Comparing ATPase activity of ATP-binding cassette subfamily C member 4, lamprey CFTR, and human CFTR using an antimony-phosphomolybdate assay.

Introduction: ATP-binding cassette (ABC) transporters use the hydrolysis of ATP to power the active transport of molecules, but paradoxically the cystic fibrosis transmembrane regulator (CFTR, ABCC7) forms an ion channel. We previously showed that ATP-binding cassette subfamily C member 4 (ABCC4) is the closest mammalian paralog to CFTR, compared to other ABC transporters. In addition, Lamprey CFTR (Lp-CFTR) is the oldest known CFTR ortholog and has unique structural and functional features compared to human CFTR (hCFTR). The availability of these evolutionarily distant orthologs gives us the opportunity to study the changes in ATPase activity that may be related to their disparate functions. Methods: We utilized the baculovirus expression system with Sf9 insect cells and made use of the highly sensitive antimony-phosphomolybdate assay for testing the ATPase activity of human ABCC4 (hABCC4), Lp-CFTR, and hCFTR under similar experimental conditions. This assay measures the production of inorganic phosphate (Pi) in the nanomolar range. Results: Crude plasma membranes were purified, and protein concentration, determined semi-quantitatively, of hABCC4, Lp-CFTR, and hCFTR ranged from 0.01 to 0.36 µg/µL. No significant difference in expression level was found although hABCC4 trended toward the highest level. hABCC4 was activated by ATP with the equilibrium constant (Kd) 0.55 ± 0.28 mM (n = 8). Estimated maximum ATPase rate (Vmax) for hABCC4 was about 0.2 nmol/µg/min when the protein was activated with 1 mM ATP at 37°C (n = 7). Estimated maximum ATPase rate for PKA-phosphorylated Lp-CFTR reached about half of hCFTR levels in the same conditions. Vmax for both Lp-CFTR and hCFTR were significantly increased in high PKA conditions compared to low PKA conditions. Maximum intrinsic ATPase rate of hABCC4 in the absence of substrate was twice that of hCFTR when activated in 1 mM ATP. Conclusion: The findings here suggest that while both ABCC4 and hCFTR bear one consensus and one degenerate ATPase site, the hCFTR exhibited a reduced intrinsic ATPase activity. In addition, ATPase activity in the CFTR lineage increased from Lp-CFTR to hCFTR. Finally, the studies pave the way to purify hABCC4, Lp-CFTR, and hCFTR from Sf9 cells for their structural investigation, including by cryo-EM, and for studies of evolution in the ABC transporter superfamily.

Mathematical models of cystic fibrosis as a systemic disease.

Cystic fibrosis (CF) is widely known as a disease of the lung, even though it is in truth a systemic disease, whose symptoms typically manifest in gastrointestinal dysfunction first. CF ultimately impairs not only the pancreas and intestine but also the lungs, gonads, liver, kidneys, bones, and the cardiovascular system. It is caused by one of several mutations in the gene of the epithelial ion channel protein CFTR. Intense research and improved antimicrobial treatments during the past eight decades have steadily increased the predicted life expectancy of a person with CF (pwCF) from a few weeks to over 50 years. Moreover, several drugs ameliorating the sequelae of the disease have become available in recent years, and notable treatments of the root cause of the disease have recently generated substantial improvements in health for some but not all pwCF. Yet, numerous fundamental questions remain unanswered. Complicating CF, for instance in the lung, is the fact that the associated insufficient chloride secretion typically perturbs the electrochemical balance across epithelia and, in the airways, leads to the accumulation of thick, viscous mucus and mucus plaques that cannot be cleared effectively and provide a rich breeding ground for a spectrum of bacterial and fungal communities. The subsequent infections often become chronic and respond poorly to antibiotic treatments, with outcomes sometimes only weakly correlated with the drug susceptibility of the target pathogen. Furthermore, in contrast to rapidly resolved acute infections with a single target pathogen, chronic infections commonly involve multi-species bacterial communities, called "infection microbiomes," that develop their own ecological and evolutionary dynamics. It is presently impossible to devise mathematical models of CF in its entirety, but it is feasible to design models for many of the distinct drivers of the disease. Building upon these growing yet isolated modeling efforts, we discuss in the following the feasibility of a multi-scale modeling framework, known as template-and-anchor modeling, that allows the gradual integration of refined sub-models with different granularity. The article first reviews the most important biomedical aspects of CF and subsequently describes mathematical modeling approaches that already exist or have the potential to deepen our understanding of the multitude aspects of the disease and their interrelationships. The conceptual ideas behind the approaches proposed here do not only pertain to CF but are translatable to other systemic diseases. This article is categorized under: Congenital Diseases > Computational Models.

A transistor model for the cystic fibrosis transmembrane conductance regulator.

In this paper we present a transistor circuit model for cystic fibrosis transmembrane conductance regulator (CFTR) that seeks to map the functional form of CFTR both in wild type and mutants. The circuit architecture is configured so that the function, and as much as possible the form, faithfully represents what is known about CFTR from cryo-electron microscopy and molecular dynamics. The model is a mixed analog-digital topology with an AND gate receiving the input from two separate ATP-nucleotide-binding domain binding events. The analog portion of the circuit takes the output from the AND gate as its input. The input to the circuit model and its noise characteristics are extracted from single-channel patch-clamp experiments. The chloride current predicted by the model is then compared with single-channel patch-clamp recordings for wild-type CFTR. We also consider the patch-clamp recordings from CFTR with a G551D point mutation, a clinically relevant mutant that is responsive to therapeutic management. Our circuit model approach enables bioengineering approaches to CFTR and allows biophysicists to use efficient circuit simulation tools to analyze its behavior.

Permissive and nonpermissive channel closings in CFTR revealed by a factor graph inference algorithm.

The closing of the gated ion channel in the cystic fibrosis transmembrane conductance regulator can be categorized as nonpermissive to reopening, which involves the unbinding of ADP or ATP, or permissive, which does not. Identifying the type of closing is of interest as interactions with nucleotides can be affected in mutants or by introducing agonists. However, all closings are electrically silent and difficult to differentiate. For single-channel patch-clamp traces, we show that the type of the closing can be accurately determined by an inference algorithm implemented on a factor graph, which we demonstrate using both simulated and lab-obtained patch-clamp traces.

Bacteriophage-Loaded Poly(lactic-co-glycolic acid) Microparticles Mitigate Staphylococcus aureus Infection and Cocultures of Staphylococcus aureus and Pseudomonas aeruginosa.

Lung infections caused by Gram-positive Staphylococcus aureus (S. aureus) and coinfections caused by S. aureus and Gram-negative Pseudomonas aeruginosa (P. aeruginosa) are challenging to treat, especially with the rise in the number of antibiotic-resistant strains of these pathogens. Bacteriophage (phage) are bacteria-specific viruses that can infect and lyse bacteria, providing a potentially effective therapy for bacterial infections. However, the development of bacteriophage therapy is impeded by limited suitable biomaterials that can facilitate effective delivery of phage to the lung. Here, the ability of porous microparticles engineered from poly(lactic-co-glycolic acid) (PLGA), a biodegradable polyester, to effectively deliver phage to the lung, is demonstrated. The phage-loaded microparticles (phage-MPs) display potent antimicrobial efficacy against various strains of S. aureus in vitro and in vivo, and arrest the growth of a clinical isolate of S. aureus in the presence of sputum supernatant obtained from cystic fibrosis patients. Moreover, phage-MPs efficiently mitigate in vitro cocultures of S. aureus and P. aeruginosa and display excellent cytocompatibility with human lung epithelial cells. Therefore, phage-MPs represents a promising therapy to treat bacterial lung infection.

The molecular evolution of function in the CFTR chloride channel.

The ATP-binding cassette (ABC) transporter superfamily includes many proteins of clinical relevance, with genes expressed in all domains of life. Although most members use the energy of ATP binding and hydrolysis to accomplish the active import or export of various substrates across membranes, the cystic fibrosis transmembrane conductance regulator (CFTR) is the only known animal ABC transporter that functions primarily as an ion channel. Defects in CFTR, which is closely related to ABCC subfamily members that bear function as bona fide transporters, underlie the lethal genetic disease cystic fibrosis. This article seeks to integrate structural, functional, and genomic data to begin to answer the critical question of how the function of CFTR evolved to exhibit regulated channel activity. We highlight several examples wherein preexisting features in ABCC transporters were functionally leveraged as is, or altered by molecular evolution, to ultimately support channel function. This includes features that may underlie (1) construction of an anionic channel pore from an anionic substrate transport pathway, (2) establishment and tuning of phosphoregulation, and (3) optimization of channel function by specialized ligand-channel interactions. We also discuss how divergence and conservation may help elucidate the pharmacology of important CFTR modulators.

Mechanistic analysis and significance of sphingomyelinase-mediated decreases in transepithelial CFTR currents in nHBEs.

Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). In the lungs, this manifests as immune cell infiltration and bacterial infections, leading to tissue destruction. Previous work has determined that acute bacterial sphingomyelinase (SMase) decreases CFTR function in bronchial epithelial cells from individuals without CF (nHBEs) and with CF (cfHBEs, homozygous ΔF508-CFTR mutation). This study focuses on exploring the mechanisms underlying this effect. SMase increased the abundance of dihydroceramides, a result mimicked by blockade of ceramidase enzyme using ceranib-1, which also decreased CFTR function. The SMase-mediated inhibitory mechanism did not involve the reduction of cellular CFTR abundance or removal of CFTR from the apical surface, nor did it involve the activation of 5' adenosine monophosphate-activated protein kinase. In order to determine the pathological relevance of these sphingolipid imbalances, we evaluated the sphingolipid profiles of cfHBEs and cfHNEs (nasal) as compared to non-CF controls. Sphingomyelins, ceramides, and dihydroceramides were largely increased in CF cells. Correction of ΔF508-CFTR trafficking with VX445 + VX661 decreased some sphingomyelins and all ceramides, but exacerbated increases in dihydroceramides. Additional treatment with the CFTR potentiator VX770 did not affect these changes, suggesting rescue of misfolded CFTR was sufficient. We furthermore determined that cfHBEs express more acid-SMase protein than nHBEs. Lastly, we determined that airway-like neutrophils, which are increased in the CF lung, secrete acid-SMase. Identifying the mechanism of SMase-mediated inhibition of CFTR will be important, given the imbalance of sphingolipids in CF cells and the secretion of acid-SMase from cell types relevant to CF.

Sphingomyelinase decreases transepithelial anion secretion in airway epithelial cells in part by inhibiting CFTR-mediated apical conductance.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel whose dysfunction causes cystic fibrosis (CF). The loss of CFTR function in pulmonary epithelial cells causes surface dehydration, mucus build-up, inflammation, and bacterial infections that lead to lung failure. Little has been done to evaluate the effects of lipid perturbation on CFTR activity, despite CFTR residing in the plasma membrane. This work focuses on the acute effects of sphingomyelinase (SMase), a bacterial virulence factor secreted by CF relevant airway bacteria which degrades sphingomyelin into ceramide and phosphocholine, on the electrical circuitry of pulmonary epithelial monolayers. We report that basolateral SMase decreases CFTR-mediated transepithelial anion secretion in both primary bronchial and tracheal epithelial cells from explant tissue, with current CFTR modulators unable to rescue this effect. Focusing on primary cells, we took a holistic ion homeostasis approach to determine a cause for reduced anion secretion following SMase treatment. Using impedance analysis, we determined that basolateral SMase inhibits apical and basolateral conductance in non-CF primary cells without affecting paracellular permeability. In CF primary airway cells, correction with clinically relevant CFTR modulators did not prevent SMase-mediated inhibition of CFTR currents. Furthermore, SMase was found to inhibit only apical conductance in these cells. Future work should determine the mechanism for SMase-mediated inhibition of CFTR currents, and further explore the clinical relevance of SMase and sphingolipid imbalances.

Alteration of Membrane Cholesterol Content Plays a Key Role in Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Channel Activity.

Altered cholesterol homeostasis in cystic fibrosis patients has been reported, although controversy remains. As a major membrane lipid component, cholesterol modulates the function of multiple ion channels by complicated mechanisms. However, whether cholesterol directly modulates cystic fibrosis transmembrane conductance regulator (CFTR) channel function remains unknown. To answer this question, we determined the effects of changing plasma membrane cholesterol levels on CFTR channel function utilizing polarized fischer rat thyroid (FRT) cells and primary human bronchial epithelial (HBE) cells. Treatment with methyl-ß-cyclodextrin (MßCD) significantly reduced total cholesterol content in FRT cells, which significantly decreased forskolin (FSK)-mediated activation of both wildtype (WT-) and P67L-CFTR. This effect was also seen in HBE cells expressing WT-CFTR. Cholesterol modification by cholesterol oxidase and cholesterol esterase also distinctly affected activation of CFTR by FSK. In addition, alteration of cholesterol increased the potency of VX-770, a clinically used potentiator of CFTR, when both WT- and P67L-CFTR channels were activated at low FSK concentrations; this likely reflects the apparent shift in the sensitivity of WT-CFTR to FSK after alteration of membrane cholesterol. These results demonstrate that changes in the plasma membrane cholesterol level significantly modulate CFTR channel function and consequently may affect sensitivity to clinical therapeutics in CF patients.

Electrophysiological Approaches for the Study of Ion Channel Function.

Ion channels play crucial roles in cell physiology, and are a major class of targets for clinically relevant pharmaceuticals. Because they carry ionic current, the function and pharmacology of ion channels can be studied using electrophysiological approaches that range in resolution from the single molecule to many millions of molecules. This chapter describes electrophysiological approaches for the study of one representative ion channel that is defective in a genetic disease, and that is the target of so-called highly effective modulator therapies now used in the clinic: the cystic fibrosis transmembrane conductance regulator (CFTR). Protocols are provided for studying CFTR expressed heterologously, for CFTR expressed in situ in airway epithelial cells, and for purified or partially purified CFTR protein reconstituted into planar lipid bilayers.

The bidirectional relationship between CFTR and lipids.

Cystic Fibrosis (CF) is the most common life-shortening genetic disease among Caucasians, resulting from mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). While work to understand this protein has resulted in new treatment strategies, it is important to emphasize that CFTR exists within a complex lipid bilayer - a concept largely overlooked when performing structural and functional studies. In this review we discuss cellular lipid imbalances in CF, mechanisms by which lipids affect membrane protein activity, and the specific impact of detergents and lipids on CFTR function.

Early Detection of Cystic Fibrosis Acute Pulmonary Exacerbations by Exhaled Breath Condensate Metabolomics.

The most common cause of death in cystic fibrosis (CF) patients is progressive lung function decline, which is punctuated by acute pulmonary exacerbations (APEs). A major challenge is to discover biomarkers for detecting an oncoming APE and allow for pre-emptive clinical interventions. Metabolic profiling of exhaled breath condensate (EBC) samples collected from CF patients before, during, and after APEs and under stable conditions (n = 210) was performed using ultraperformance liquid chromatography (UPLC) coupled to Orbitrap mass spectrometry (MS). Negative ion mode MS data showed that classification between metabolic profiles from "pre-APE" (pending APE before the CF patient had any signs of illness) and stable CF samples was possible with good sensitivities (85.7 and 89.5%), specificities (88.4 and 84.1%), and accuracies (87.7 and 85.7%) for pediatric and adult patients, respectively. Improved classification performance was achieved by combining positive with negative ion mode data. Discriminant metabolites included two potential biomarkers identified in a previous pilot study: lactic acid and 4-hydroxycyclohexylcarboxylic acid. Some of the discriminant metabolites had microbial origins, indicating a possible role of bacterial metabolism in APE progression. The results show promise for detecting an oncoming APE using EBC metabolites, thus permitting early intervention to abort such an event.

An Ancient CFTR Ortholog Informs Molecular Evolution in ABC Transporters.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel central to the development of secretory diarrhea and cystic fibrosis. The oldest CFTR ortholog identified is from dogfish shark, which retains similar structural and functional characteristics to the mammalian protein, thereby highlighting CFTR's critical role in regulating epithelial ion transport in vertebrates. However, the identification of an early CFTR ortholog with altered structure or function would provide critical insight into the evolution of epithelial anion transport. Here, we describe the earliest known CFTR, expressed in sea lamprey (Petromyzon marinus), with unique structural features, altered kinetics of activation and sensitivity to inhibition, and altered single-channel conductance compared to human CFTR. Our data provide the earliest evolutionary evidence of CFTR, offering insight regarding changes in gene and protein structure that underpin evolution from transporter to anion channel. Importantly, these data provide a unique platform to enhance our understanding of vertebrate phylogeny over a critical period of evolutionary expansion.

VX-770-mediated potentiation of numerous human CFTR disease mutants is influenced by phosphorylation level.

VX-770 (ivacaftor) is approved for clinical use in CF patients bearing multiple CFTR mutations. VX-770 potentiated wildtype CFTR and several disease mutants expressed in oocytes in a manner modulated by PKA-mediated phosphorylation. Potentiation of some other mutants, including G551D-CFTR, was less dependent upon the level of phosphorylation, likely related to the severe gating defects in these mutants exhibited in part by a shift in PKA sensitivity to activation, possibly due to an electrostatic interaction of D551 with K1250. Phosphorylation-dependent potentiation of wildtype CFTR and other variants also was observed in epithelial cells. Hence, the efficacy of potentiators may be obscured by a ceiling effect when drug screening is performed under strongly phosphorylating conditions. These results should be considered in campaigns for CFTR potentiator discovery, and may enable the expansion of VX-770 to CF patients bearing ultra-orphan CFTR mutations.

ATP-Dependent Signaling in Simulations of a Revised Model of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily that has uniquely evolved to function as a chloride channel. It binds and hydrolyzes ATP at its nucleotide binding domains to form a pore providing a diffusive pathway within its transmembrane domains. CFTR is the only known protein from the ABC superfamily with channel activity, and its dysfunction causes the disease cystic fibrosis. While much is known about the functional aspects of CFTR, significant gaps remain, such as the structure-function relationship underlying signaling of ATP binding. In the present work, we refined an existing homology model using an intermediate-resolution (9 Å) published cryo-electron microscopy map. The newly derived models have been simulated in equilibrium molecular dynamics simulations for a total of 2.5 µs in multiple ATP-occupancy states. Putative conformational movements connecting ATP binding with pore formation are elucidated and quantified. Additionally, new interdomain interactions between E543, K968, and K1292 have been identified and confirmed experimentally; these interactions may be relevant for signaling ATP binding and hydrolysis to the transmembrane domains and induction of pore opening.

Zebrafish aversive taste co-receptor is expressed in both chemo- and mechanosensory cells and plays a role in lateral line development.

Fishes rely on both chemical and tactile senses to orient themselves to avoid predators, and to detect and taste food. This is likely achieved by highly coordinated reception of signals by mechano- and chemosensory receptors in fish. A small co-receptor from zebrafish, receptor activity modifying protein (RAMP)-like triterpene glycoside receptor (RL-TGR), was previously found to be involved in recognition of triterpene glycosides, a family of naturally occurring compounds that act as chemical defenses in various prey species. However, its localization, function, and how it impacts sensory organ development in vivo is not known. Here we show that RL-TGR is expressed in zebrafish in both i) apical microvilli of the chemosensory cells of taste buds including the epithelium of lips and olfactory epithelium, and ii) mechanosensory cells of neuromasts belonging to the lateral line system. Loss-of-function analyses of RL-TGR resulted in significantly decreased number of neuromasts in the posterior lateral line system and decreased body length, suggesting that RL-TGR is involved in deposition and migration of the neuromasts. Collectively, these results provide the first in vivo genetic evidence of sensory cell-specific expression of this unusual co-receptor and reveal its additional role in the lateral line development in zebrafish.

Molecular Dynamics Flexible Fitting Simulations Identify New Models of the Closed State of the Cystic Fibrosis Transmembrane Conductance Regulator Protein.

Cystic fibrosis (CF) is a lethal, genetic disease found in particular in humans of European origin which is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The search for CF therapies acting by modulating the impaired function of mutant CFTR will be greatly advanced by high resolution structures of CFTR in different states. To date, two medium resolution electron microscopy (EM) structures of CFTR are available (one of a distant zebrafish (Danio rerio) CFTR ortholog and one of human CFTR). The two models are nearly identical to one another, and both correspond to the inward-facing, nucleotide binding domains (NBDs) separated, closed state of the channel. In addition, lower resolution structural data are available for human CFTR in an alternative conformation which likely features associated NBDs and thus geometrically resembles the conducting state of the channel. Multiple homology models of human CFTR in multiple states have been developed over the years, yet their correspondence to the existing structural information is unexplored. In this work we use molecular dynamics flexible fitting (MDFF) simulations to refine two previously described CFTR models based on the available cryo-EM map of the human protein. This map was recorded in the absence of ATP and consequently represents closed-state CFTR yet its features likely correspond to an NBD associated conformation of the protein. Accordingly, the resulting models feature dimerized NBDs yet with no membrane traversing pore. Moreover, the open probability of the new models as deduced from the MDFF trajectories is significantly lower than that deduced from control MD trajectories initiated from the starting models. We propose that the new models correspond to a CFTR conformation which to date was largely unexplored yet is one that is relevant to the gating cycle of the protein. In particular this conformation may participate in rapid channel opening and closing through small allosteric movements controlled by nucleotide binding and dissociation events. Analyzing the resulting trajectories (and not only the final models as is usually the case), we demonstrate that the refined models have good stereochemical properties and are also in favorable agreement with multiple experimental data. Moreover, despite different starting points, the final models share many common features. Finally, we propose that the combination of high resolution cryo-EM maps, which are currently emerging from multiple laboratories, and MDFF simulations will be of value for the development of yet more reliable CFTR models as well as for the identification of binding sites for CFTR modulators.

Comparison of Ambient and Atmospheric Pressure Ion Sources for Cystic Fibrosis Exhaled Breath Condensate Ion Mobility-Mass Spectrometry Metabolomics.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The vast majority of the mortality is due to progressive lung disease. Targeted and untargeted CF breath metabolomics investigations via exhaled breath condensate (EBC) analyses have the potential to expose metabolic alterations associated with CF pathology and aid in assessing the effectiveness of CF therapies. Here, transmission-mode direct analysis in real time traveling wave ion mobility spectrometry time-of-flight mass spectrometry (TM-DART-TWIMS-TOF MS) was tested as a high-throughput alternative to conventional direct infusion (DI) electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) methods, and a critical comparison of the three ionization methods was conducted. EBC was chosen as the noninvasive surrogate for airway sampling over expectorated sputum as EBC can be collected in all CF subjects regardless of age and lung disease severity. When using pooled EBC collected from a healthy control, ESI detected the most metabolites, APCI a log order less, and TM-DART the least. TM-DART-TWIMS-TOF MS was used to profile metabolites in EBC samples from five healthy controls and four CF patients, finding that a panel of three discriminant EBC metabolites, some of which had been previously detected by other methods, differentiated these two classes with excellent cross-validated accuracy. Graphical Abstract ᅟ.

Feasibility of Early Detection of Cystic Fibrosis Acute Pulmonary Exacerbations by Exhaled Breath Condensate Metabolomics: A Pilot Study.

Progressive lung function decline and, ultimately, respiratory failure are the most common cause of death in patients with cystic fibrosis (CF). This decline is punctuated by acute pulmonary exacerbations (APEs), and in many cases, there is a failure to return to baseline lung function. Ultraperformance liquid chromatography quadrupole-time-of-flight mass spectrometry was used to profile metabolites in exhaled breath condensate (EBC) samples from 17 clinically stable CF patients, 9 CF patients with an APE severe enough to require hospitalization (termed APE), 5 CF patients during recovery from a severe APE (termed post-APE), and 4 CF patients who were clinically stable at the time of collection but in the subsequent 1-3 months developed a severe APE (termed pre-APE). A panel containing two metabolic discriminant features, 4-hydroxycyclohexylcarboxylic acid and pyroglutamic acid, differentiated the APE samples from the stable CF samples with 84.6% accuracy. Pre-APE samples were distinguished from stable CF samples by lactic acid and pyroglutamic acid with 90.5% accuracy and in general matched the APE signature when projected onto the APE vs stable CF model. Post-APE samples were on average more similar to stable CF samples in terms of their metabolomic signature. These results show the feasibility of detecting and predicting an oncoming APE or monitoring APE treatment using EBC metabolites.