involved in CFTR functional divergence, by virtue of being both CFTR-specific and conserved among all CFTR orthologs, was R352 in the sixth transmembrane helix (TM6). Patch-clamp experiments show that R352 interacts with D993 in TM9 to stabilize the open-channel state; D993 is absolutely conserved between CFTRs and ABCC4s. These data suggest that CFTR channel activity evolved, at least in part, by converting the conformational changes associated with binding and hydrolysis of ATP, as are found in true ABC Transporters, into an open permeation pathway by means of intraprotein interactions that stabilize the open state. This analysis sets the stage for understanding the evolutionary and functional relationships that make CFTR a unique ABC transporter protein.