Semi-automated non-target processing in GC × GC–MS metabolomics analysis: applicability for biomedical studies

Due to the complexity of typical metabolomics samples and the many steps required to obtain quantitative data in GC × GC–MS consisting of deconvolution, peak picking, peak merging, and integration, the unbiased non-target quantification of GC × GC–MS data still poses a major challenge in metabolomics analysis. The feasibility of using commercially available software for non-target processing of GC × GC–MS data was assessed. For this purpose a set of mouse liver samples (24 study samples and five quality control (QC) samples prepared from the study samples) were measured with GC × GC–MS and GC–MS to study the development and progression of insulin resistance, a primary characteristic of diabetes type 2. A total of 170 and 691 peaks were quantified in, respectively, the GC–MS and GC × GC–MS data for all study and QC samples. The quantitative results for the QC samples were compared to assess the quality of semi-automated GC × GC–MS processing compared to targeted GC–MS processing which involved time-consuming manual correction of all wrongly integrated metabolites and was considered as golden standard. The relative standard deviations (RSDs) obtained with GC × GC–MS were somewhat higher than with GC–MS, due to less accurate processing. Still, the biological information in the study samples was preserved and the added value of GC × GC–MS was demonstrated; many additional candidate biomarkers were found with GC × GC–MS compared to GC–MS

Integrin affinity modulation and survival signalling.

Integrins are heterodimeric transmembrane proteins that provide a bi-directional link between the cell’s internal biological mechanisms and the extracellular environment. During inside-out signalling, intracellular messages converge on the integrin cytoplasmic domain to induce a conformational change. This is transmitted to the extracellular domain where it results in an alteration in affinity for integrin ligands such as fibronectin and laminin. In this way the cell has developed the ability to modulate the critical functions of adhesion and cell movement. In outside-in signalling, the integrin performs a more complex function than simple adhesion; upon binding to ligand, the integrin extracellular domain undergoes a conformational change which is transmitted to the cytoplasmic domain. This alters the integrin’s cytoplasmic domain affinity for intracellular signalling proteins and results in the activation of intracellular second messenger pathways. In this way, the extracellular milieu is able to influence intracellular signalling including those involved in apoptosis. This thesis demonstrates data which provide original insights into bi-directional integrin signalling: Inside-out signalling: Constitutively active Notch1 increases β3-integrin affinity and abrogates Hras-mediated integrin suppression without increasing expression of β3- integrin. Dominant-Negative Rras blocks Notch-mediated integrin activation and Notch1-mediated reversal of Hras and Raf-mediated integrin suppression and this is independent of erk phosphorylation. Notch1 induces Rras activation. Functional adhesion assays confirm that Notch1IC increases K562 adhesion in a β1-integrin dependent manner and this is abrogated by Dominant-Negative Rras. This data supports a mechanism in which Notch1 increases integrin affinity via activation of Rras. Outside-in signalling: Evidence is presented demonstrating that extracellular matrix proteins, laminin and fibronectin, activate β1-integrins to protect SCLC cells against the apoptotic effects of etoposide and ionizing radiation via PI3Kinase activation. This occurs in two ways: 1) PI3Kinase-dependent β1-integrin signalling resulting in phosphorylation of Bad and reduced caspase-9 cleavage and 2) a β1-integrinmediated over-riding of etoposide and radiotherapy-induced cell cycle S phase delay and G2/M arrest. β1-integrin-mediated outside-in survival signalling was investigated further in the in vivo setting; MatrigelTM, a basement membrane product rich in extracellular matrix proteins, promoted SCLC xenograft survival and growth in a β1-integrin and tyrosine kinase-dependent manner. This data provides novel insights into the critical functions that integrins play in adhesion and survival signalling

Fishmeal supplementation during ovine pregnancy and lactation protects against maternal stress-induced programming of the offspring immune system

BACKGROUND: Prenatally stressed offspring exhibit increased susceptibility to inflammatory disorders due to in utero programming. Research into the effects of n-3 PUFAs shows promising results for the treatment and prevention of these disorders. The purpose of this study was to investigate whether maternal fishmeal supplementation during pregnancy and lactation protects against programming of the offspring’s immune response following simulated maternal infection. METHODS: In order to accomplish this, 53 ewes were fed a diet supplemented with fishmeal (FM; rich in n-3 PUFA) or soybean meal (SM; rich in n-6 PUFAs) from day 100 of gestation (gd 100) through lactation. On gd135, half the ewes from each dietary group were challenged with either 1.2 μg/kg Escherichia coli lipopolysaccharide (LPS) endotoxin to simulate a bacterial infection, or saline as the control. At 4.5 months of age the offspring’s dermal immune response was assessed by cutaneous hypersensitivity testing with ovalbumin (OVA) and candida albicans (CAA) 21 days after sensitization. Skinfold measurements were taken and serum blood samples were also collected to assess the primary and secondary antibody immune response. RESULTS: Offspring born to SM + LPS mothers had a significantly greater change in skinfold thickness in response to both antigens as well as a greater secondary antibody response to OVA compared to all treatments. CONCLUSIONS: Supplementation during pregnancy with FM appears to protect against adverse fetal programming that may occur during maternal infection and this may reduce the risk of atopic disease later in life

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Th1 and Th2 chemokines, vaccine induced 1 immunity and allergic disease in infants after maternal ω-3 fatty acid supplementation during pregnancy and lactatio

Th1 and Th2 chemokines, vaccine induced 1 immunity and allergic disease in infants after maternal ω-3 fatty acid supplementation during pregnancy and lactation

We investigated whether the previously reported preventive effect of maternal ω-3 fatty acid supplementation on IgE-associated allergic disease in infancy may be mediated by facilitating a balanced circulating Th2/Th1 chemokine profile in the infant. Vaccine-induced immune responses at 2 y of age were also evaluated. Pregnant women, at risk of having an allergic infant, were randomized to daily supplementation with 1.6 g eicosapentaenoic acid and 1.1 g docosahexaenoic acid or placebo from the 25th gestational week through 3.5 mo of breastfeeding. Infant plasma was analyzed for chemokines (cord blood, 3, 12, 24 mo) and anti-tetanus and anti-diphtheria IgG (24 mo). High Th2-associated CC-chemokine ligand 17 (CCL17) levels were associated with infant allergic disease (p < 0.05). In infants without, but not with, maternal history of allergy, the ω-3 supplementation was related to lower CCL17/CXC-chemokine ligand 11 (CXCL11) (Th2/Th1) ratios (p < 0.05). Furthermore, in nonallergic, but not in allergic infants, ω-3 supplementation was linked with higher Th1-associated CXCL11 levels (p < 0.05), as well as increased IgG titers to diphtheria (p = 0.01) and tetanus (p = 0.05) toxins. Thus, the prospect of balancing the infant immune system toward a less Th2-dominated response, by maternal ω-3 fatty acid supplementation, seems to be influenced by allergic status