Pseudomonas aeruginosa is the main contributor to the morbidity and mortality of cystic fibrosis (CF) patients. Chronic respiratory infections are rarely eradicated due to protection from CF mucus and the biofilm matrix. The composition of the biofilm matrix determines its viscoelastic properties and affects antibiotic efficacy. Nitric oxide (NO) can both disrupt the physical structure of the biofilm and eradicate interior colonies. The effects of a CF-like growth environment on P. aeruginosa biofilm susceptibility to NO were investigated using parallel plate macrorheology and particle tracking microrheology. Biofilms grown in the presence of mucins and DNA contained greater concentrations of DNA in the matrix and exhibited concomitantly larger viscoelastic moduli compared to those grown in tryptic soy broth. Greater viscoelastic moduli correlated with increased tolerance to tobramycin and colistin. Remarkably, NO-releasing cyclodextrins eradicated all biofilms at the same concentration. The capacity of NO-releasing cyclodextrins to eradicate P. aeruginosa biofilms irrespective of matrix composition suggests that NO-based therapies may be superior antibiofilm treatments compared to conventional antibiotics.
Loss of long-chain acyl-CoA synthetase isoform-1 (ACSL1) in mouse skeletal muscle (Acsl1 M؊/؊ ) severely reduces acyl-CoA synthetase activity and fatty acid oxidation. However, the effects of decreased fatty acid oxidation on skeletal muscle function, histology, use of alternative fuels, and mitochondrial function and morphology are unclear. We observed that Acsl1 M؊/؊ mice have impaired voluntary running capacity and muscle grip strength and that their gastrocnemius muscle contains myocytes with central nuclei, indicating muscle regeneration. We also found that plasma creatine kinase and aspartate aminotransferase levels in Acsl1 M؊/؊ mice are 3.4-and 1.5-fold greater, respectively, than in control mice (Acsl1 flox/flox ), indicating muscle damage, even without exercise, in the Acsl1 M؊/؊ mice. Moreover, caspase-3 protein expression exclusively in Acsl1 M؊/؊ skeletal muscle and the presence of cleaved caspase-3 suggested myocyte apoptosis. Mitochondria in Acsl1 M؊/؊ skeletal muscle were swollen with abnormal cristae, and mitochondrial biogenesis was increased. Glucose uptake did not increase in Acsl1 M؊/؊ skeletal muscle, and pyruvate oxidation was similar in gastrocnemius homogenates from Acsl1 M؊/؊ and control mice. The rate of protein synthesis in Acsl1 M؊/؊ glycolytic muscle was 2.1-fold greater 30 min after exercise than in the controls, suggesting resynthesis of proteins catabolized for fuel during the exercise. At this time, mTOR complex 1 was activated, and autophagy was blocked. These results suggest that fatty acid oxidation is critical for normal skeletal muscle homeostasis during both rest and exercise. We conclude that ACSL1 deficiency produces an overall defect in muscle fuel metabolism that increases protein catabolism, resulting in exercise intolerance, muscle weakness, and myocyte apoptosis.
Across the globe, millions of people are affected by muco-obstructive pulmonary diseases like cystic fibrosis, asthma, and chronic obstructive pulmonary disease. In MOPDs, the airway mucus becomes hyperconcentrated, increasing viscoelasticity and impairing mucus clearance. Research focused on treatment of MOPDs requires relevant sources of airway mucus both as a control sample type and as a basis for manipulation to study the effects of additional hyperconcentration, inflammatory milieu, and biofilm growth on the biochemical and biophysical properties of mucus. Endotracheal tube mucus has been identified as a prospective source of native airway mucus given its several advantages over sputum and airway cell culture mucus such as ease of access and in vivo production that includes surface airway and submucosal gland secretions. Still, many ETT samples suffer from altered tonicity and composition from either dehydration, salivary dilution, or other contamination. Herein, the biochemical compositions of ETT mucus from healthy human subjects were determined. Samples were characterized in terms of tonicity, pooled, and restored to normal tonicity. Salt-normalized ETT mucus exhibited similar concentration-dependent rheologic properties as originally isotonic mucus. This rheology agreed across spatial scales and with previous reports of the biophysics of ETT mucus. This work affirms previous reports of the importance of salt concentration on mucus rheology and presents methodology to increase yield native airway mucus samples for laboratory use and manipulation.
Skeletal muscle exhibits plasticity and complexity in the choice of fuel substrate. During starvation, muscle initiates protein degradation to provide amino acids for liver gluconeogenesis, during acute exercise, muscle uses glucose as the primary fuel, while during rest or prolonged exercise, muscle switches to use primarily fatty acids. Defective fatty acid metabolism is one of the leading causes of metabolic myopathy. Long chain acyl‐CoA synthetase isoform‐1 (ACSL1) converts long chain fatty acids to long chain acyl‐CoAs, before acyl‐CoAs enter mitochondria for β‐oxidation or are esterified to form complex lipids. Although 5 Acsl isoforms exist, deletion of Acsl1 reduces 90% of ACS activity and fatty acid oxidation (FAO) in gastrocnemius, indicating that ACSL1 is the predominant ACSL isoform in the muscle. The objective of this study was to examine how reduced FAO through deletion of Acsl1 affects muscle physiology. We hypothesized that ACSL1‐directed fatty acid metabolism is essential for energy balance in muscle, and that lack of ACSL1 causes myopathy. To test this hypothesis, we used a mouse model in which ACSL1 is specifically deleted in skeletal muscle (Acsl1M−/−). Compared to control mice (Acsl1flox/flox), Acsl1M−/− mice demonstrated 3.4‐ and 1.5‐fold increases in plasma creatine kinase and aspartate aminotransferase activities, indicating that deletion of Acsl1 led to muscle damage. Gastrocnemius from Acsl1M−/− mice also showed the presence of central nuclei, a marker of muscle regeneration. In addition, we detected expression of caspase 3 protein only in Acsl1M−/− muscle, suggesting that the apoptosis pathway was involved. Different muscle fiber types prefer to metabolize different substrates. To investigate the consequence of Acsl1 deletion on muscle fiber type, succinate dehydrogenase staining was performed and it showed a significantly increased percentage of oxidative fibers in Acsl1M−/− gastrocnemius compared to controls. In addition, gene expression of myosin heavy chain I, an oxidative muscle fiber marker, was 6 fold higher in Acsl1M−/− glycolytic fibers than in controls, suggesting a switch from glycolytic to oxidative fibers. We further hypothesized that the fiber switch in Acsl1M−/− gastrocnemius muscle was caused by increased damage to glycolytic fibers compared to oxidative fibers. In summary, deletion of Acsl1 caused muscle damage and regeneration and promoted a glycolytic to oxidative fiber switch. Ongoing studies are examining protein turnover to determine its contribution to muscle damage.Support or Funding InformationSupported by DK56598.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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