Taphonomic experiments reveal authentic molecular signals for fossil melanins and verify preservation of phaeomelanin in fossils.

Melanin pigments play a critical role in physiological processes and shaping animal behaviour. Fossil melanin is a unique resource for understanding the functional evolution of melanin but the impact of fossilisation on molecular signatures for eumelanin and, especially, phaeomelanin is not fully understood. Here we present a model for the chemical taphonomy of fossil eumelanin and phaeomelanin based on thermal maturation experiments using feathers from extant birds. Our results reveal which molecular signatures are authentic signals for thermally matured eumelanin and phaeomelanin, which signatures are artefacts derived from the maturation of non-melanin molecules, and how these chemical data are impacted by sample preparation. Our model correctly predicts the molecular composition of eumelanins in diverse vertebrate fossils from the Miocene and Cretaceous and, critically, identifies direct molecular evidence for phaeomelanin in these fossils. This taphonomic framework adds to the geochemical toolbox that underpins reconstructions of melanin evolution and of melanin-based coloration in fossil vertebrates.

An ancestral hard-shelled sea turtle with a mosaic of soft skin and scutes.

The transition from terrestrial to marine environments by secondarily aquatic tetrapods necessitates a suite of adaptive changes associated with life in the sea, e.g., the scaleless skin in adult individuals of the extant leatherback turtle. A partial, yet exceptionally preserved hard-shelled (Pan-Cheloniidae) sea turtle with extensive soft-tissue remains, including epidermal scutes and a virtually complete flipper outline, was recently recovered from the Eocene Fur Formation of Denmark. Examination of the fossilized limb tissue revealed an originally soft, wrinkly skin devoid of scales, together with organic residues that contain remnant eumelanin pigment and inferred epidermal transformation products. Notably, this stem cheloniid-unlike its scaly living descendants-combined scaleless limbs with a bony carapace covered in scutes. Our findings show that the adaptive transition to neritic waters by the ancestral pan-chelonioids was more complex than hitherto appreciated, and included at least one evolutionary lineage with a mosaic of integumental features not seen in any living turtle.

3D Molecular Imaging of Stratum Corneum by Mass Spectrometry Suggests Distinct Distribution of Cholesteryl Esters Compared to Other Skin Lipids.

The crucial barrier properties of the stratum corneum (SC) depend critically on the design and integrity of its layered molecular structure. However, analysis methods capable of spatially resolved molecular characterization of the SC are scarce and fraught with severe limitations, e.g., regarding molecular specificity or spatial resolution. Here, we used 3D time-of-flight secondary ion mass spectrometry to characterize the spatial distribution of skin lipids in corneocyte multilayer squams obtained by tape stripping. Depth profiles of specific skin lipids display an oscillatory behavior that is consistent with successive monitoring of individual lipid and corneocyte layers of the SC structure. Whereas the most common skin lipids, i.e., ceramides, C24:0 and C26:0 fatty acids and cholesteryl sulfate, are similarly organized, a distinct 3D distribution was observed for cholesteryl oleate, suggesting a different localization of cholesteryl esters compared to the lipid matrix separating the corneocyte layers. The possibility to monitor the composition and spatial distribution of endogenous lipids as well as active drug and cosmetic substances in individual lipid and corneocyte layers has the potential to provide important contributions to the basic understanding of barrier function and penetration in the SC.

Preservation and Taphonomy of Fossil Insects from the Earliest Eocene of Denmark.

Marine sediments of the lowermost Eocene Stolleklint Clay and Fur Formation of north-western Denmark have yielded abundant well-preserved insects. However, despite a long history of research, in-depth information pertaining to preservational modes and taphonomic pathways of these exceptional animal fossils remains scarce. In this paper, we use a combination of scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX), transmission electron microscopy (TEM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) to assess the ultrastructural and molecular composition of three insect fossils: a wasp (Hymenoptera), a damselfly (Odonata) and a pair of beetle elytra (Coleoptera). Our analyses show that all specimens are preserved as organic remnants that originate from the exoskeleton, with the elytra displaying a greater level of morphological fidelity than the other fossils. TEM analysis of the elytra revealed minute features, including a multilayered epicuticle comparable to those nanostructures that generate metallic colors in modern insects. Additionally, ToF-SIMS analyses provided spectral evidence for chemical residues of the pigment eumelanin as part of the cuticular remains. To the best of our knowledge, this is the first occasion where both structural colors and chemical traces of an endogenous pigment have been documented in a single fossil specimen. Overall, our results provide novel insights into the nature of insect body fossils and additionally shed light on exceptionally preserved terrestrial insect faunas found in marine paleoenvironments.

Chemical Evaluation of Eumelanin Maturation by ToF-SIMS and Alkaline Peroxide Oxidation HPLC Analysis.

Residual melanins have been detected in multimillion-year-old animal body fossils; however, confident identification and characterization of these natural pigments remain challenging due to loss of chemical signatures during diagenesis. Here, we simulate this post-burial process through artificial maturation experiments using three synthetic and one natural eumelanin exposed to mild (100 °C/100 bar) and harsh (250 °C/200 bar) environmental conditions, followed by chemical analysis employing alkaline hydrogen peroxide oxidation (AHPO) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Our results show that AHPO is sensitive to changes in the melanin molecular structure already during mild heat and pressure treatment (resulting, e.g., in increased C-C cross-linking), whereas harsh maturation leads to extensive loss of eumelanin-specific chemical markers. In contrast, negative-ion ToF-SIMS spectra are considerably less affected by mild maturation conditions, and eumelanin-specific features remain even after harsh treatment. Detailed analysis of ToF-SIMS spectra acquired prior to experimental treatment revealed significant differences between the investigated eumelanins. However, systematic spectral changes upon maturation reduced these dissimilarities, indicating that intense heat and pressure treatment leads to the formation of a common, partially degraded, eumelanin molecular structure. Our findings elucidate the complementary nature of AHPO and ToF-SIMS during chemical characterization of eumelanin traces in fossilized organismal remains.

Omega-3 Polyunsaturated Fatty Acids Decrease Aortic Valve Disease Through the Resolvin E1 and ChemR23 Axis.

BACKGROUND: Aortic valve stenosis (AVS), which is the most common valvular heart disease, causes a progressive narrowing of the aortic valve as a consequence of thickening and calcification of the aortic valve leaflets. The beneficial effects of omega-3 polyunsaturated fatty acids (n-3 PUFAs) in cardiovascular prevention have recently been demonstrated in a large randomized, controlled trial. In addition, n-3 PUFAs serve as the substrate for the synthesis of specialized proresolving mediators, which are known by their potent beneficial anti-inflammatory, proresolving, and tissue-modifying properties in cardiovascular disease. However, the effects of n-3 PUFA and specialized proresolving mediators on AVS have not yet been determined. The aim of this study was to identify the role of n-3 PUFA-derived specialized proresolving mediators in relation to the development of AVS. METHODS: Lipidomic and transcriptomic analyses were performed in human tricuspid aortic valves. Apoe-/- mice and wire injury in C57BL/6J mice were used as models for mechanistic studies. RESULTS: We found that n-3 PUFA incorporation into human stenotic aortic valves was higher in noncalcified regions compared with calcified regions. Liquid chromatography tandem mass spectrometry-based lipid mediator lipidomics identified that the n-3 PUFA-derived specialized proresolving mediator resolvin E1 was dysregulated in calcified regions and acted as a calcification inhibitor. Apoe-/- mice expressing the Caenorhabditis elegans Fat-1 transgene (Fat-1tg×Apoe-/-), which enables the endogenous synthesis of n-3 PUFA and increased valvular n-3 PUFA content, exhibited reduced valve calcification, lower aortic valve leaflet area, increased M2 macrophage polarization, and improved echocardiographic parameters. Finally, abrogation of the resolvin E1 receptor ChemR23 enhanced disease progression, and the beneficial effects of Fat-1tg were abolished in the absence of ChemR23. CONCLUSIONS: n-3 PUFA-derived resolvin E1 and its receptor ChemR23 emerge as a key axis in the inhibition of AVS progression and may represent a novel potential therapeutic opportunity to be evaluated in patients with AVS.

Fossil insect eyes shed light on trilobite optics and the arthropod pigment screen.

Fossilized eyes permit inferences of the visual capacity of extinct arthropods1-3. However, structural and/or chemical modifications as a result of taphonomic and diagenetic processes can alter the original features, thereby necessitating comparisons with modern species. Here we report the detailed molecular composition and microanatomy of the eyes of 54-million-year-old crane-flies, which together provide a proxy for the interpretation of optical systems in some other ancient arthropods. These well-preserved visual organs comprise calcified corneal lenses that are separated by intervening spaces containing eumelanin pigment. We also show that eumelanin is present in the facet walls of living crane-flies, in which it forms the outermost ommatidial pigment shield in compound eyes incorporating a chitinous cornea. To our knowledge, this is the first record of melanic screening pigments in arthropods, and reveals a fossilization mode in insect eyes that involves a decay-resistant biochrome coupled with early diagenetic mineralization of the ommatidial lenses. The demonstrable secondary calcification of lens cuticle that was initially chitinous has implications for the proposed calcitic corneas of trilobites, which we posit are artefacts of preservation rather than a product of in vivo biomineralization4-7. Although trilobite eyes might have been partly mineralized for mechanical strength, a (more likely) organic composition would have enhanced function via gradient-index optics and increased control of lens shape.

Triglyceride-Rich Lipoproteins as a Source of Proinflammatory Lipids in the Arterial Wall.

Apolipoprotein B -containing lipoproteins include triglyceride-rich lipoproteins (chylomicrons and their remnants, and very low-density lipoproteins and their remnants) and cholesterol-rich low-density lipoprotein particles. Of these, lipoproteins having sizes below 70-80 nm may enter the arterial wall, where they accumulate and induce the formation of atherosclerotic lesions. The processes that lead to accumulation of lipoprotein-derived lipids in the arterial wall have been largely studied with a focus on the low-density lipoprotein particles. However, recent observational and genetic studies have discovered that the triglyceriderich lipoproteins and their remnants are linked with cardiovascular disease risk. In this review, we describe the potential mechanisms by which the triglyceride-rich remnant lipoproteins can contribute to the development of atherosclerotic lesions, and highlight the differences in the atherogenicity between low-density lipoproteins and the remnant lipoproteins.

Soft-tissue evidence for homeothermy and crypsis in a Jurassic ichthyosaur.

Ichthyosaurs are extinct marine reptiles that display a notable external similarity to modern toothed whales. Here we show that this resemblance is more than skin deep. We apply a multidisciplinary experimental approach to characterize the cellular and molecular composition of integumental tissues in an exceptionally preserved specimen of the Early Jurassic ichthyosaur Stenopterygius. Our analyses recovered still-flexible remnants of the original scaleless skin, which comprises morphologically distinct epidermal and dermal layers. These are underlain by insulating blubber that would have augmented streamlining, buoyancy and homeothermy. Additionally, we identify endogenous proteinaceous and lipid constituents, together with keratinocytes and branched melanophores that contain eumelanin pigment. Distributional variation of melanophores across the body suggests countershading, possibly enhanced by physiological adjustments of colour to enable photoprotection, concealment and/or thermoregulation. Convergence of ichthyosaurs with extant marine amniotes thus extends to the ultrastructural and molecular levels, reflecting the omnipresent constraints of their shared adaptation to pelagic life.

Imaging the distribution of skin lipids and topically applied compounds in human skin using mass spectrometry.

The barrier functions of skin against water loss, microbial invasion and penetration of xenobiotics rely, in part, on the spatial distribution of the biomolecular constituents in the skin structure, particularly its horny layer (stratum corneum). However, all skin layers are important to describe normal and dysfunctional skin conditions, and to develop adapted therapies or skin care products. In this work, time-of-flight secondary ion mass spectrometry (ToF-SIMS) combined with scanning electron microscopy (SEM) was used to image the spatial distribution of a variety of molecular species, from stratum corneum down to dermis, in cross-section samples of human abdominal skin. The results demonstrate the expected localization of ceramide and saturated long-chain fatty acids in stratum corneum (SC) and cholesterol sulfate in the upper part of the viable epidermis. The localization of exogenous compounds is demonstrated by the detection and imaging of carvacrol (a constituent of oregano or thyme essential oil) and ceramide, after topical application onto ex vivo human skin. Carvacrol showed pronounced accumulation to triglyceride-containing structures in the deeper parts of dermis. In contrast, the exogenous ceramide was found to be localized in SC. Furthermore, the complementary character of this approach with classical ex vivo skin absorption analysis methods is demonstrated.

Preservation potential of keratin in deep time.

Multiple fossil discoveries and taphonomic experiments have established the durability of keratin. The utility and specificity of antibodies to identify keratin peptides has also been established, both in extant feathers under varying treatment conditions, and in feathers from extinct organisms. Here, we show localization of feather-keratin antibodies to control and heat-treated feathers, testifying to the repeatability of initial data supporting the preservation potential of keratin. We then show new data at higher resolution that demonstrates the specific response of these antibodies to the feather matrix, we support the presence of protein in heat-treated feathers using ToF-SIMS, and we apply these methods to a fossil feather preserved in the unusual environment of sinter hot springs. We stress the importance of employing realistic conditions such as sediment burial when designing experiments intended as proxies for taphonomic processes occurring in the fossil record. Our data support the hypothesis that keratin, particularly the ß-keratin that comprises feathers, has potential to preserve in fossil remains.

Structure and Composition of Native Membrane Derived Polymer-Supported Lipid Bilayers.

Over the last two decades, supported lipid bilayers (SLBs) have been extensively used as model systems to study cell membrane structure and function. While SLBs have been traditionally produced from simple lipid mixtures, there has been a recent surge in compositional complexity to better mimic cellular membranes and thereby bridge the gap between classic biophysical approaches and cell experiments. To this end, native cellular membrane derived SLBs (nSLBs) have emerged as a new category of SLBs. As a new type of biomimetic material, an analytical workflow must be designed to characterize its molecular composition and structure. Herein, we demonstrate how a combination of fluorescence microscopy, neutron reflectometry, and secondary ion mass spectrometry offers new insights on structure, composition, and quality of nSLB systems formed using so-called hybrid vesicles, which are a mixture of native membrane material and synthetic lipids. With this approach, we demonstrate that the nSLB formed a continuous structure with complete mixing of the synthetic and native membrane components and a molecular stoichiometry that essentially mirrors that of the hybrid vesicles. Furthermore, structural investigation of the nSLB revealed that PEGylated lipids do not significantly thicken the hydration layer between the bilayer and substrate when on silicon substrates; however, nSLBs do have more topology than their simpler, purely synthetic counterparts. Beyond new insights regarding the structure and composition of nSLB systems, this work also serves to guide future researchers in producing and characterizing nSLBs from their cellular membrane of choice.

Toward multiplexed quantification of biomolecules on surfaces using time-of-flight secondary ion mass spectrometry.

Accurate detection and quantification of individual molecules is important for the development of improved diagnostic methods as well as biochemical characterization of disease progression and treatments. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a surface analysis technique capable of imaging the distribution of specific molecules on surfaces with a high spatial resolution (<1 µm) and high sensitivity. ToF-SIMS is particularly suitable for detection of molecules up to ∼2 kDa, including lipids, whereas larger molecules, such as peptides and proteins, are fragmented during analysis, which makes them difficult to identify. In this study, an approach for extending the molecular detection capability of ToF-SIMS is presented, based on the specific binding of functionalized liposomes to molecular targets on the sample surface and subsequent detection of the liposomes by ToF-SIMS. Furthermore, by using different recognition elements conjugated to liposomes with different lipid compositions, simultaneous detection of different targets was accomplished. This multiplexing capability was investigated for two types of recognition elements (antibodies and cholera toxin) and for target molecules immobilized on surfaces using two frequently applied surface functionalization strategies: a supported lipid bilayer aimed to mimic a cell membrane and a polyethylene glycol modified surface, commonly employed in bioanalytical sensor applications. The efficacy of the conjugation protocols and the specificity of the recognition mechanism were confirmed using quartz crystal microbalance with dissipation monitoring, while fluorescence microscopy was used to validate the ToF-SIMS data and the reliability of the freeze-drying step required for ToF-SIMS analysis. The results demonstrated specific binding of the two types of liposomes to each target and showed a concentration-dependent binding to the targets on the different model surfaces. In particular, the possibility to use the contrasts in the mass spectra of SIMS to identify the concentration dependent coverage of different liposomes opens up new opportunities for multiplexed detection and quantification of molecules at biotechnology relevant interfaces.

Nanometer-scale molecular organization in lipid membranes studied by time-of-flight secondary ion mass spectrometry.

The organization of lipid membranes plays an important role in a wide range of biological processes at different length scales. Herein, the authors present a procedure based on time-of-flight secondary ion mass spectrometry (ToF-SIMS) to characterize the nanometer-scale ordering of lipids in lipid membrane structures on surfaces. While ToF-SIMS is a powerful tool for label-free analysis of lipid-containing samples, its limited spatial resolution prevents in-depth knowledge of how lipid properties affect the molecular assembly of the membrane. The authors overcome this limitation by measuring the formation of lipid dimers, originating in the same nanometer-sized primary ion impact areas. The lipid dimers reflect the local lipid environment and thus allow us to characterize the membrane miscibility on the nanometer level. Using this technique, the authors show that the chemical properties of the constituting lipids are critical for the structure and organization of the membrane on both the nanometer and micrometer length scales. Our results show that even at lipid surface compositions favoring two-phase systems, lipids are still extracted from solid, gel phase, domains into the surrounding fluid supported lipid bilayer surrounding the gel phase domains. The technique offers a means to obtain detailed knowledge of the chemical composition and organization of lipid membranes with potential application in systems where labeling is not possible, such as cell-derived supported lipid bilayers.

Biochemistry and adaptive colouration of an exceptionally preserved juvenile fossil sea turtle.

The holotype (MHM-K2) of the Eocene cheloniine Tasbacka danica is arguably one of the best preserved juvenile fossil sea turtles on record. Notwithstanding compactional flattening, the specimen is virtually intact, comprising a fully articulated skeleton exposed in dorsal view. MHM-K2 also preserves, with great fidelity, soft tissue traces visible as a sharply delineated carbon film around the bones and marginal scutes along the edge of the carapace. Here we show that the extraordinary preservation of the type of T. danica goes beyond gross morphology to include ultrastructural details and labile molecular components of the once-living animal. Haemoglobin-derived compounds, eumelanic pigments and proteinaceous materials retaining the immunological characteristics of sauropsid-specific ß-keratin and tropomyosin were detected in tissues containing remnant melanosomes and decayed keratin plates. The preserved organics represent condensed remains of the cornified epidermis and, likely also, deeper anatomical features, and provide direct chemical evidence that adaptive melanism - a biological means used by extant sea turtle hatchlings to elevate metabolic and growth rates - had evolved 54 million years ago.

Probing amyloid-ß pathology in transgenic Alzheimer's disease (tgArcSwe) mice using MALDI imaging mass spectrometry.

The pathological mechanisms underlying Alzheimer's disease (AD) are still not understood. The disease pathology is characterized by the accumulation and aggregation of amyloid-ß (Aß) peptides into extracellular plaques, however the factors that promote neurotoxic Aß aggregation remain elusive. Imaging mass spectrometry (IMS) is a powerful technique to comprehensively elucidate the spatial distribution patterns of lipids, peptides and proteins in biological tissues. In the present study, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS)-based imaging was used to study Aß deposition in transgenic mouse brain tissue and to elucidate the plaque-associated chemical microenvironment. The imaging experiments were performed in brain sections of transgenic Alzheimer's disease mice carrying the Arctic and Swedish mutation of amyloid-beta precursor protein (tgArcSwe). Multivariate image analysis was used to interrogate the IMS data for identifying pathologically relevant, anatomical features based on their chemical identity. This include cortical and hippocampal Aß deposits, whose amyloid peptide content was further verified using immunohistochemistry and laser microdissection followed by MALDI MS analysis. Subsequent statistical analysis on spectral data of regions of interest revealed brain region-specific differences in Aß peptide aggregation. Moreover, other plaque-associated protein species were identified including macrophage migration inhibitory factor suggesting neuroinflammatory processes and glial cell reactivity to be involved in AD pathology. The presented data further highlight the potential of IMS as a powerful approach in neuropathology. Hanrieder et al. described an imaging mass spectrometry based study on comprehensive spatial profiling of C-terminally truncated Aß species within individual plaques in tgArcSwe mice. Here, brain region-dependent differences in Aß truncation and other plaque-associated proteins, such as macrophage migration inhibitory factor, were observed. The data shed further light on plaque-associated molecular mechanisms implicated in Alzheimer's pathogenesis. Cover image for this issue: doi: 10.1111/jnc.13328.

Imaging of amyloid-ß in Alzheimer's disease transgenic mouse brains with ToF-SIMS using immunoliposomes.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been proven to successfully image different kinds of molecules, especially a variety of lipids, in biological samples. Proteins, however, are difficult to detect as specific entities with this method due to extensive fragmentation. To circumvent this issue, the authors present in this work a method developed for detection of proteins using antibody-conjugated liposomes, so called immunoliposomes, which are able to bind to the specific protein of interest. In combination with the capability of ToF-SIMS to detect native lipids in tissue samples, this method opens up the opportunity to analyze many different biomolecules, both lipids and proteins, at the same time, with high spatial resolution. The method has been applied to detect and image the distribution of amyloid-ß (Aß), a biologically relevant peptide in Alzheimer's disease (AD), in transgenic mouse brain tissue. To ensure specific binding, the immunoliposome binding was verified on a model surface using quartz crystal microbalance with dissipation monitoring. The immunoliposome binding was also investigated on tissue sections with fluorescence microscopy, and compared with conventional immunohistochemistry using primary and secondary antibodies, demonstrating specific binding to Aß. Using ToF-SIMS imaging, several endogenous lipids, such as cholesterol and sulfatides, were also detected in parallel with the immunoliposome-labeled Aß deposits, which is an advantage compared to fluorescence microscopy. This method can thus potentially provide further information about lipid-protein interactions, which is important to understand the mechanisms of neurodegeneration in AD.

Matrix effects in nilotinib formulations with pH-responsive polymer produced by carbon dioxide-mediated precipitation.

Factors determining the pH-controlled dissolution kinetics of nilotinib formulations with the pH-titrable polymer hydroxypropyl methylcellulose phthalate, obtained by carbon dioxide-mediated precipitation, were mechanistically examined in acid and neutral environment. The matrix effect, modulating the drug dissolution, was characterized with a battery of physicochemical methodologies, including ToF-SIMS for surface composition, SAXS/WAXS and modulated DSC for crystallization characterization, and simultaneous UV-imaging and Raman spectroscopy for monitoring the dissolution process in detail. The hybrid particle formulations investigated consisted of amorphous nilotinib embedded in a polymer matrix in single continuous phase, displaying extended retained amorphicity also under wet conditions. It was demonstrated by Raman and FTIR spectroscopy that the efficient drug dispersion and amorphization in the polymer matrix were mediated by hydrogen bonding between the drug and the phthalate groups on the polymer. Simultaneous Raman and UV-imaging studies of the effect of drug load on the swelling and dissolution of the polymer matrix revealed that high nilotinib load prevented matrix swelling on passage from acid to neutral pH, thereby preventing re-precipitation and re-crystallization of incorporated nilotinib. These findings provide a mechanistic foundation of formulation development of nilotinib and other protein kinase inhibitors, which are now witnessing an intense therapeutic and industrial attention due to the difficulty in formulating these compounds so that efficient oral bioavailability is reached.

Molecular composition and ultrastructure of Jurassic paravian feathers.

Feathers are amongst the most complex epidermal structures known, and they have a well-documented evolutionary trajectory across non-avian dinosaurs and basal birds. Moreover, melanosome-like microbodies preserved in association with fossil plumage have been used to reconstruct original colour, behaviour and physiology. However, these putative ancient melanosomes might alternatively represent microorganismal residues, a conflicting interpretation compounded by a lack of unambiguous chemical data. We therefore used sensitive molecular imaging, supported by multiple independent analytical tests, to demonstrate that the filamentous epidermal appendages in a new specimen of the Jurassic paravian Anchiornis comprise remnant eumelanosomes and fibril-like microstructures, preserved as endogenous eumelanin and authigenic calcium phosphate. These results provide novel insights into the early evolution of feathers at the sub-cellular level, and unequivocally determine that melanosomes can be preserved in fossil feathers.