Dedreia Tull

Evidence that asthma is a developmental origin disease influenced by maternal diet and bacterial metabolites

Asthma is prevalent in Western countries, and recent explanations have evoked the actions of the gut microbiota. Here we show that feeding mice a high-fibre diet yields a distinctive gut microbiota, which increases the levels of the short-chain fatty acid, acetate. High-fibre or acetate-feeding led to marked suppression of allergic airways disease (AAD, a model for human asthma), by enhancing T-regulatory cell numbers and function. Acetate increases acetylation at the Foxp3 promoter, likely through HDAC9 inhibition. Epigenetic effects of fibre/acetate in adult mice led us to examine the influence of maternal intake of fibre/acetate. High-fibre/acetate feeding of pregnant mice imparts on their adult offspring an inability to develop robust AAD. High fibre/acetate suppresses expression of certain genes in the mouse fetal lung linked to both human asthma and mouse AAD. Thus, diet acting on the gut microbiota profoundly influences airway responses, and may represent an approach to prevent asthma, including during pregnancy.

VEGF-D Promotes Tumor Metastasis by Regulating Prostaglandins Produced by the Collecting Lymphatic Endothelium

Lymphatic metastasis is facilitated by lymphangiogenic growth factors VEGF-C and VEGF-D that are secreted by some primary tumors. We identified regulation of PGDH, the key enzyme in prostaglandin catabolism, in endothelial cells of collecting lymphatics, as a key molecular change during VEGF-D-driven tumor spread. The VEGF-D-dependent regulation of the prostaglandin pathway was supported by the finding that collecting lymphatic vessel dilation and subsequent metastasis were affected by nonsteroidal anti-inflammatory drugs (NSAIDs), known inhibitors of prostaglandin synthesis. Our data suggest a control point for cancer metastasis within the collecting lymphatic endothelium, which links VEGF-D/VEGFR-2/VEGFR-3 and the prostaglandin pathways. Collecting lymphatics therefore play an active and important role in metastasis and may provide a therapeutic target to restrict tumor spread.

The Reductase That Catalyzes Mycolic Motif Synthesis Is Required for Efficient Attachment of Mycolic Acids to Arabinogalactan

Mycolic acids are essential components of the cell walls of bacteria belonging to the suborder Corynebacterineae, including the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. Mycolic acid biosynthesis is complex and the target of several frontline antimycobacterial drugs. The condensation of two fatty acids to form a 2-alkyl-3-keto mycolate precursor and the subsequent reduction of this precursor represent two key and highly conserved steps in this pathway. Although the enzyme catalyzing the condensation step has recently been identified, little is known about the putative reductase. Using an extensive bioinformatic comparison of the genomes of M. tuberculosis and Corynebacterium glutamicum, we identified NCgl2385, the orthologue of Rv2509 in M. tuberculosis, as a potential reductase candidate. Deletion of the gene in C. glutamicum resulted in a slow growing strain that was deficient in arabinogalactan-linked mycolates and synthesized abnormal forms of the mycolate-containing glycolipids trehalose dicorynomycolate and trehalose monocorynomycolate. Analysis of the native and acetylated trehalose glycolipids by MALDI-TOF mass spectrometry indicated that these novel glycolipids contained an unreduced β-keto ester. This was confirmed by analysis of sodium borodeuteride-reduced mycolic acids by gas chromatography mass spectrometry. Reintroduction of the NCgl2385 gene into the mutant restored the transfer of mature mycolic acids to both the trehalose glycolipids and cell wall arabinogalactan. These data indicate that NCgl2385, which we have designated CmrA, is essential for the production of mature trehalose mycolates and subsequent covalent attachment of mycolic acids onto the cell wall, thus representing a focus for future structural and pathogenicity studies.

Secondary Acylation of Klebsiella pneumoniae Lipopolysaccharide Contributes to Sensitivity to Antibacterial Peptides

Klebsiella pneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K. pneumoniae disease pathogenesis by using a mutation of lpxM (msbB/waaN), which encodes the enzyme responsible for late secondary acylation of immature lipid A molecules. A K. pneumoniae B5055 (K2:O1) lpxM mutant was found to be attenuated for growth in the lungs in a mouse pneumonia model leading to reduced lethality of the bacterium. B5055ΔlpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055ΔlpxM showed increased permeability of the outer membrane and an increased susceptibility to certain antibacterial peptides suggesting that in vivo attenuation may be due in part to sensitivity to antibacterial peptides present in the lungs of BALB/c mice. These data support the view that lipopolysaccharide acylation plays a important role in providing Gram-negative bacteria some resistance to structural and innate defenses and especially the antibacterial properties of detergents (e.g. bile) and cationic defensins.

Normalizing and integrating metabolomics data.

Metabolomics research often requires the use of multiple analytical platforms, batches of samples, and laboratories, any of which can introduce a component of unwanted variation. In addition, every experiment is subject to within-platform and other experimental variation, which often includes unwanted biological variation. Such variation must be removed in order to focus on the biological information of interest. We present a broadly applicable method for the removal of unwanted variation arising from various sources for the identification of differentially abundant metabolites and, hence, for the systematic integration of data on the same quantities from different sources. We illustrate the versatility and the performance of the approach in four applications, and we show that it has several advantages over the existing normalization methods.

Human inflammatory and resolving lipid mediator responses to resistance exercise and ibuprofen treatment

Classical proinflammatory eicosanoids, and more recently discovered lipid mediators with anti-inflammatory and proresolving bioactivity, exert a complex role in the initiation, control, and resolution of inflammation. Using a targeted lipidomics approach, we investigated circulating lipid mediator responses to resistance exercise and treatment with the NSAID ibuprofen. Human subjects undertook a single bout of unaccustomed resistance exercise (80% of one repetition maximum) following oral ingestion of ibuprofen (400 mg) or placebo control. Venous blood was collected during early recovery (0-3 h and 24 h postexercise), and serum lipid mediator composition was analyzed by LC-MS-based targeted lipidomics. Postexercise recovery was characterized by elevated levels of cyclooxygenase (COX)-1 and 2-derived prostanoids (TXB2, PGE2, PGD2, PGF2α, and PGI2), lipooxygenase (5-LOX, 12-LOX, and 15-LOX)-derived hydroxyeicosatetraenoic acids (HETEs), and leukotrienes (e.g., LTB4), and epoxygenase (CYP)-derived epoxy/dihydroxy eicosatrienoic acids (EpETrEs/DiHETrEs). Additionally, we detected elevated levels of bioactive lipid mediators with anti-inflammatory and proresolving properties, including arachidonic acid-derived lipoxins (LXA4 and LXB4), and the EPA (E-series) and DHA (D-series)-derived resolvins (RvD1 and RvE1), and protectins (PD1 isomer 10S, 17S-diHDoHE). Ibuprofen treatment blocked exercise-induced increases in COX-1 and COX-2-derived prostanoids but also resulted in off-target reductions in leukotriene biosynthesis, and a diminished proresolving lipid mediator response. CYP pathway product metabolism was also altered by ibuprofen treatment, as indicated by elevated postexercise serum 5,6-DiHETrE and 8,9-DiHETrE only in those receiving ibuprofen. These findings characterize the blood inflammatory lipid mediator response to unaccustomed resistance exercise in humans and show that acute proinflammatory signals are mechanistically linked to the induction of a biological active inflammatory resolution program, regulated by proresolving lipid mediators during postexercise recovery.

Compartmentalization of lipid biosynthesis in mycobacteria.

The plasma membrane of Mycobacterium sp. is the site of synthesis of several distinct classes of lipids that are either retained in the membrane or exported to the overlying cell envelope. Here, we provide evidence that enzymes involved in the biosynthesis of two major lipid classes, the phosphatidylinositol mannosides (PIMs) and aminophospholipids, are compartmentalized within the plasma membrane. Enzymes involved in the synthesis of early PIM intermediates were localized to a membrane subdomain termed PMf, that was clearly resolved from the cell wall by isopyknic density centrifugation and amplified in rapidly dividing Mycobacterium smegmatis. In contrast, the major pool of apolar PIMs and enzymes involved in polar PIM biosynthesis were localized to a denser fraction that contained both plasma membrane and cell wall markers (PM-CW). Based on the resistance of the PIMs to solvent extraction in live but not lysed cells, we propose that polar PIM biosynthesis occurs in the plasma membrane rather than the cell wall component of the PM-CW. Enzymes involved in phosphatidylethanolamine biosynthesis also displayed a highly polarized distribution between the PMf and PM-CW fractions. The PMf was greatly reduced in non-dividing cells, concomitant with a reduction in the synthesis and steady-state levels of PIMs and amino-phospholipids and the redistribution of PMf marker enzymes to non-PM-CW fractions. The formation of the PMf and recruitment of enzymes to this domain may thus play a role in regulating growth-specific changes in the biosynthesis of membrane and cell wall lipids.

Mechanical cell disruption for lipid extraction from microalgal biomass

Cell disruption is an integral part of the downstream operation required to produce biodiesel from microalgae. This study investigated the use of ultrasonication and high-pressure homogenization (HPH) as cell disruption methods for two microalgal species, Tetraselmis suecica (TS) and Chlorococcum sp. (C sp.). The kinetics of cell disruption followed a first-order model (0.65<R(2)<1.00). Disruption rate constant for ultrasonication was directly proportional to power level and followed a parabolic relationship with initial cell concentration, while that for HPH was directly proportional to operating pressure and inversely proportional to initial cell concentration. Mean disruption rate constant for HPH was approximately seven times that for ultrasonication. Mean disruption rate constant for TS cells was roughly 20% higher than that for C sp. cells. Subjecting TS culture to cell disruption prior to lipid extraction resulted in 5-8-fold increase in lipid yield and 3-5-fold increase in triglyceride yield.

A Unique Thymic Fibroblast Population Revealed by the Monoclonal Antibody MTS-15

T cell differentiation in the thymus is dependent upon signals from thymic stromal cells. Most studies into the nature of these signals have focused only on the support provided by the thymic epithelium, but there is an emerging view that other stromal cells such as mesenchymal fibroblasts may also be involved. Study of the latter has been hindered by a lack of appropriate markers, particularly those allowing their isolation. In this study, we describe a new surface marker of thymic stroma, MTS-15, and demonstrate its specificity for fibroblasts and a subset of endothelial cells. Coculture experiments showed that the determinant could be transferred between cells. Extensive biochemical analysis demonstrated that the Ag bound by MTS-15 was the glycosphingolipid Forssman determinant, consistent with the distribution observed. Transcriptional analysis of purified MTS-15+ thymic fibroblasts revealed a unique expression profile for a number of chemokines and growth factors important to thymocyte and epithelial cell development. In a model of cyclophosphamide-induced thymic involution and regeneration, fibroblasts were found to expand extensively and express growth factors important to epithelial proliferation and increased T cell production just before thymic regeneration. Overall, this study identifies a useful marker of thymic fibroblasts and highlights this subpopulation as a key player in thymic function by virtue of their support of both thymocytes and epithelial cells.

Specific plasma lipid classes and phospholipid fatty acids indicative of dairy food consumption associate with insulin sensitivity

BACKGROUND Reports have suggested that the consumption of dairy foods may reduce risk of type 2 diabetes on the basis of evidence of raised circulating ruminant fatty acids. OBJECTIVE We determined whether certain phospholipid species and fatty acids that are associated with full-fat dairy consumption may also be linked to diminished insulin resistance. DESIGN Four variables of insulin resistance and sensitivity were defined from oral-glucose-tolerance tests in 86 overweight and obese subjects with metabolic syndrome. Plasma phospholipids, sphingolipids, and fatty acids were determined by using a lipidomic analysis and gas chromatography-mass spectrometry to provide objective markers of dairy consumption. Food records provided information on dairy products. Associations were determined by using linear regression analyses adjusted for potential confounders age, sex, systolic blood pressure, waist:hip ratio, or body mass index (BMI) and corrected for multiple comparisons. RESULTS Lysophosphatidylcholine, lyso-platelet-activating factor, and several phospholipid fatty acids correlated directly with the number of servings of full-fat dairy foods. Lysophosphatidylcholine and lyso-platelet-activating factor were also associated directly with insulin sensitivity when accounting for the waist:hip ratio (Matsuda index unadjusted, P < 0.001 for both; adjusted for multiple comparisons, P < 0.02 for both) and inversely with insulin resistance (fasting insulin unadjusted, P < 0.001 for both; adjusted, P = 0.04 and P < 0.05, respectively; homeostasis model assessment of insulin resistance adjusted, P = 0.04 for both; post-glucose insulin area under the plasma insulin curve during the 120 min of the test adjusted, P < 0.01 for both). The substitution of BMI for the waist:hip ratio attenuated associations modestly. Phospholipid fatty acid 17:0 also tended to be associated directly with insulin sensitivity and inversely with resistance. CONCLUSION Variables of insulin resistance were lower at higher concentrations of specific plasma phospholipids that were also indicators of full-fat dairy consumption. This trial was registered at clinicaltrials.gov as NCT00163943.