The spatial distributions of various specific lipids in freeze-dried mouse brain sections were monitored using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Mouse brain sections were prepared by cryosectioning, rinsing in 0.15 M NH3HCOO, and freeze-drying, after which the samples were analyzed directly by TOF-SIMS, using Au3+ ions as primary ions. Positive and negative TOF-SIMS spectra of the tissue surface contained peaks from quasimolecular ions of a variety of specific lipids, including cholesterol, sulfatides, phosphatidylinositols, and phosphatidylcholines. Images showing the spatial signal intensity distributions of specific ions were recorded across analysis areas ranging from 100 x 100 microm(2) to 9 x 9 mm(2). The results demonstrate a highly complementary localization of cholesterol and phosphatidylcholine over dimensions from millimeter to micrometer range. Characteristic spatial distributions of several other lipids, including sulfatides and phosphatidylinositols, were observed. Principal component analysis was used to localize regions of the sample surface that show common spectral features. Spectra from different such regions showed large variations in lipid ion signals, indicating large variations in the lipid composition in different regions.
Background n-3 polyunsaturated fatty acids, namely docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), reduce the risk of cardiovascular disease and can ameliorate many of obesity-associated disorders. We hypothesised that the latter effect will be more pronounced when DHA/EPA is supplemented as phospholipids rather than as triglycerides.Methodology/Principal FindingsIn a ‘prevention study’, C57BL/6J mice were fed for 9 weeks on either a corn oil-based high-fat obesogenic diet (cHF; lipids ∼35% wt/wt), or cHF-based diets in which corn oil was partially replaced by DHA/EPA, admixed either as phospholipids or triglycerides from marine fish. The reversal of obesity was studied in mice subjected to the preceding cHF-feeding for 4 months. DHA/EPA administered as phospholipids prevented glucose intolerance and tended to reduce obesity better than triglycerides. Lipemia and hepatosteatosis were suppressed more in response to dietary phospholipids, in correlation with better bioavailability of DHA and EPA, and a higher DHA accumulation in the liver, white adipose tissue (WAT), and muscle phospholipids. In dietary obese mice, both DHA/EPA concentrates prevented a further weight gain, reduced plasma lipid levels to a similar extent, and tended to improve glucose tolerance. Importantly, only the phospholipid form reduced plasma insulin and adipocyte hypertrophy, while being more effective in reducing hepatic steatosis and low-grade inflammation of WAT. These beneficial effects were correlated with changes of endocannabinoid metabolome in WAT, where phospholipids reduced 2-arachidonoylglycerol, and were more effective in increasing anti-inflammatory lipids such as N-docosahexaenoylethanolamine.Conclusions/SignificanceCompared with triglycerides, dietary DHA/EPA administered as phospholipids are superior in preserving a healthy metabolic profile under obesogenic conditions, possibly reflecting better bioavalability and improved modulation of the endocannabinoid system activity in WAT.
Fossil feathers, hairs and eyes are regularly preserved as carbonized traces comprised of masses of micrometre-sized bodies that are spherical, oblate or elongate in shape. For a long time, these minute structures were regarded as the remains of biofilms of keratinophilic bacteria, but recently they have been reinterpreted as melanosomes; that is, colour-bearing organelles. Resolving this fundamental difference in interpretation is crucial: if endogenous then the fossil microbodies would represent a significant advancement in the fields of palaeontology and evolutionary biology given, for example, the possibility to reconstruct integumentary colours and plumage colour patterns. It has previously been shown that certain trace elements occur in fossils as organometallic compounds, and hence may be used as biomarkers for melanin pigments. Here we expand this knowledge by demonstrating the presence of molecularly preserved melanin in intimate association with melanosome-like microbodies isolated from an argentinoid fish eye from the early Eocene of Denmark.
We describe a new method for simultaneously collecting particles in exhaled air for subsequent chemical analysis and measuring their size distribution. After forced exhalation, particles were counted and collected in spots on silicon wafers with a cascade impactor. Several phospholipids were identified by time-of-flight secondary ion mass spectrometric analysis of the collected spots, suggesting that the particles originated from the lower airways. The amount of particles collected in ten exhalations was sufficient for characterizing the phospholipid composition. The feasibility of the technique in respiratory research is demonstrated by analysis of the phospholipid composition of exhaled particles from healthy controls, patients with asthma, and patients with cystic fibrosis. We believe this technology will be useful for monitoring patients with respiratory disease and has a high potential to detect new biomarkers in exhaled air.
Throughout the animal kingdom, adaptive colouration serves critical functions ranging from inconspicuous camouflage to ostentatious sexual display, and can provide important information about the environment and biology of a particular organism. The most ubiquitous and abundant pigment, melanin, also has a diverse range of non-visual roles, including thermoregulation in ectotherms. However, little is known about the functional evolution of this important biochrome through deep time, owing to our limited ability to unambiguously identify traces of it in the fossil record. Here we present direct chemical evidence of pigmentation in fossilized skin, from three distantly related marine reptiles: a leatherback turtle, a mosasaur and an ichthyosaur. We demonstrate that dark traces of soft tissue in these fossils are dominated by molecularly preserved eumelanin, in intimate association with fossilized melanosomes. In addition, we suggest that contrary to the countershading of many pelagic animals, at least some ichthyosaurs were uniformly dark-coloured in life. Our analyses expand current knowledge of pigmentation in fossil integument beyond that of feathers, allowing for the reconstruction of colour over much greater ranges of extinct taxa and anatomy. In turn, our results provide evidence of convergent melanism in three disparate lineages of secondarily aquatic tetrapods. Based on extant marine analogues, we propose that the benefits of thermoregulation and/or crypsis are likely to have contributed to this melanisation, with the former having implications for the ability of each group to exploit cold environments.
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a promising tool for subcellular chemical analysis of biological cells. However, to obtain relevant information, the method used for sample preparation is critical. In this work, we have used TOF-SIMS, scanning electron microscopy (SEM), and interference reflection microscopy (IRM) to study the effects of different fixation and drying methods on the morphology and chemical structure of human fibroblast cells (hTERT) adhered to a silicon surface. Specifically, two fixation techniques (chemical fixation with glutaraldehyde and cryofixation by plunge freezing) and two drying techniques (freeze drying and alcohol substitution drying) were investigated. Cryofixation followed by freeze drying was determined to produce dried cells with preserved cell morphology, intact cell membranes, and retained sodium/potassium ion concentration gradients across the plasma membrane. By washing samples in an aqueous solution of ammonium formate (AF) before cryofixation, the accumulation of salts on the sample surface during drying could be suppressed. IRM measurements showed that the cell morphology was preserved during washing with ammonium formate, although some swelling occurred. Compared with cryofixation, cells fixed with glutaraldehyde showed finer structures on the cell surface in SEM and similar lipid distributions in TOF-SIMS, but the sodium/potassium ion gradients were not retained. Alcohol drying was determined to remove cell membrane phospholipids significantly, although the use of osmium tetroxide as a post-fixative was shown to decrease this effect.
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