Advances in proteomics methods for the analysis of exhaled breath condensate.

The analysis of exhaled breath condensate (EBC) demonstrates a promising avenue of minimally invasive biopsies for diagnostics. EBC is obtained by cooling exhaled air and collecting the condensation to be utilized for downstream analysis using various analytical methods. The aqueous phase of breath contains a large variety of miscible small compounds including polar electrolytes, amino acids, cytokines, chemokines, peptides, small proteins, metabolites, nucleic acids, and lipids/eicosanoids-however, these analytes are typically present at minuscule levels in EBC, posing a considerable technical challenge. Along with recent improvements in devices for breath collection, the sensitivity and resolution of liquid chromatography coupled to online mass spectrometry-based proteomics has attained subfemtomole sensitivity, vastly enhancing the quality of EBC sample analysis. As a result, proteomics analysis of EBC has been expanding the field of breath biomarker research. We present an au courant overview of the achievements in proteomics of EBC, the advancement of EBC collection devices, and the current and future applications for EBC biomarker analysis.

Myocardial Metabolomics of Human Heart Failure With Preserved Ejection Fraction.

BACKGROUND: The human heart primarily metabolizes fatty acids, and this decreases as alternative fuel use rises in heart failure with reduced ejection fraction (HFrEF). Patients with severe obesity and diabetes are thought to have increased myocardial fatty acid metabolism, but whether this is found in those who also have heart failure with preserved ejection fraction (HFpEF) is unknown. METHODS: Plasma and endomyocardial biopsies were obtained from HFpEF (n=38), HFrEF (n=30), and nonfailing donor controls (n=20). Quantitative targeted metabolomics measured organic acids, amino acids, and acylcarnitines in myocardium (72 metabolites) and plasma (69 metabolites). The results were integrated with reported RNA sequencing data. Metabolomics were analyzed using agnostic clustering tools, Kruskal-Wallis test with Dunn test, and machine learning. RESULTS: Agnostic clustering of myocardial but not plasma metabolites separated disease groups. Despite more obesity and diabetes in HFpEF versus HFrEF (body mass index, 39.8 kg/m2 versus 26.1 kg/m2; diabetes, 70% versus 30%; both P<0.0001), medium- and long-chain acylcarnitines (mostly metabolites of fatty acid oxidation) were markedly lower in myocardium from both heart failure groups versus control. In contrast, plasma levels were no different or higher than control. Gene expression linked to fatty acid metabolism was generally lower in HFpEF versus control. Myocardial pyruvate was higher in HFpEF whereas the tricarboxylic acid cycle intermediates succinate and fumarate were lower, as were several genes controlling glucose metabolism. Non-branched-chain and branched-chain amino acids (BCAA) were highest in HFpEF myocardium, yet downstream BCAA metabolites and genes controlling BCAA metabolism were lower. Ketone levels were higher in myocardium and plasma of patients with HFrEF but not HFpEF. HFpEF metabolomic-derived subgroups were differentiated by only a few differences in BCAA metabolites. CONCLUSIONS: Despite marked obesity and diabetes, HFpEF myocardium exhibited lower fatty acid metabolites compared with HFrEF. Ketones and metabolites of the tricarboxylic acid cycle and BCAA were also lower in HFpEF, suggesting insufficient use of alternative fuels. These differences were not detectable in plasma and challenge conventional views of myocardial fuel use in HFpEF with marked diabetes and obesity and suggest substantial fuel inflexibility in this syndrome.

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue.

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

Cardiovascular Disease and Severe Hypoxemia Are Associated With Higher Rates of Noninvasive Respiratory Support Failure in Coronavirus Disease 2019 Pneumonia.

Acute hypoxemic respiratory failure is the major complication of coronavirus disease 2019, yet optimal respiratory support strategies are uncertain. We aimed to describe outcomes with high-flow oxygen delivered through nasal cannula and noninvasive positive pressure ventilation in coronavirus disease 2019 acute hypoxemic respiratory failure and identify individual factors associated with noninvasive respiratory support failure. DESIGN: Retrospective cohort study to describe rates of high-flow oxygen delivered through nasal cannula and/or noninvasive positive pressure ventilation success (live discharge without endotracheal intubation). Fine-Gray subdistribution hazard models were used to identify patient characteristics associated with high-flow oxygen delivered through nasal cannula and/or noninvasive positive pressure ventilation failure (endotracheal intubation and/or in-hospital mortality). SETTING: One large academic health system, including five hospitals (one quaternary referral center, a tertiary hospital, and three community hospitals), in New York City. PATIENTS: All hospitalized adults 18-100 years old with coronavirus disease 2019 admitted between March 1, 2020, and April 28, 2020. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 331 and 747 patients received high-flow oxygen delivered through nasal cannula and noninvasive positive pressure ventilation as the highest level of noninvasive respiratory support, respectively; 154 (46.5%) in the high-flow oxygen delivered through nasal cannula cohort and 167 (22.4%) in the noninvasive positive pressure ventilation cohort were successfully discharged without requiring endotracheal intubation. In adjusted models, significantly increased risk of high-flow oxygen delivered through nasal cannula and noninvasive positive pressure ventilation failure was seen among patients with cardiovascular disease (subdistribution hazard ratio, 1.82; 95% CI, 1.17-2.83 and subdistribution hazard ratio, 1.40; 95% CI, 1.06-1.84, respectively). Conversely, a higher peripheral blood oxygen saturation to Fio2 ratio at high-flow oxygen delivered through nasal cannula and noninvasive positive pressure ventilation initiation was associated with reduced risk of failure (subdistribution hazard ratio, 0.32; 95% CI, 0.19-0.54, and subdistribution hazard ratio 0.34; 95% CI, 0.21-0.55, respectively). CONCLUSIONS: A significant proportion of patients receiving noninvasive respiratory modalities for coronavirus disease 2019 acute hypoxemic respiratory failure achieved successful hospital discharge without requiring endotracheal intubation, with lower success rates among those with comorbid cardiovascular disease or more severe hypoxemia. The role of high-flow oxygen delivered through nasal cannula and noninvasive positive pressure ventilation in coronavirus disease 2019-related acute hypoxemic respiratory failure warrants further consideration.

TGF-ß1 Evokes Human Airway Smooth Muscle Cell Shortening and Hyperresponsiveness via Smad3.

Transforming growth factor ß1 (TGF-ß1), a cytokine whose levels are elevated in the airways of patients with asthma, perpetuates airway inflammation and modulates airway structural cell remodeling. However, the role of TGF-ß1 in excessive airway narrowing in asthma, or airway hyperresponsiveness (AHR), remains unclear. In this study, we set out to investigate the direct effects of TGF-ß1 on human airway smooth muscle (HASM) cell shortening and hyperresponsiveness. The dynamics of AHR and single-cell excitation-contraction coupling were measured in human precision-cut lung slices and in isolated HASM cells using supravital microscopy and magnetic twisting cytometry, respectively. In human precision-cut lung slices, overnight treatment with TGF-ß1 significantly augmented basal and carbachol-induced bronchoconstriction. In isolated HASM cells, TGF-ß1 increased basal and methacholine-induced cytoskeletal stiffness in a dose- and time-dependent manner. TGF-ß1-induced single-cell contraction was corroborated by concomitant increases in myosin light chain and myosin phosphatase target subunit 1 phosphorylation levels, which were attenuated by small interfering RNA-mediated knockdown of Smad3 and pharmacological inhibition of Rho kinase. Strikingly, these physiological effects of TGF-ß1 occurred through a RhoA-independent mechanism, with little effect on HASM cell [Ca2+]i levels. Together, our data suggest that TGF-ß1 enhances HASM excitation-contraction coupling pathways to induce HASM cell shortening and hyperresponsiveness. These findings reveal a potential link between airway injury-repair responses and bronchial hyperreactivity in asthma, and define TGF-ß1 signaling as a potential target to reduce AHR in asthma.

Gα12 facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3-kinase-mediated activation in a RhoA-dependent manner.

BACKGROUND AND PURPOSE: PI3K-dependent activation of Rho kinase (ROCK) is necessary for agonist-induced human airway smooth muscle cell (HASMC) contraction, and inhibition of PI3K promotes bronchodilation of human small airways. The mechanisms driving agonist-mediated PI3K/ROCK axis activation, however, remain unclear. Given that G12 family proteins activate ROCK pathways in other cell types, their role in M3 muscarinic acetylcholine receptor-stimulated PI3K/ROCK activation and contraction was examined. EXPERIMENTAL APPROACH: Gα12 coupling was evaluated using co-immunoprecipitation and serum response element (SRE)-luciferase reporter assays. siRNA and pharmacological approaches, as well as overexpression of a regulator of G-protein signaling (RGS) proteins were applied in HASMCs. Phosphorylation levels of Akt, myosin phosphatase targeting subunit-1 (MYPT1), and myosin light chain-20 (MLC) were measured. Contraction and shortening were evaluated using magnetic twisting cytometry (MTC) and micro-pattern deformation, respectively. Human precision-cut lung slices (hPCLS) were utilized to evaluate bronchoconstriction. KEY RESULTS: Knockdown of M3 receptors or Gα12 attenuated activation of Akt, MYPT1, and MLC phosphorylation. Gα12 coimmunoprecipitated with M3 receptors, and p115RhoGEF-RGS overexpression inhibited carbachol-mediated induction of SRE-luciferase reporter. p115RhoGEF-RGS overexpression inhibited carbachol-induced activation of Akt, HASMC contraction, and shortening. Moreover, inhibition of RhoA blunted activation of PI3K. Lastly, RhoA inhibitors induced dilation of hPCLS. CONCLUSIONS AND IMPLICATIONS: Gα12 plays a crucial role in HASMC contraction via RhoA-dependent activation of the PI3K/ROCK axis. Inhibition of RhoA activation induces bronchodilation in hPCLS, and targeting Gα12 signaling may elucidate novel therapeutic targets in asthma. These findings provide alternative approaches to the clinical management of airway obstruction in asthma.

Phosphoinositide 3-Kinase in Asthma: Novel Roles and Therapeutic Approaches.

Asthma manifests as airway hyperresponsiveness and inflammation, including coughing, wheezing, and shortness of breath. Immune cells and airway structural cells orchestrate asthma pathophysiology, leading to mucus secretion, airway narrowing, and obstruction. Phosphoinositide 3-kinase, a lipid kinase, plays a crucial role in many of the cellular and molecular mechanisms driving asthma pathophysiology and represents an attractive therapeutic target. Here, we summarize the diverse roles of phosphoinositide 3-kinase in the pathogenesis of asthma and discuss novel therapeutic approaches to treatment.

Transforming Growth Factor ß1 Function in Airway Remodeling and Hyperresponsiveness. The Missing Link?

The pathogenesis of asthma includes a complex interplay among airway inflammation, hyperresponsiveness, and remodeling. Current evidence suggests that airway structural cells, including bronchial smooth muscle cells, myofibroblasts, fibroblasts, and epithelial cells, mediate all three aspects of asthma pathogenesis. Although studies show a connection between airway remodeling and changes in bronchomotor tone, the relationship between the two remains unclear. Transforming growth factor ß1 (TGF-ß1), a growth factor elevated in the airway of patients with asthma, plays a role in airway remodeling and in the shortening of various airway structural cells. However, the role of TGF-ß1 in mediating airway hyperresponsiveness remains unclear. In this review, we summarize the literature addressing the role of TGF-ß1 in airway remodeling and shortening. Through our review, we aim to further elucidate the role of TGF-ß1 in asthma pathogenesis and the link between airway remodeling and airway hyperresponsiveness in asthma and to define TGF-ß1 as a potential therapeutic target for reducing asthma morbidity and mortality.

Inhibition of PI3K promotes dilation of human small airways in a rho kinase-dependent manner.

BACKGROUND AND PURPOSE: Asthma manifests as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyperresponsiveness (AHR). Although the molecular mechanisms remain unclear, activation of specific PI3K isoforms mediate inflammation and AHR. We aimed to determine whether inhibition of PI3Kδ evokes dilation of airways and to elucidate potential mechanisms. EXPERIMENTAL APPROACH: Human precision cut lung slices from non-asthma donors and primary human airway smooth muscle (HASM) cells from both non-asthma and asthma donors were utilized. Phosphorylation of Akt, myosin phosphatase target subunit 1 (MYPT1) and myosin light chain (MLC) were assessed in HASM cells following either PI3K inhibitor or siRNA treatment. HASM relaxation was assessed by micro-pattern deformation. Reversal of constriction of airways was assessed following stimulation with PI3K or ROCK inhibitors. KEY RESULTS: Soluble inhibitors or PI3Kδ knockdown reversed carbachol-induced constriction of human airways, relaxed agonist-contracted HASM and inhibited pAkt, pMYPT1 and pMLC in HASM. Similarly, inhibition of Rho kinase also dilated human PCLS airways and suppressed pMYPT1 and pMLC. Baseline pMYPT1 was significantly elevated in HASM cells derived from asthma donors in comparison with non-asthma donors. After desensitization of the ß2 -adrenoceptors, a PI3Kδ inhibitor remained an effective dilator. In the presence of IL-13, dilation by a ß agonist, but not PI3K inhibitor, was attenuated. CONCLUSION AND IMPLICATIONS: PI3Kδ inhibitors act as dilators of human small airways. Taken together, these findings provide alternative approaches to the clinical management of airway obstruction in asthma.

Formaldehyde Induces Rho-Associated Kinase Activity to Evoke Airway Hyperresponsiveness.

Formaldehyde, a common indoor air pollutant, exacerbates asthma and synergizes with allergen to induce airway hyperresponsiveness (AHR) in animal models. The mechanisms mediating formaldehyde-induced AHR remain poorly understood. We posit that formaldehyde modulates agonist-induced contractile response of human airway smooth muscle (HASM) cells to elicit AHR. HASM cells were exposed to formaldehyde or vehicle and agonist-induced intracellular Ca2+ ([Ca2+]i) and myosin light-chain phosphatase (MYPT1) phosphorylation were determined. Air-liquid interface-differentiated human bronchial epithelial (HBE) cells were exposed to formaldehyde or vehicle and cocultured with HASM cells. Agonist-induced [Ca2+]i and MYPT1 phosphorylation were determined in the cocultured HASM cells. Precision-cut human lung slices were exposed to PBS or varying concentrations of formaldehyde, and then carbachol-induced airway narrowing was determined 24 hours after exposure. HASM cells were transfected with nontargeting or nuclear factor erythroid-derived 2, like 2 (Nrf-2)-targeting small interfering RNA and exposed to formaldehyde or vehicle, followed by determination of antioxidant response (quinone oxido-reductase 1 and thioredoxin 1) and basal and agonist-induced MYPT1 phosphorylation. Formaldehyde enhanced the basal Rho-kinase activity and MYPT1 phosphorylation with little effect on agonist-induced [Ca2+]i in HASM cells. Formaldehyde induced Nrf-2-dependent antioxidant response in HASM cells, although the MYPT1 phosphorylation was independent of Nrf-2 induction. Although HBE cells exposed to formaldehyde had little effect on agonist-induced [Ca2+]i or MYPT1 phosphorylation in cocultured HASM cells, formaldehyde enhanced carbachol-induced airway responsiveness in precision-cut human lung slices. In conclusion, formaldehyde induces phosphorylation of the regulatory subunit of MYPT1, independent of formaldehyde-induced Nrf-2 activation in HASM cells. The findings suggest that the Rho kinase-dependent Ca2+ sensitization pathway plays a role in formaldehyde-induced AHR.

Epithelium-generated neuropeptide Y induces smooth muscle contraction to promote airway hyperresponsiveness.

Asthma is one of the most common chronic diseases globally and can be divided into presenting with or without an immune response. Current therapies have little effect on nonimmune disease, and the mechanisms that drive this type of asthma are poorly understood. Here, we have shown that loss of the transcription factors forkhead box P1 (Foxp1) and Foxp4, which are critical for lung epithelial development, in the adult airway epithelium evokes a non-Th2 asthma phenotype that is characterized by airway hyperresponsiveness (AHR) without eosinophilic inflammation. Transcriptome analysis revealed that loss of Foxp1 and Foxp4 expression induces ectopic expression of neuropeptide Y (Npy), which has been reported to be present in the airways of asthma patients, but whose importance in disease pathogenesis remains unclear. Treatment of human lung airway explants with recombinant NPY increased airway contractility. Conversely, loss of Npy in Foxp1- and Foxp4-mutant airway epithelium rescued the AHR phenotype. We determined that NPY promotes AHR through the induction of Rho kinase activity and phosphorylation of myosin light chain, which induces airway smooth muscle contraction. Together, these studies highlight the importance of paracrine signals from the airway epithelium to the underlying smooth muscle to induce AHR and suggest that therapies targeting epithelial induction of this phenotype may prove useful in treatment of noneosinophilic asthma.

Dynamics of goat distal hind limb muscle-tendon function in response to locomotor grade.

The functional roles of the lateral gastrocnemius (LG), medial gastrocnemius (MG) and superficial digital flexor (SDF) muscle-tendon units (MTUs) in domestic goats (N=6) were studied as a function of locomotor grade, testing the hypothesis that changes in distal limb muscle work would reflect changes in mechanical work requirements while goats walked or trotted on the level, 15 deg. decline and 15 deg. incline. As steep terrain-adapted animals, changes in muscle work output are expected to be particularly important for goats. In vivo muscle-tendon forces, fascicle length changes and muscle activation were recorded via tendon force buckles, sonomicrometry and electromyography to evaluate the work performance and elastic energy recovery of the three distal MTUs. These recordings confirmed that fascicle strain and force within goat distal hind limb muscles are adjusted in response to changes in mechanical work demand associated with locomotor grade. In general, muscle work was modulated most consistently by changes in fascicle strain, with increased net shortening (P<0.001) observed as goats switched from decline to level to incline locomotion. Peak muscle stresses increased as goats increased speed from a walk to a trot within each grade condition (P<0.05), and also increased significantly with grade (P<0.05 to P<0.01). Due to the increase in net fascicle shortening and muscle force, net muscle work per cycle also increased significantly (P<0.05 to P<0.005) as goats switched from decline to level to incline conditions (LG work: 20 mJ to 56 mJ to 209 mJ; MG work: -7 mJ to 34 mJ to 179 mJ; SDF work: -42 mJ to 14 mJ to 71 mJ, at a 2.5 ms(-1) trot). Although muscle work was modulated in response to changes in grade, the amount of work produced by these three distal pennate muscles was small (being <3%) in comparison with the change in mechanical energy required of the limb as a whole. Elastic energy recovery in the SDF and gastrocnemius (GA) tendons was substantial across all three grades, with the SDF tendon recovering 2.4 times more energy, on average, than the GA tendon. In parallel with the increase in muscle-tendon force, tendon energy recovery also increased as goats increased speed and changed gait, reaching the highest levels when goats trotted on an incline at 2.5 ms(-1) (GA: 173 mJ; SDF: 316 mJ). In general, tendon elastic energy exceeded net muscle work across all grade and gait conditions. These results demonstrate, for the first time in a quadruped, similar findings to those observed in ankle extensor muscles in humans, wallabies, turkeys and guinea fowl, suggesting that distal muscle-tendon architecture more generally favors a design for economic force production and tendon elastic energy recovery, with the majority of limb work during incline or decline running performed by larger proximal muscles.

Compliance, actuation, and work characteristics of the goat foreleg and hindleg during level, uphill, and downhill running.

We model the action of muscle-tendon system(s) about a given joint as a serial actuator and spring. By this technique, the experimental joint moment is imposed while the combined angular deflection of the actuator and spring are constrained to match the experimental joint angle throughout the stance duration. The same technique is applied to the radial leg (i.e., shoulder/hip-to-foot). The spring constant that minimizes total actuator work is considered optimal, and this minimum work is expressed as a fraction of total joint/radial leg work, yielding an actuation ratio (AR; 1 = pure actuation and 0 = pure compliance). To address work modulation, we determined the specific net work (SNW), the absolute value of net divided by total work. This ratio is unity when only positive or negative work is done and zero when equal energy is absorbed and returned. Our proximodistal predictions of joint function are supported during level and 15 degrees grade running. The greatest AR and SNW are found in the proximal leg joints (elbow and knee). The ankle joint is the principal spring of the hindleg and shows no significant change in SNW with grade, reflecting the true compliance of the common calcaneal tendon. The principal foreleg spring is the metacarpophalangeal joint. The observed pattern of proximal actuation and distal compliance, as well as the substantial SNW at proximal joints, minimal SNW at intermediate joints, and variable energy absorption at distal joints, may emerge as general principles in quadruped limb mechanics and help to inform the leg designs of highly capable running robots.