Detection and Degradation of Stalled Nascent Chains via Ribosome-Associated Quality Control.

Stalled protein synthesis produces defective nascent chains that can harm cells. In response, cells degrade these nascent chains via a process called ribosome-associated quality control (RQC). Here, we review the irregularities in the translation process that cause ribosomes to stall as well as how cells use RQC to detect stalled ribosomes, ubiquitylate their tethered nascent chains, and deliver the ubiquitylated nascent chains to the proteasome. We additionally summarize how cells respond to RQC failure.

A new way of D/Ealing with protein misfolding.

Huang et al. (2021) show that proteins containing aspartate- and glutamate-rich stretches represent a putative new class of ATP-independent molecular chaperones that operate on diverse client proteins in vitro and protect bona fide interactors against aggregation in cells.

Mechanisms of gelofusine protection in an in vitro model of polymyxin B-associated renal injury.

Polymyxins are a last-resort treatment option for multidrug-resistant gram-negative bacterial infections, but they are associated with nephrotoxicity. Gelofusine was previously shown to reduce polymyxin-associated kidney injury in an animal model. However, the mechanism(s) of renal protection has not been fully elucidated. Here, we report the use of a cell culture model to provide insights into the mechanisms of renal protection. Murine epithelial proximal tubular cells were exposed to polymyxin B. Cell viability, lactate dehydrogenase (LDH) release, polymyxin B uptake, mitochondrial superoxide production, nuclear morphology, and apoptosis activation were evaluated with or without concomitant gelofusine. A megalin knockout cell line was used as an uptake inhibition control. Methionine was included in selected experiments as an antioxidant control. A polymyxin B concentration-dependent reduction in cell viability was observed. Increased viability was observed in megalin knockout cells following comparable polymyxin B exposures. Compared with polymyxin B exposure alone, concomitant gelofusine significantly increased cell viability as well as reduced LDH release, polymyxin B uptake, mitochondrial superoxide, and apoptosis. Gelofusine and methionine were more effective at reducing renal cell injury in combination than either agent alone. In conclusion, the mechanisms of renal protection by gelofusine involve decreasing cellular drug uptake, reducing subsequent oxidative stress and apoptosis activation. These findings would be valuable for translational research into clinical strategies to attenuate drug-associated acute kidney injury.NEW & NOTEWORTHY Gelofusine is a gelatinous saline solution with the potential to attenuate polymyxin-associated nephrotoxicity. We demonstrated that the mechanisms of gelofusine renal protection involve reducing polymyxin B uptake by proximal tubule cells, limiting subsequent oxidative stress and apoptosis activation. In addition, gelofusine was more effective at reducing cellular injury than a known antioxidant control, methionine, and a megalin knockout cell line, indicating that gelofusine likely has additional pharmacological properties besides only megalin inhibition.

A Minimal Nontrivial Example of Higher Order Evidence.

Higher order evidence (evidence about evidence) allows epidemiologists and other health data scientists to account for measurement error in validation data. Here, to illustrate the use of higher order evidence, we provide a minimal nontrivial example of estimating the proportion and show how higher order evidence can be used to construct sensitivity analyses. The proposed method provides a flexible approach to account for multiple levels of distortion in the results of epidemiologic studies.

Getting Precise about Gender and Sex Measurement: A Primer for Epidemiologists.

Accurately measuring gender and sex is crucial in public health and epidemiology. Iteratively reexamining how variables-including gender and sex-are conceptualized and operationalized is necessary to achieve impactful research. Reexamining gender and sex advances epidemiology toward its goals of health promotion and disease elimination. While we cannot reduce the complexities of sex and gender to simply an issue of measurement, striving to capture these concepts and experiences accurately must be an ongoing dialogue and practice-to the benefit of the field and population health. We assert that epidemiology must counteract misconceptions and accurately measure gender and sex in epidemiology. We aim to summarize existing critiques and guiding principles in measuring gender and sex that can be applied in practice.

Systemic Arterial Oxygen Levels Differentiate Pre- and Post-capillary Predominant Hemodynamic Abnormalities During Exercise in Undifferentiated Dyspnea on Exertion.

BACKGROUND: Whether systemic oxygen levels (SaO2) during exercise can provide a window into invasively derived exercise hemodynamic profiles in patients with undifferentiated dyspnea on exertion is unknown. METHODS: We performed cardiopulmonary exercise testing with invasive hemodynamic monitoring and arterial blood gas sampling in individuals referred for dyspnea on exertion. Receiver operator analysis was performed to distinguish heart failure with preserved ejection fraction from pulmonary arterial hypertension. RESULTS: Among 253 patients (mean ± SD, age 63 ± 14 years, 55% female, arterial O2 [PaO2] 87 ± 14 mmHg, SaO2 96% ± 4%, resting pulmonary capillary wedge pressure [PCWP] 18 ± 4mmHg, and pulmonary vascular resistance [PVR] 2.7 ± 1.2 Wood units), there was no exercise PCWP threshold, measured up to 49 mmHg, above which hypoxemia was consistently observed. Exercise PaO2 was not correlated with exercise PCWP (rho = 0.04; P = 0.51) but did relate to exercise PVR (rho = -0.46; P < 0.001). Exercise PaO2 and SaO2 levels distinguished left-heart-predominant dysfunction from pulmonary-vascular-predominant dysfunction with an area under the curve of 0.89 and 0.89, respectively. CONCLUSION: Systemic O2 levels during exercise distinguish relative pre- and post-capillary pulmonary hemodynamic abnormalities in patients with undifferentiated dyspnea. Hypoxemia during upright exercise should not be attributed to isolated elevation in left heart filling pressures and should prompt consideration of pulmonary vascular dysfunction.

Investigating the Validity of the MMPI-3 Eating Concerns (EAT) scale across Men and Women in a University Sample.

Disordered eating is a major health epidemic that occurs at disproportionate rates among young adults and for which gender plays a major role in symptom presentation. Broadband psychological instruments have historically not included disordered eating as a core scale construct. The recent release of the Minnesota Multiphasic Personality Inventory-3 (MMPI-3) offers an opportunity to address this shortcoming through the newly developed Eating Concerns Scale (EAT) for which the existing literature is promising but limited. This study expands research on EAT by investigating its validity and comparing findings across gender. In 345 college students (102 men, 243 women), we examined gender differences between men and women in the EAT scale's structure, item endorsement rates, mean scores, and correlations with measures of body image and eating pathology. Differences emerged in item endorsement rate, scale score elevation rate, and correlation magnitudes. Broadly, findings further support EAT's use in detecting eating pathology and highlight ways in which the EAT scale may not effectively capture masculine expressions of eating pathology, namely binging and purging behaviors. To assess eating pathology more comprehensively, clinicians and researchers should consider including assessments of eating pathology inclusive of masculine eating patterns. Limitations and future research directions are also discussed.

Assessment of PTSD and Trauma Symptoms With the MMPI-3 in College Students: Validity and Incremental Utility of the Anxiety Related Experiences (ARX) Scale.

Post Traumatic Stress Disorder (PTSD) is heterogeneous in nature, which complicates diagnostic efforts and makes accurate assessment tools critical. The MMPI family of instruments are widely used broadband measures of psychopathology, including trauma symptomology. The MMPI-3's Anxiety Related Experiences scale (ARX) is an expansion of the MMPI-2-RF Anxiety (AXY) scale which has historically represented the MMPI family's best measure of trauma symptoms. This study expands research on ARX in 2 samples of college students (n = 332 [PCL-5 Criterion] & n = 58 [CAPS-5 Criterion]) by examining ARX's incremental, criterion, and classification validity. ARX incrementally predicted PCL-5 total and cluster scores beyond that accounted for by AXY (R2Δ = .01-.09). ARX accounted for the most unique variance, beyond RCd and RC7, in CAPS-5 interview ratings of intrusion symptoms (R2Δ = .16). ARX was strongly related to trauma symptomology broadly (r = .42-.58) and demonstrated strong screening ability at T65 (sensitivity = .37-.40; specificity = .91-.92) and stronger diagnostic screening at T75 (sensitivity = .31; specificity = .93). We discuss clinical considerations when using ARX for assessing PTSD.

Determining the MMPI-3 SUI scale's cross-sectional and prospective utility in suicide risk assessment.

OBJECTIVE: In-depth suicide risk assessments are particularly important to long-term suicide prevention. Broadband measures of psychopathology, such as the Minnesota Multiphasic Personality Inventory (MMPI) instruments, assess suicide risk factors and various mental health comorbidities. With the recent release of the MMPI-3, the Suicidal/Death Ideation (SUI) scale underwent revisions to improve its construct validity and detection of suicide risk factors. Thus, we hypothesized the MMPI-3 SUI scale would demonstrate medium to large associations with suicidal experience and behaviors, future ideation, and interpersonal risk factors of suicide. METHODS: A sample of 124 college students screened for elevated depressive symptoms completed a brief longitudinal study. Participants completed a baseline session including the MMPI-3 and criterion measures and three brief follow-ups every 2 weeks. RESULTS: SUI scores were most robustly associated with increased risk for past suicidal ideation, planning, and perceived burdensomeness. Prospectively assessed suicidal ideation was also meaningfully associated with SUI. SUI scale elevations indicate an increased risk of suicide-related risk factors. CONCLUSION: The MMPI-3 is a valuable tool to inform long-term suicide prevention for those experiencing elevated depressive symptoms as the SUI scale can assess past, current, and future suicide-related risk factors, including suicidal ideation and behaviors.

Cross validation of the Personality Assessment Inventory (PAI) Cognitive Bias Scale of Scales (CB-SOS) over-reporting indicators in a military sample.

Following the development of the Cognitive Bias Scale (CBS), three other cognitive over-reporting indicators were created. This study cross-validates these new Cognitive Bias Scale of Scales (CB-SOS) measurements in a military sample and contrasts their performance to the CBS. We analyzed data from 288 active-duty soldiers who underwent neuropsychological evaluation. Groups were established based on performance validity testing (PVT) failure. Medium effects (d = .71 to .74) were observed between those passing and failing PVTs. The CB-SOS scales have high specificity (≥.90) but low sensitivity across the suggested cut scores. While all CB-SOS were able to achieve .90, lower scores were typically needed. CBS demonstrated incremental validity beyond CB-SOS-1 and CB-SOS-3; only CB-SOS-2 was incremental beyond CBS. In a military sample, the CB-SOS scales have more limited sensitivity than in its original validation, indicating an area of limited utility despite easier calculation. The CBS performs comparably, if not better, than CB-SOS scales. CB-SOS-2's differences in performance in this study and its initial validation suggest that its psychometric properties may be sample dependent. Given their ease of calculation and relatively high specificity, our study supports the interpretation of elevated CB-SOS scores indicating those who are likely to fail concurrent PVTs.

Behavioral and transcriptomic analyses of mecp2 function in zebrafish.

Rett syndrome (RTT), a human neurodevelopmental disorder characterized by severe cognitive and motor impairments, is caused by dysfunction of the conserved transcriptional regulator Methyl-CpG-binding protein 2 (MECP2). Genetic analyses in mouse Mecp2 mutants, which exhibit key features of human RTT, have been essential for deciphering the mechanisms of MeCP2 function; nonetheless, our understanding of these complex mechanisms is incomplete. Zebrafish mecp2 mutants exhibit mild behavioral deficits but have not been analyzed in depth. Here, we combine transcriptomic and behavioral assays to assess baseline and stimulus-evoked motor responses and sensory filtering in zebrafish mecp2 mutants from 5 to 7 days post-fertilization (dpf). We show that zebrafish mecp2 function is required for normal thigmotaxis but is dispensable for gross movement, acoustic startle response, and sensory filtering (habituation and sensorimotor gating), and reveal a previously unknown role for mecp2 in behavioral responses to visual stimuli. RNA-seq analysis identified a large gene set that requires mecp2 function for correct transcription at 4 dpf, and pathway analysis revealed several pathways that require MeCP2 function in both zebrafish and mammals. These findings show that MeCP2's function as a transcriptional regulator is conserved across vertebrates and supports using zebrafish to complement mouse modeling in elucidating these conserved mechanisms.

Zileuton ameliorates aminoglycoside and polymyxin-associated acute kidney injury in an animal model.

OBJECTIVES: Aminoglycosides and polymyxins are antibiotics with in vitro activity against MDR Gram-negative bacteria. However, their clinical use is hindered by dose-limiting nephrotoxicity. The objective of this project was to determine if zileuton can reduce nephrotoxicity associated with amikacin and polymyxin B in a rat model of acute kidney injury. METHODS: Sprague Dawley rats (n = 10, both genders) were administered either amikacin (300 mg/kg) or polymyxin B (20 mg/kg) daily for 10 days. Zileuton (4 and 10 mg/kg) was delivered intraperitoneally 15 min before antibiotic administration. Blood samples were collected at baseline and daily to determine serum creatinine concentration. Nephrotoxicity was defined as a ≥2× elevation of baseline serum creatinine. Time-to-event analysis and log rank test were used to compare the onset of nephrotoxicity in different cohorts. Histopathological analysis was also conducted to characterize the extent of kidney injury. RESULTS: Animals receiving amikacin or polymyxin B alone had nephrotoxicity rates of 90% and 100%, respectively. The overall rate was reduced to 30% in animals receiving adjuvant zileuton. The onset of nephrotoxicity associated with amikacin and polymyxin B was also significantly delayed by zileuton at 4 and 10 mg/kg, respectively. Histopathology confirmed reduced kidney injury in animals receiving amikacin concomitant with zileuton. CONCLUSIONS: Our pilot data suggest that zileuton has the potential to attenuate nephrotoxicity associated with last-line antibiotics. This would allow these antibiotics to treat MDR Gram-negative bacterial infections optimally without dose-limiting constraints. Further studies are warranted to optimize drug delivery and dosing in humans.

Accuracy of portable ultrasound for obstetric biometry.

OBJECTIVE: We assessed the accuracy of two portable ultrasound machines (PUM) in obtaining fetal biometry and estimating gestational age. METHODS: We analyzed data from the Fetal Age Machine Learning Initiative, an observational study of pregnant women in the United States and Zambia. Each participant underwent assessment by an experienced sonographer using both a high-specification ultrasound machine (HSUM) and a PUM (either Butterfly iQ or Clarius C3) to measure fetal biometry and calculate estimated gestational age (EGA) at each visit. Through comparison of paired PUM-HSUM scans, we estimated agreement between individual biometry measurements and aggregate gestational age estimates by reporting mean difference, along with intraclass correlation coefficient (ICC) and Bland-Altman plots, adjusting for trend. RESULTS: 881 participants contributed 1386 paired PUM-HSUM ultrasound studies between April and December 2021. PUM studies included 991 Butterfly and 395 Clarius. Gestational age at scan ranged from 7 to 38 weeks. Compared to HSUM, the Butterfly PUM had a mean difference of -0.20 days (95%CI±0.40) in the 1st trimester and -0.68 days (95%CI±0.68) in the 2nd/3rd trimesters. Also compared to HSUM, the Clarius PUM had a mean difference of 0.47 days (95%CI±0.64) in the 1st trimester and -1.67 days (95%CI±0.43) in the 2nd/3rd trimesters. ICCs were 0.989 or greater throughout. Increasing gestational age was associated with increasing error and absolute error. Both PUM devices demonstrated a modest trend toward underestimation of EGA at advancing gestational ages in 2nd/3rd trimester scans, compared to HSUM. CONCLUSION: Both the Butterfly iQ and Clarius C3 PUM devices were highly accurate in performing fetal biometry in a diverse population from the US and Zambia. This article is protected by copyright. All rights reserved.

Optimal ceftazidime/avibactam dosing exposure against KPC-producing Klebsiella pneumoniae.

OBJECTIVES: Infections due to carbapenem-resistant Enterobacterales are considered urgent public health threats and often treated with a ß-lactam/ß-lactamase inhibitor combination. However, clinical treatment failure and resistance emergence have been attributed to inadequate dosing. We used a novel framework to provide insights of optimal dosing exposure of ceftazidime/avibactam. METHODS: Seven clinical isolates of Klebsiella pneumoniae producing different KPC variants were examined. Ceftazidime susceptibility (MIC) was determined by broth dilution using escalating concentrations of avibactam. The observed MICs were characterized as response to avibactam concentrations using an inhibitory sigmoid Emax model. Using the best-fit parameter values, %fT>MICi was estimated for various dosing regimens of ceftazidime/avibactam. A hollow-fibre infection model (HFIM) was subsequently used to ascertain the effectiveness of selected regimens over 120 h. The drug exposure threshold associated with bacterial suppression was identified by recursive partitioning. RESULTS: In all scenarios, ceftazidime MIC reductions were well characterized with increasing avibactam concentrations. In HFIM, bacterial regrowth over time correlated with emergence of resistance. Overall, suppression of bacterial regrowth was associated with %fT>MICi ≥ 76.1% (100% versus 18.2%; P < 0.001). Using our framework, the optimal drug exposure could be achieved with ceftazidime/avibactam 2.5 g every 12 h in 5 out of 7 isolates. Furthermore, ceftazidime/avibactam 2.5 g every 8 h can suppress an isolate deemed resistant based on conventional susceptibility testing method. CONCLUSIONS: An optimal drug exposure to suppress KPC-producing bacteria was identified. The novel framework is informative and may be used to guide optimal dosing of other ß-lactam/ß-lactamase inhibitor combinations. Further in vivo investigations are warranted.

Adverse childhood experiences and transcriptional response in school-age children.

This study evaluated whether children with higher adverse childhood experiences (ACE) scores had alterations in immune cell gene expression profiles. RNA sequencing was conducted on dried blood spot samples from 37 generally healthy English-speaking children (age 5-11) who were recruited from well-child visits at a university-affiliated pediatric practice. The Whole Child Assessment was used to assess ACE exposure. Primary analyses examined an a priori-specified composite of 19 pro-inflammatory gene transcripts. Secondary analyses examined a 34-gene composite assessing Type I interferon response, and used Transcript Origin Analyses to identify cellular mechanisms. After controlling for age, body mass index percentile, sex, race/ethnicity, current insurance status, and household smoking exposure, pro-inflammatory gene expression was elevated by 0.094 log2 RNA expression units with each Child-ACE total score point (p = .019). Type I interferon gene expression was similarly upregulated (0.103; p = .008). Transcript origin analyses implicated CD8+ T cell as the primary sources of gene transcripts upregulated, and nonclassical (CD16+) monocytes as sources of downregulated transcripts. These preliminary analyses suggest that parent-reported ACE exposures are associated with increased expression of both inflammatory and interferon gene transcripts in children's circulating blood cells.

The Influence of Service Era: Comparing Personality Assessment Inventory (PAI) Scale Scores Within a Posttraumatic Stress Disorder Treatment Clinic (PCT).

Research is mixed on the role of service era in symptom endorsement among Veterans, with differences emerging depending on the instrument evaluated. This study compares Personality Assessment Inventory (PAI) scale scores of VA test-takers who served during the Vietnam, Desert Storm, or Post-9/11 service eras. The sample was collected at a VA Posttraumatic Stress Disorder Clinical Team. Associations between gender and combat exposure were also examined as covariates. Results suggest that Veterans' self-report on the PAI is influenced by service era, even after accounting for gender and combat exposure during deployment. The largest differences were between Vietnam or Post-9/11 Veterans and those from the Gulf War era. Symptom differences typically varied across scales commonly associated with symptoms of trauma exposure/posttraumatic stress disorder. Implications for the clinical use of, and research with, the PAI and other broadband personality assessments within the VA healthcare system and trauma treatment settings are discussed.

Deja Vu: semantics-aware recording and replay of high-speed eye tracking and interaction data to support cognitive studies of software engineering tasks-methodology and analyses.

The paper introduces a fundamental technological problem with collecting high-speed eye tracking data while studying software engineering tasks in an integrated development environment. The use of eye trackers is quickly becoming an important means to study software developers and how they comprehend source code and locate bugs. High quality eye trackers can record upwards of 120 to 300 gaze points per second. However, it is not always possible to map each of these points to a line and column position in a source code file (in the presence of scrolling and file switching) in real time at data rates over 60 gaze points per second without data loss. Unfortunately, higher data rates are more desirable as they allow for finer granularity and more accurate study analyses. To alleviate this technological problem, a novel method for eye tracking data collection is presented. Instead of performing gaze analysis in real time, all telemetry (keystrokes, mouse movements, and eye tracker output) data during a study is recorded as it happens. Sessions are then replayed at a much slower speed allowing for ample time to map gaze point positions to the appropriate file, line, and column to perform additional analysis. A description of the method and corresponding tool, Deja Vu, is presented. An evaluation of the method and tool is conducted using three different eye trackers running at four different speeds (60 Hz, 120 Hz, 150 Hz, and 300 Hz). This timing evaluation is performed in Visual Studio, Eclipse, and Atom IDEs. Results show that Deja Vu can playback 100% of the data recordings, correctly mapping the gaze to corresponding elements, making it a well-founded and suitable post processing step for future eye tracking studies in software engineering. Finally, a proof of concept replication analysis of four tasks from two previous studies is performed. Due to using the Deja Vu approach, this replication resulted in richer collected data and improved on the number of distinct syntactic categories that gaze was mapped on in the code.

Holstein Friesian dairy cattle edited for diluted coat color as a potential adaptation to climate change.

BACKGROUND: High-producing Holstein Friesian dairy cattle have a characteristic black and white coat, often with large proportions of black. Compared to a light coat color, black absorbs more solar radiation which is a contributing factor to heat stress in cattle. To better adapt dairy cattle to rapidly warming climates, we aimed to lighten their coat color by genome editing. RESULTS: Using gRNA/Cas9-mediated editing, we introduced a three bp deletion in the pre-melanosomal protein 17 gene (PMEL) proposed as causative variant for the semi-dominant color dilution phenotype observed in Galloway and Highland cattle. Calves generated from cells with homozygous edits revealed a strong color dilution effect. Instead of the characteristic black and white markings of control calves generated from unedited cells, the edited calves displayed a novel grey and white coat pattern. CONCLUSION: This, for the first time, verified the causative nature of the PMEL mutation for diluting the black coat color in cattle. Although only one of the calves was healthy at birth and later succumbed to a naval infection, the study showed the feasibility of generating such edited animals with the possibility to dissect the effects of the introgressed edit and other interfering allelic variants that might exist in individual cattle and accurately determine the impact of only the three bp change.

Functions of spontaneous and voluntary future thinking: evidence from subjective ratings.

Future thinking is defined as the ability to withdraw from reality and mentally project oneself into the future. The primary aim of the present study was to examine whether functions of future thoughts differed depending on their mode of elicitation (spontaneous or voluntary) and an attribute of goal-relatedness (selected-goal-related or selected-goal-unrelated). After producing spontaneous and voluntary future thoughts in a laboratory paradigm, participants provided ratings on four proposed functions of future thinking (self, directive, social, and emotional regulation). Findings showed that spontaneous and voluntary future thoughts were rated similarly on all functions except the directive function, which was particularly relevant to spontaneous future thoughts. Future thoughts classed as goal-related (selected-goal-related) were rated higher across all functions, and there was largely no interaction between mode of elicitation and goal-relatedness. A higher proportion of spontaneous future thoughts were selected-goal-related compared with voluntary future thoughts. In general, these results indicate that future thinking has significant roles across affective, behavioural, self and social functions, and supports theoretical views that implicate spontaneous future thought in goal-directed cognition and behaviour.

Differential Clinical Profiles, Exercise Responses, and Outcomes Associated With Existing HFpEF Definitions.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is common, yet there is currently no consensus on how to define HFpEF according to various society and clinical trial criteria. How clinical and hemodynamic profiles of patients vary across definitions is unclear. We sought to determine clinical characteristics, as well as physiologic and prognostic implications of applying various criteria to define HFpEF. METHODS: We examined consecutive patients with chronic exertional dyspnea (New York Heart Association class II to IV) and ejection fraction ≥50% referred for comprehensive cardiopulmonary exercise testing with invasive hemodynamic monitoring. We applied societal and clinical trial HFpEF definitions and compared clinical profiles, exercise responses, and cardiovascular outcomes. RESULTS: Of 461 patients (age 58±15 years, 62% women), 416 met American College of Cardiology/American Heart Association (ACC/AHA), 205 met European Society of Cardiology (ESC), and 55 met Heart Failure Society of America (HFSA) criteria for HFpEF. Clinical profiles and exercise capacity varied across definitions, with peak oxygen uptake of 16.2±5.2 (ACC/AHA), 14.1±4.2 (ESC), and 12.7±3.1 mL·kg-1·min-1 (HFSA). A total of 243 patients had hemodynamic evidence of HFpEF (abnormal rest or exercise filling pressures), of whom 222 met ACC/AHA, 161 met ESC, and 41 met HFSA criteria. Over a mean follow-up of 3.8 years, the incidence of cardiovascular outcomes ranged from 75 (ACC/AHA) to 298 events per 1000 person-years (HFSA). Application of clinical trial definitions of HFpEF similarly resulted in distinct patient classification and prognostication. CONCLUSIONS: Use of different HFpEF classifications variably enriches for future cardiovascular events, but at the expense of not including up to 85% of individuals with physiologic evidence of HFpEF. Comprehensive phenotyping of patients with suspected heart failure highlights the limitations and heterogeneity of current HFpEF definitions and may help to refine HFpEF subgrouping to test therapeutic interventions.