Cholesteryl Esters Are Elevated in the Lipid Fraction of Bronchoalveolar Lavage Fluid Collected from Pediatric Cystic Fibrosis Patients

Background

Host-derived lipids including cholesteryl esters (CEs) such as cholesteryl linoleate have emerged as important antibacterial effectors of innate immunity in the airways and cholesteryl linoleate has been found elevated in the context of inflammation. Cystic fibrosis (CF) patients suffer from chronic infection and severe inflammation in the airways. Here, we identified and quantified CEs in bronchoalveolar lavage fluid (BALF) from CF patients and non-CF disease controls, and tested whether CE concentrations are linked to the disease.

Materials and Methods

CEs in BALF from 6 pediatric subjects with CF and 7 pediatric subjects with non-CF chronic lung disease were quantified by mass spectral analysis using liquid chromatography coupled with tandem mass spectrometry and multiple reaction monitoring. BALFs were also examined for total lipid, total protein, albumin, and, as a marker for inflammation, human neutrophil peptide (HNP) 1–3 concentrations. Statistical analysis was conducted after log 10 transformation of the data.

Results

Total lipid/protein ratio was reduced in CF BALF (p = 0.018) but the concentrations of CEs, including cholesteryl linoleate, were elevated in the total lipid fraction in CF BALF compared to non-CF disease controls (p < 0.050). In addition, the concentrations of CEs and HNP1-3 correlated with one another (p < 0.050).

Conclusions

The data suggests that the lipid composition of BALF is altered in CF with less total lipid relative to protein but with increased CE concentrations in the lipid fraction, likely contributed by inflammation. Future longitudinal studies may reveal the suitability of CEs as a novel biomarker for CF disease activity which may provide new information on the lipid mediated pathophysiology of the disease.     

Tests for inbreeding and outbreeding depression and estimation of population differentiation in the bird-pollinated shrub Grevillea mucronulata

Background and Aims

Plants show patterns of spatial genetic differentiation reflecting gene flow mediated by pollen and seed dispersal and genotype × environment interactions. If patterns of genetic structure are determined largely by gene flow then they may be useful in predicting the likelihood of inbreeding or outbreeding depression but should be less useful if there is strong site-specific selection. For many Australian plants little is known about either their population genetics or the effects on mating systems of variation in pollen transfer distances. Experimental pollinations were used to compare the reproductive success of bird-adapted Grevillea mucronulata plants mated with individuals from a range of spatial scales. A hierarchical survey of microsatellite DNA variation was also conducted to describe the scale of population differentiation for neutral markers.

Methods

The effects of four pollen treatments on reproductive performance were compared. These treatments were characterized by transfer of pollen from (a) neighbouring adults; (b) an adjacent cluster of adults (30–50 m distant); (c) a distant cluster (>5 km distant); and (d) open pollination. Sets of 17·9 ± 3·3 leaves from each of 15 clusters of plants were genotyped and spatial autocorrelation and F statistics were used to describe patterns of genetic structure.

Key Results

Grevillea mucronulata displayed evidence of both inbreeding and outbreeding depression, with ‘intermediate’ pollen producing consistently superior outcomes for most aspects of fitness including seed set, seed size, germination and seedling growth. Significant genotypic structuring was detected within clusters (spatial autocorrelation) and among adjacent clusters and clusters separated by >5 km distance (F_ST = 0·07 and 0·10).

Conclusions

The superior outcome of intermediate pollen transfer and genetic differentiation of adjacent clusters suggests that G. mucronulata selection disfavours matings among closely and distantly related neighbours. Moreover, the performance of open-pollinated seedlings was poor, implying that current mating patterns are suboptimal.     

Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of male germ cells. During spermatogenesis, apoptosis occurs in >50% of total germ cells. Sertoli cells phagocytose these apoptotic germ cells and degrade their components using lysosomal enzymes. Here we demonstrated that SGG was a physiological substrate of Sertoli lysosomal arylsulfatase A (ARSA). SGG accumulated in Sertoli cells of Arsa(-/-) mice, and at 8 months of age, this buildup led to lysosomal swelling and other cellular abnormalities typical of a lysosomal storage disorder. This disorder likely compromised Sertoli cell functions, manifesting as impaired spermatogenesis and production of sperm with near-zero fertilizing ability in vitro. Fecundity of Arsa(-/-) males was thus reduced when they were older than 5 months. Sperm SGG is known for its roles in fertilization. Therefore, the minimal sperm fertilizing ability of 8-month-old Arsa(-/-) males may be explained by the 50% reduction of their sperm SGG levels, a result that was also observed in testicular germ cells. These unexpected decreases in SGG levels might be partly due to depletion of the backbone lipid palmitylpalmitoylglycerol that is generated from the SGG degradation pathway in Sertoli cells and normally recycled to new generations of primary spermatocytes for SGG synthesis.     

Shifts in reproductive assurance strategies and inbreeding costs associated with habitat fragmentation in Central American mahogany

The influence of habitat fragmentation on mating patterns and progeny fitness in trees is critical for understanding the long-term impact of contemporary landscape change on the sustainability of biodiversity. We examined the relationship between mating patterns, using microsatellites, and fitness of progeny, in a common garden trial, for the insect-pollinated big-leaf mahogany, Swietenia macrophylla King, sourced from forests and isolated trees in 16 populations across Central America. As expected, isolated trees had disrupted mating patterns and reduced fitness. However, for dry provenances, fitness was negatively related to correlated paternity, while for mesic provenances, fitness was correlated positively with outcrossing rate and negatively with correlated paternity. Poorer performance of mesic provenances is likely because of reduced effective pollen donor density due to poorer environmental suitability and greater disturbance history. Our results demonstrate a differential shift in reproductive assurance and inbreeding costs in mahogany, driven by exploitation history and contemporary landscape context.     

Metal-free Superoxide Dismutase-1 and Three Different Amyotrophic Lateral Sclerosis Variants Share a Similar Partially Unfolded ��-Barrel at Physiological Temperature

The structure and unfolding of metal-free (apo) human wild-type SOD1 and three pathogenic variants of SOD1 (A4V, G93R, and H48Q) that cause familial amyotrophic lateral sclerosis have been studied with amide hydrogen/deuterium exchange and mass spectrometry. The results indicate that a significant proportion of each of these proteins exists in solution in a conformation in which some strands of the β-barrel (i.e. β2) are well protected from exchange at physiological temperature (37 °C), whereas other strands (i.e. β3 and β4) appear to be unprotected from hydrogen/deuterium exchange. Moreover, the thermal unfolding of these proteins does not result in the uniform incorporation of deuterium throughout the polypeptide but involves the local unfolding of different residues at different temperatures. Some regions of the proteins (i.e. the “Greek key” loop, residues 104–116) unfold at a significantly higher temperature than other regions (i.e. β3 and β4, residues 21–53). Together, these results show that human wild-type apo-SOD1 and variants have a partially unfolded β-barrel at physiological temperature and unfold non-cooperatively.     

Pore dilation occurs in TRPA1 but not in TRPM8 channels

Pain-related sensitization and synaptic plasticity in the central nucleus of the amygdala (CeA) depend on the endogenous activation of NMDA receptors and phosphorylation of the NR1 subunit through a PKA-dependent mechanism. Functional NMDA receptors are heteromeric assemblies of NR1 with NR2A-D or NR3A, B subunits. NMDA receptors composed of NR1 and NR2B subunits have been implicated in neuroplasticity and are present in the CeA. Here we used a selective NR2B antagonist (Ro-256981) to determine the contribution of NR2B-containing NMDA receptors to pain-related sensitization of CeA neurons. Extracellular single-unit recordings were made from CeA neurons in anesthetized adult male rats before and during the development of an acute arthritis. Arthritis was induced in one knee joint by intraarticular injections of kaolin and carrageenan. Brief (15 s) mechanical stimuli of innocuous (100–500 g/30 mm²) and noxious (1000–2000 g/30 mm²) intensity were applied to the knee and other parts of the body. In agreement with our previous studies, all CeA neurons developed increased background and evoked activity after arthritis induction. Ro-256981 (1, 10 and 100 μM; 15 min each) was administered into the CeA by microdialysis 5–6 h postinduction of arthritis. Ro-256981 concentration-dependently decreased evoked responses, but not background activity. This pattern of effect is different from that of an NMDA receptor antagonist (AP5) in our previous studies. AP5 (100 μM – 5 mM) inhibited background activity and evoked responses. The differential effects of AP5 and Ro-256981 may suggest that NMDA receptors containing the NR2B subunit are important but not sole contributors to pain-related changes of CeA neurons.