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Cyclooxygenases are known as important regulators of metabolism and immune processes via conversion of C20 fatty acids into various regulatory lipid mediators, and cyclooxygenase activity has been implicated in browning of white adipose tissues. We generated transgenic (TG) C57BL/6 mice expressing the Ptgs2 gene encoding cyclooxygenase-2 (COX-2) in mature adipocytes. TG mice fed a high-fat diet displayed marginally lower weight gain with less hepatic steatosis and a slight improvement in insulin sensitivity, but no difference in glucose tolerance. Compared to littermate wildtype mice, TG mice selectively reduced inguinal white adipose tissue (iWAT) mass and fat cell size, whereas the epididymal (eWAT) fat depot remained unchanged. The changes in iWAT were accompanied by increased levels of specific COX-derived lipid mediators and increased mRNA levels of interleukin-33, interleukin-4 and arginase-1, but not increased expression of uncoupling protein 1 or increased energy expenditure. Epididymal WAT (eWAT) in TG mice exhibited few changes except from increased infiltration with eosinophils. Our findings suggest a role for COX-2-derived lipid mediators from adipocytes in mediating type 2 immunity cues in subcutaneous WAT associated with decreased hepatic steatosis, but with no accompanying induction of browning and increased energy expenditure.

Cellular fatty acid (FA) profiles have been acknowledged as biomarkers in various human diseases. Nevertheless, common FA analysis by gas chromatography mass spectrometry (GC-MS) requires long analysis time. Hence, there is a need for feasible methods for high throughput analysis in clinical studies. FA was extracted from red blood cells (RBC) and derivatized to fatty acid methyl esters (FAME). A method using gas chromatography tandem mass spectrometry (GC-MS/MS) with ammonia-induced chemical ionization (CI) was developed for the analysis of FA profiles in human RBC. We compared this method with classical single GC-MS using electron impact ionization (EI). The FA profiles of 703 RBC samples were determined by GC-MS/MS. In contrast to EI ammonia-induced CI resulted in adequate amounts of molecular ions for further fragmentation of FAME. Specific fragments for confident quantification and fragmentation were determined for 45 FA. The GC-MS/MS method has a total run time of 9min compared to typical analysis times of up to 60min in conventional GC-MS. Intra and inter assay variations were <10% for all FA analyzed. Analysis of RBC FA composition revealed an age-dependent increase of the omega-3 eicosapentaenoic and docosahexaenoic acid, and a decline of the omega-6 linoleic acid with a corresponding rise of the omega-3 index. The combination of ammonia-induced CI and tandem mass spectrometry after GC separation allows for high-throughput, robust and confident analysis of FA profiles in the clinical laboratory., (Copyright © 2016. Published by Elsevier B.V.)

Background: Living on a farm has repeatedly been shown to protect children from asthma and allergies. A major factor involved in this effect is consumption of unprocessed cow's milk obtained directly from a farm. However, this phenomenon has never been shown in a longitudinal design, and the responsible milk components are still unknown., Objectives: We sought to assess the asthma-protective effect of unprocessed cow's milk consumption in a birth cohort and to determine whether the differences in the fatty acid (FA) composition of unprocessed farm milk and industrially processed milk contributed to this effect., Methods: The Protection Against Allergy-Study in Rural Environments (PASTURE) study followed 1133 children living in rural areas in 5 European countries from birth to age 6 years. In 934 children milk consumption was assessed by using yearly questionnaires, and samples of the "usually" consumed milk and serum samples of the children were collected at age 4 years. Doctor-diagnosed asthma was parent reported at age 6 years. In a nested case-control study of 35 asthmatic and 49 nonasthmatic children, 42 FAs were quantified in milk samples., Results: The risk of asthma at 6 years of age was reduced by previous consumption of unprocessed farm milk compared with shop milk (adjusted odds ratio for consumption at 4 years, 0.26; 95% CI, 0.10-0.67). Part of the effect was explained by the higher fat content of farm milk, particularly the higher levels of ω-3 polyunsaturated FAs (adjusted odds ratio, 0.29; 95% CI, 0.11-0.81)., Conclusion: Continuous farm milk consumption in childhood protects against asthma at school age partially by means of higher intake of ω-3 polyunsaturated FAs, which are precursors of anti-inflammatory mediators., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Soluble protein and lipid mediators play essential roles in the tumor environment, but their cellular origins, targets, and clinical relevance are only partially known. We have addressed this question for the most abundant cell types in human ovarian carcinoma ascites, namely tumor cells and tumor-associated macrophages., Results: Transcriptome-derived datasets were adjusted for errors caused by contaminating cell types by an algorithm using expression data derived from pure cell types as references. These data were utilized to construct a network of autocrine and paracrine signaling pathways comprising 358 common and 58 patient-specific signaling mediators and their receptors. RNA sequencing based predictions were confirmed for several proteins and lipid mediators. Published expression microarray results for 1018 patients were used to establish clinical correlations for a number of components with distinct cellular origins and target cells. Clear associations with early relapse were found for STAT3-inducing cytokines, specific components of WNT and fibroblast growth factor signaling, ephrin and semaphorin axon guidance molecules, and TGFβ/BMP-triggered pathways. An association with early relapse was also observed for secretory macrophage-derived phospholipase PLA2G7, its product arachidonic acid (AA) and signaling pathways controlled by the AA metabolites PGE2, PGI2, and LTB4. By contrast, the genes encoding norrin and its receptor frizzled 4, both selectively expressed by cancer cells and previously not linked to tumor suppression, show a striking association with a favorable clinical course., Conclusions: We have established a signaling network operating in the ovarian cancer microenvironment with previously unidentified pathways and have defined clinically relevant components within this network.

Based on 3-(((4-(hexylamino)-2-methoxyphenyl)amino)sulfonyl)-2-thiophenecarboxylic acid methyl ester (ST247, compound 2), a recently described peroxisome proliferator-activated receptor (PPAR)β/δ-selective inverse agonist, we designed and synthesized a series of structurally related ligands. The structural modifications presented herein ultimately resulted in a series of ligands that display increased cellular activity relative to 2. Moreover, with methyl 3-(N-(2-(2-ethoxyethoxy)-4-(hexylamino)phenyl)sulfamoyl)thiophene-2-carboxylate (PT-S264, compound 9 u), biologically relevant plasma concentrations in mice were achieved. The compounds presented in this study will provide useful novel tools for future investigations addressing the role of PPARβ/δ in physiological and pathophysiological processes., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a lipid ligand-inducible transcription factor associated with macrophage polarization. However, its function in tumor-associated macrophages (TAMs) has not been investigated to date. Here, we report the PPARβ/δ-regulated transcriptome and cistrome for TAMs from ovarian carcinoma patients. Comparison with monocyte-derived macrophages shows that the vast majority of direct PPARβ/δ target genes are upregulated in TAMs and largely refractory to synthetic agonists, but repressible by inverse agonists. Besides genes with metabolic functions, these include cell type-selective genes associated with immune regulation and tumor progression, e.g., LRP5, CD300A, MAP3K8 and ANGPTL4. This deregulation is not due to increased expression of PPARβ/δ or its enhanced recruitment to target genes. Instead, lipidomic analysis of malignancy-associated ascites revealed high concentrations of polyunsaturated fatty acids, in particular linoleic acid, acting as potent PPARβ/δ agonists in macrophages. These fatty acid ligands accumulate in lipid droplets in TAMs, thereby providing a reservoir of PPARβ/δ ligands. These observations suggest that the deregulation of PPARβ/δ target genes by ligands of the tumor microenvironment contributes to the pro-tumorigenic polarization of ovarian carcinoma TAMs. This conclusion is supported by the association of high ANGPTL4 expression with a shorter relapse-free survival in serous ovarian carcinoma.

Plexiform neurofibromas (PNFs) are benign nerve sheath tumors mostly associated with neurofibromatosis type 1. They often extend through multiple layers of tissue and therefore cannot be treated satisfactorily by surgery. Nilotinib is a tyrosine kinase inhibitor used to treat leukemia, with advantages over the prototype imatinib in terms of potency and selectivity towards BCR-ABL, and the DDR, PDGFR, and KIT receptor kinases. In this study, we compared efficacies of the two drugs on cultured cells of PNF in vitro and on xenografted tumor fragments on sciatic nerve of athymic nude mice. Xenografts were monitored weekly using a high resolution ultrasound measurement. Treatment with nilotinib at a daily dose of 100 mg/kg for four weeks led to a reduction of the graft sizesstd by 68±7% in the 8 treated mice, significantly more than the 33±8% reduction in the 8 untreated mice (P<0.05) and the 47±15% in the 7 mice treated with imatinib (P<0.05). The peak plasma nilotinib concentration 6.6±1.1 µM is within the pharmacological range of clinical application. Imatinib, but not nilotinib significantly hindered body weight increase of the mice and elevated cytotoxicity of mouse spleen cells (P<0.05). Our results suggest that nilotinib may be more potent than imatinib for treating PNFs and may also be better tolerated. Imatinib seems to have some off-target effect in elevating immunity.

The increasing use of silver nanoparticles, (AgNPs), will inevitably result in their release into the environment and thereby cause the exposure to plants. It was claimed that using AgNPs is a safe and efficient method to preserve and treat agents of disease in agriculture. This study tries to understand the protein populations and sub-populations and follow up environmental AgNPs stresses. To accomplish these, the action of homemade spherical AgNPs colloidal suspension against Oryza sativa L. was investigated by a proteomic approach (2-DE and NanoLC/FT-ICR MS identification). Twenty-eight responsive (decrement/increment in abundance) proteins were identified. Proteomic results revealed that an exposure of O. sativa L., root with different concentrations of AgNPs resulted in an accumulation of protein precursors, indicative of the dissipation of a proton motive force. The identified proteins are involved in oxidative stress tolerance, Ca(2+) regulation and signaling, transcription and protein degradation, cell wall and DNA/RNA/protein direct damage, cell division and apoptosis. The expression pattern of these proteins and their possible involvement in the nontoxicity mechanisms were discussed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Emerging technologies in functional genomics and proteomics provide a way of achieving high-throughput analyses, understanding effects on protein populations and sub-populations and follow up environmental stresses. To accomplish these, the action of homemade spherical Silver nanoparticles colloidal suspension (AgNPs) against Bacillus thuringiensis (isolate from Oryza sativa L. rhizosphere) was investigated by a proteomic approach (2-DE and NanoLC/FT-ICR MS identification). Thirty four responsive (up/down regulated) proteins were identified. Proteomic results revealed that an exposure of B. thuringiensis cells with different concentrations of AgNPs resulted in an accumulation of envelope protein precursors, indicative of the dissipation of a proton motive force. Identified proteins are involved in oxidative stress tolerance, metal detoxification, transcription and elongation processes, protein degradation, cytoskeleton remodeling and cell division. The expression pattern of these proteins and their possible involvement in the nontoxicity mechanisms were discussed., (© 2013 Elsevier Inc. All rights reserved.)

The vast majority of organisms produce ATP by a membrane-bound rotating protein complex, termed F-ATP synthase. In chloroplasts, the corresponding enzyme generates ATP by using a transmembrane proton gradient generated during photosynthesis, a process releasing high amounts of molecular oxygen as a natural byproduct. Due to its chemical properties, oxygen can be reduced incompletely which generates several highly reactive oxygen species (ROS) that are able to oxidize a broad range of biomolecules. In extension to previous studies it could be shown that ROS dramatically decreased ATP synthesis in situ and affected the CF1 portion in vitro. A conserved cluster of three methionines and a cysteine on the chloroplast γ subunit could be identified by mass spectrometry to be oxidized by ROS. Analysis of amino acid substitutions in a hybrid F1 assembly system indicated that these residues were exclusive catalytic targets for hydrogen peroxide and singlet oxygen, although it could be deduced that additional unknown amino acid targets might be involved in the latter reaction. The cluster was tightly integrated in catalytic turnover since mutants varied in MgATPase rates, stimulation by sulfite and chloroplast-specific γ subunit redox-modulation. Some partial disruptions of the cluster by mutagenesis were dominant over others regarding their effects on catalysis and response to ROS., (© 2012 Elsevier B.V. All rights reserved.)

Rationale: The analysis of proteins by mass spectrometry imaging is an important biomedical application as spatial distributions can be used to identify markers for pathological processes. The direct detection and identification of proteins on tissue can be hindered by a number of factors including limited mass range and fragmentation efficiency as well as incompatibility with formalin-fixed samples., Methods: To overcome some of these limitations, on-tissue digestion of proteins was followed by detection of the resulting peptides. Trypsin was applied by a spraying device. Matrix-assisted laser desorption/ionization (MALDI) imaging experiments were performed with a home-built atmospheric-pressure imaging source attached to a LTQ Orbitrap mass spectrometer. The mass accuracy under imaging conditions was better than 3 ppm RMS. This allowed for confident identification of tryptic peptides by comparison with liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) measurements of an adjacent mouse brain section., Results: A spatial resolution of 50 µm was obtained for tryptic peptides on tissue. Several tryptic peptides of myelin showed matching spatial distributions, and numerous tryptic peptides of other proteins were identified. MS images were generated with a bin size (mass range used for image generation) of Δm/z = 0.01 u. Examples demonstrate that MS images with lower selectivity can result in misleading information about the spatial distribution of tryptic peptides., Conclusions: The presented method combines a significantly improved spatial resolution for tryptic peptides with low-ppm mass accuracy in a single experiment and thus provides highly reliable and specific information., (Copyright © 2012 John Wiley & Sons, Ltd.)

The spatial distribution of proteins in tissue sections can be used to identify potential markers for pathological processes. Tissue sections are often subjected to enzymatic digestion before matrix-assisted laser desorption/ionization (MALDI) imaging. This study is targeted at improving the on-tissue identification of tryptic peptides by accurate mass measurements and complementary off-line liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analysis. Two adjacent mouse brain sections were analyzed in parallel. The first section was spotted with trypsin and analyzed by MALDI imaging. Direct on-tissue MS/MS experiments of this section resulted in the identification of 14 peptides (originating from 4 proteins). The second tissue section was homogenized, fractionated by ultracentrifugation and digested with trypsin prior to LC/ESI-MS/MS analysis. The number of identified peptides was increased to 153 (corresponding to 106 proteins) by matching imaged mass peaks to peptides which were identified in these LC/ESI-MS/MS experiments. All results (including MALDI imaging data) were based on accurate mass measurements (RMS <2 ppm) and allow a confident identification of tryptic peptides. Measurements based on lower accuracy would have led to ambiguous or misleading results. MS images of identified peptides were generated with a bin width (mass range used for image generation) of Δm/z = 0.01. The application of accurate mass measurements and additional LC/MS measurements increased both the quality and the number of peptide identifications. The advantages of this approach for the analysis of biological tissue sections are demonstrated and discussed in detail. Results indicate that accurate mass measurements are needed for confident identification and specific image generation of tryptic peptides in tissue sections., (Copyright © 2011 John Wiley & Sons, Ltd.)