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Elemental characterisation of melanin in feathers via synchrotron X-ray imaging and absorption spectroscopy
Scientific Reports
Abstract
Melanin is a critical component of biological systems, but the exact chemistry of melanin is still imprecisely known. This is partly due to melanin’s complex heterogeneous nature and partly because many studies use synthetic analogues and/or pigments extracted from their natural biological setting, which may display important differences from endogenous pigments. Here we demonstrate how synchrotron X-ray analyses can non-destructively characterise the elements associated with melanin pigment in situ within extant feathers. Elemental imaging shows that the distributions of Ca, Cu and Zn are almost exclusively controlled by melanin pigment distribution. X-ray absorption spectroscopy demonstrates that the atomic coordination of zinc and sulfur is different within eumelanised regions compared to pheomelanised regions. This not only impacts our fundamental understanding of pigmentation in extant organisms but also provides a significant contribution to the evidence-based colour palette available for reconstructing the appearance of fossil organisms.
Supplementary resource (1)
Data
September 2016
... [16][17][18][19][20][21] However, with the use of new imaging and analytical methods such as X-ray spectroscopy, Xray fluorescence (XRF) analyses, secondary-ion mass spectroscopy, and Raman spectroscopy, interdisciplinary studies utilizing chemistry to investigate taphonomy are becoming increasingly common. [22][23][24][25][26][27][28][29][30][31][32][33][34] XRF is a method that uses X-rays to excite elements in a sample, providing compositional data that can be qualitatively and quantitatively analyzed to better understand taphonomic pathways in fossils. XRF has been applied to a wide range of fossil groups including vertebrates, invertebrates, and plants. ...
... 32,34 Recent application of this technology to the study of extinct paravians have provided insights into the fossilization process of bones, feathers, and other soft tissues. 23,[26][27][28][29][30][31][32][33][34] Soft tissues such as visceral organs and integument (e.g., feathers and scales, etc.) preserved in exceptional specimens of feathered non-avialan dinosaurs, fossil birds, and primitive mammals have provided irreplaceable information for understanding the biological and ecological evolution of these animals. 23,[42][43][44][45][46][47] Taphonomic studies link the preservation of these soft tissues to the deposition of finely layered shale or mudstone interbedded with ash layers resulting from volcanic eruptions. ...
... Ca, Cu, and Ni are associated with melanin pigmentation in modern and fossil feathers. 22,29,30,39,58 As previously documented in other paravian fossils such as Jianianhualong, Anchiornis, Archaeopteryx, and Gansus, as well as other extinct and living birds, Cu levels usually vary between different feather samples within an individual and are generally higher in the feathers than in bone . 26,28,32,39,58 Cu is distributed fairly evenly throughout the preserved feathers of F4282 and is relatively enriched in the tail feathers, but does not show any significant between feather tracts. ...
... The cystine peak is strong in white and especially black feathers. Spectra for both feather colours also show a weak, broad peak for cysteic acid (centred at 2,480.92 eV; sulfoxide; S=O) and a weak shoulder for benzothiazole (centred at 2,476 eV; S-heterocycle) 22 . ...
... eV) (ref. 22). ...
... Similar to cysteine, benzothiazole sulfur has an excitation energy of 2,474.12 eV and can be observed in spectra only via the presence of a shoulder feature at 2,476 eV 22 . For details on the diagnostic X-ray energies (that is, usually the energy corresponding to the first sharp peak in a XANES spectrum) for analytical standards of selected sulfur-bearing compounds, see Extended Data Fig. 4 and Extended Data Table 3. ...
Fossil proteins are valuable tools in evolutionary biology. Recent technological advances and better integration of experimental methods have confirmed the feasibility of biomolecular preservation in deep time, yielding new insights into the timing of key evolutionary transitions. Keratins (formerly α-keratins) and corneous β-proteins (CBPs, formerly β-keratins) are of particular interest as they define tissue structures that underpin fundamental physiological and ecological strategies and have the potential to inform on the molecular evolution of the vertebrate integument. Reports of CBPs in Mesozoic fossils, however, appear to conflict with experimental evidence for CBP degradation during fossilization. Further, the recent model for molecular modification of feather chemistry during the dinosaur–bird transition does not consider the relative preservation potential of different feather proteins. Here we use controlled taphonomic experiments coupled with infrared and sulfur X-ray spectroscopy to show that the dominant β-sheet structure of CBPs is progressively altered to α-helices with increasing temperature, suggesting that (α-)keratins and α-helices in fossil feathers are most likely artefacts of fossilization. Our analyses of fossil feathers shows that this process is independent of geological age, as even Cenozoic feathers can comprise primarily α-helices and disordered structures. Critically, our experiments show that feather CBPs can survive moderate thermal maturation. As predicted by our experiments, analyses of Mesozoic feathers confirm that evidence of feather CBPs can persist through deep time.
... Paleontological objects (fossilized remains of animals and plants) have the potential to preserve remnant chemistry of the original organism (some constrained by specific biomarkers). Such fossils include mineralized soft Sciau et al. (2006), Liu et al. (2007), Lynch et al. (2007), Mirguet (2009), Leon et al. (2010, Jackson et al. (2013Jackson et al. ( , 2014Jackson et al. ( , 2017, Dejoie et al. (2014aDejoie et al. ( , b, 2015, Leon et al. (2015), and Bertrand et al. (2017) Advanced Photon Source ( 1-5 μm 2 Dredge et al. (2015Dredge et al. ( , 2017, MacLeod (2015), Popelka-Filcoff et al. (2015, Jones et al. (2016), Thurrowgood et al. (2016, Alfeld andde Viguerie (2017), de Jonge et al. (2017), Sharp-Paul (2017), Kirkwood et al. (2018), Ryan et al. ( , 2014Ryan et al. ( , 2018, Paterson et al. (2011), andHoward et al. (2012) XAS Riesemeier et al. (2005), Reiche et al. (2006Reiche et al. ( , 2011, and Radtke et al. (2013Radtke et al. ( , 2016 CHESS μm Swanston et al. (2012Swanston et al. ( , 2015, Michelin et al. (2013), Rouchon and Bernard (2015), and Alleon et al. (2016aand Alleon et al. ( , 2017 Canadian Light Source ( Michelin et al. (2013), Rouchon and Bernard (2015), Alleon et al. (2016aAlleon et al. ( , 2017, and Gueriau et al. (2017) 11ID-1 SGM ∼20-100 μm diameter Bergmann ( , 2007Bergmann ( , 2011, Bergmann and Knox (2009, Larson et al. (2010), Sadeghi and Bergmann (2010), Edwards et al. (2011aEdwards et al. ( , b, 2013Edwards et al. ( , 2014Edwards et al. ( , 2016Edwards et al. ( , 2018, Wogelius et al. (2011), Manning et al. (2013), Anne et al. (2014Anne et al. ( , 2017Anne et al. ( , 2018, Barden et al. (2015a, b), Egerton et al. (2015), Harazim et al. (2015), Gueriau et al. (2017), and Gibson et al. (2018) 10-2 Bertrand et al. (2011), andHoerlé et al. (2016) tissue that is not seen in the visible light spectrum and is often overlooked in such fossils. This additional information can lead to a better understanding of the pigmentation, anatomy, development and growth stage, diet, and even health of the organism prior to death. ...
... The first SRS-XRF imaging instrument at SSRL beamline 6-2 was originally designed for imaging of the Archimedes Palimpsest (Bergmann , 2007(Bergmann , 2011Bergmann and Knox 2009). This instrument, which was operational until 2017, has since been extensively applied in the field of archaeology, medical imaging, geology, biology, and paleontology (Bergmann et al. , 2012Larson et al. 2010;Sadeghi and Bergmann 2010;Edwards et al. 2011aEdwards et al. , b, 2013Edwards et al. , 2014Edwards et al. , 2016Edwards et al. , 2018Wogelius et al. 2011;Manning et al. 2013;Anne et al. 2014Anne et al. , 2017Anne et al. , 2018Barden et al. 2015a, b;Egerton et al. 2015;Harazim et al. 2015;Gibson et al. 2018;Popescu et al. 2009a, b, c;Popescu 2009). Being optimized for high speed, one limitation of this instrument was the fact that only 16 channels were recorded for each pixel due to limits in software and hardware. ...
... Such information would allow more accurate pigment reconstruction within fossils (brown/black versus red/yellow). Therefore, we performed a study of modern feathers known to be pigmented with both eumelanin and pheomelanin (Edwards et al. 2016). The primary difference between eumelanin and pheomelanin was intimate coordination of zinc with sulfur in the pheomelanin structure (Ito et al. 2011). ...
... Paleontological objects (fossilized remains of animals and plants) have the potential to preserve remnant chemistry of the original organism (some constrained by specific biomarkers). Such fossils include mineralized soft Sciau et al. (2006), Liu et al. (2007), Lynch et al. (2007), Mirguet (2009), Leon et al. (2010, Jackson et al. (2013Jackson et al. ( , 2014Jackson et al. ( , 2017, Dejoie et al. (2014aDejoie et al. ( , b, 2015, Leon et al. (2015), and Bertrand et al. (2017) Advanced Photon Source ( 1-5 μm 2 Dredge et al. (2015Dredge et al. ( , 2017, MacLeod (2015), Popelka-Filcoff et al. (2015, Jones et al. (2016), Thurrowgood et al. (2016, Alfeld andde Viguerie (2017), de Jonge et al. (2017), Sharp-Paul (2017), Kirkwood et al. (2018), Ryan et al. ( , 2014Ryan et al. ( , 2018, Paterson et al. (2011), andHoward et al. (2012) XAS Riesemeier et al. (2005), Reiche et al. (2006Reiche et al. ( , 2011, and Radtke et al. (2013Radtke et al. ( , 2016 CHESS μm Swanston et al. (2012Swanston et al. ( , 2015, Michelin et al. (2013), Rouchon and Bernard (2015), and Alleon et al. (2016aand Alleon et al. ( , 2017 Canadian Light Source ( Michelin et al. (2013), Rouchon and Bernard (2015), Alleon et al. (2016aAlleon et al. ( , 2017, and Gueriau et al. (2017) 11ID-1 SGM ∼20-100 μm diameter Bergmann ( , 2007Bergmann ( , 2011, Bergmann and Knox (2009, Larson et al. (2010), Sadeghi and Bergmann (2010), Edwards et al. (2011aEdwards et al. ( , b, 2013Edwards et al. ( , 2014Edwards et al. ( , 2016Edwards et al. ( , 2018, Wogelius et al. (2011), Manning et al. (2013), Anne et al. (2014Anne et al. ( , 2017Anne et al. ( , 2018, Barden et al. (2015a, b), Egerton et al. (2015), Harazim et al. (2015), Gueriau et al. (2017), and Gibson et al. (2018) 10-2 Bertrand et al. (2011), andHoerlé et al. (2016) tissue that is not seen in the visible light spectrum and is often overlooked in such fossils. This additional information can lead to a better understanding of the pigmentation, anatomy, development and growth stage, diet, and even health of the organism prior to death. ...
... The first SRS-XRF imaging instrument at SSRL beamline 6-2 was originally designed for imaging of the Archimedes Palimpsest (Bergmann , 2007(Bergmann , 2011Bergmann and Knox 2009). This instrument, which was operational until 2017, has since been extensively applied in the field of archaeology, medical imaging, geology, biology, and paleontology (Bergmann et al. , 2012Larson et al. 2010;Sadeghi and Bergmann 2010;Edwards et al. 2011aEdwards et al. , b, 2013Edwards et al. , 2014Edwards et al. , 2016Edwards et al. , 2018Wogelius et al. 2011;Manning et al. 2013;Anne et al. 2014Anne et al. , 2017Anne et al. , 2018Barden et al. 2015a, b;Egerton et al. 2015;Harazim et al. 2015;Gibson et al. 2018;Popescu et al. 2009a, b, c;Popescu 2009). Being optimized for high speed, one limitation of this instrument was the fact that only 16 channels were recorded for each pixel due to limits in software and hardware. ...
... Such information would allow more accurate pigment reconstruction within fossils (brown/black versus red/yellow). Therefore, we performed a study of modern feathers known to be pigmented with both eumelanin and pheomelanin (Edwards et al. 2016). The primary difference between eumelanin and pheomelanin was intimate coordination of zinc with sulfur in the pheomelanin structure (Ito et al. 2011). ...
... In this regard, soft tissue residue is notoriously difficult to study. Synchrotron rapid scanning-X-ray fluorescence (SRS-XRF) imaging combined with X-ray absorption spectroscopy (XAS) has provided new information concerning biomarkers for pigment and other components of animal and plant tissue [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] . Integumentary pigmentation is of particular interest, as it plays key physiological and behavioral roles and can change relatively quickly on evolutionary timescales with important consequences for the species. ...
... Previously, work on fossil integument concluded that organosulfur-Zn complexes may be the residue of pheomelanin 7 ; however, detailed coordination chemistry for Zn in extant pheomelanin to use in comparison with the fossils was unavailable. Subsequently, Edwards et al. 1 applied detailed XRF and XAS to extant pheomelanin-rich feathers. The results showed that these feathers possessed a distinct chemical signature for Zn and S, with a significant portion of the Zn inventory bonded to S, almost certainly through the S contained within the pheomelanin molecule. ...
... ToF-SIMS requires destructive subsampling and can only image microscopic areas (e.g.,~10 −4 cm 2 ) of large organisms such as the avians, mammals, and fish (>10 2 cm 2 ) that are of interest in vertebrate paleontology. Previous work has indeed shown that whole-organism extrapolations from microscopic areas can be misleading 1,3 . Therefore, the work presented here seeks to test whether nondestructive and extremely sensitive synchrotron-based methods (including XAS) may be able to resolve traces of pheomelanin residue in fossil specimens and map this residue over the entire organism length scales, as we have shown is possible in extant tissue 1 . ...
Recent progress has been made in paleontology with respect to resolving pigmentation in fossil material. Morphological identification of fossilized melanosomes has been one approach, while a second methodology using chemical imaging and spectroscopy has also provided critical information particularly concerning eumelanin (black pigment) residue. In this work we develop the chemical imaging methodology to show that organosulfur-Zn complexes are indicators of pheomelanin (red pigment) in extant and fossil soft tissue and that the mapping of these residual biochemical compounds can be used to restore melanin pigment distribution in a 3 million year old extinct mammal species (Apodemus atavus). Synchotron Rapid Scanning X-ray Fluorescence imaging showed that the distributions of Zn and organic S are correlated within this fossil fur just as in pheomelanin-rich modern integument. Furthermore, Zn coordination chemistry within this fossil fur is closely comparable to that determined from pheomelanin-rich fur and hair standards. The non-destructive methods presented here provide a protocol for detecting residual pheomelanin in precious specimens.
... It induces aggregation of melanin particles, making feather more durable and resistant to mechanical stress (Niecke et al., 1999). Synchrotron X-ray analyses showed that the distribution of calcium is controlled by melanin pigment and is nonuniformly distributed throughout feather parts (Edwards et al., 2016;Howell et al., 2017). In a study performed on male Barn Owls, Roulin, Dauwe, Blust, Eens, and Beaud (2006) demonstrated a positive relationship between enhanced calcium deposition in humerus bone and plumage spottiness. ...
... It was also found in nonpigmented feather parts. Zinc appeared to have higher affinity for pheomelanin than eumelanin, and relative, not absolute concentrations correlating with pigment patterns (Edwards et al., 2016). ...
... Similarly to zinc and calcium, copper is controlled by melanin pigment patterns in feathers (Edwards et al., 2016). Both analyses in this study employed a similar sample size in the calculation of average effect size. ...
Environmental pollution, for example with metals, can significantly affect the ecosystem balance leading to severe changes. Biologically active pigments are relevant for the appearance and condition of birds. Melanin and carotenoid particles are the most frequently deposited pigments in avian integument. They are responsible for the majority of colors of bird plumage. The phenotypic expression can be affected by metal contamination. It can be manifested as color bleaching or differences in the size of plumage badges. In this study, we performed a comprehensive review of related studies in order to estimate the underlying population effect of this potential dependency. The study is based on the review of the literature regarding several avian species. It was designed to identify an area where the effect of the exposure is still poorly known. The analysis was specifically conducted to investigate the correlation between trace element concentration and eumelanin deposition. Moreover, we searched for factors that could affect spectral properties of feathers with carotenoid‐based pigmentation. As a result, we found carotenoid‐based pigmentation to be of a good use in terms of visual condition assessment. Changes in melanin‐based pattern should be analyzed separately for eu‐ and pheomelanin as well as for a range of essential and toxic elements. Comprehensive studies on the subject are still scarce. Therefore, the issue requires further investigation.
... During the process of fossilization, various kinds of released material https://onlinelibrary.wiley.com/doi/full/10.1111/iar.12495?saml_referrer 2/19 from the decayed soft tissue exchange its surrounding sediments, and remained markers and traces. The remains in sediments, such as mineralized bones, carbon membranes (Bergmann et al., 2012;Egerton et al., 2015;Oehler et al., 2006), and external molds are preserved signals of the anatomy of fossilized vertebrates, and the history of its degradation, mineralization, and diagenesis (Edwards et al., 2016;Pan et al., 2016Pan et al., , 2019. The released materials from degrading soft body provide clues for reconstructing the burial environment and form authigenic minerals, such as opal from occulated silica colloids (Chen et al., 2007;Toporski et al., 2002). ...
... surface (Edwards et al., 2016;Yang et al., 2008). However, elemental mapping by EDS and SXRF has the following limitations: the sample size was limited by the space of the vacuum chamber (Feng et al., 2007;Shackley, 2011;Zhang et al., 2019); the requirement of producing a plane and polished surface limited non-destructive analysis of fossils. ...
Vertebrate fossils usually consist only of mineralized skeletons and rarely preserve soft body parts. However, the buried soft body releases the degraded material into the surrounding sediments. The degraded material can be preserved as invisible signals of some chemicals or authigenic minerals, which can be revealed by the elemental distribution of the fossils' surface and their surrounding rock. As a method for surface elemental mapping, X-ray fluorescence (XRF) measurement requires the samples to be ground and polished as a mirror-like plane that keeps a stable distance of X-ray source-sample-detector. This destructive pretreatment is undesirable for precious fossils. To rectify this problem, a nondestructive XRF for element mapping of the fossils' 3D surface is newly invented. The new equipment can follow the 3D shape model to keep the distance for every XRF scanned point. We performed the element mapping of Ca and Fe on the surface of a marine sauropterygian fossil (Keichousaurus hui) and its surrounding sediments, and found that the soft tissue around the thoracic cage is thicker than the abdomen. The abnormal elemental distribution on the area of the angular and surangular of the skull can be explained as unprofessionally prepared. In addition, this research found that elemental mapping revealed the invisible signals of the paleoenvironment around fossils and information for the reconstruction of soft-tissue anatomy. We also give an example that based on the wide application of the “Internet of Things” and “Industrial Network,” the design and development of specialized equipment for paleontologists' requirements are going simple.
... There was a significant difference in mean Zn levels when comparing the different types Presence of muscle melanisation in fish has been associated with elevated levels of heavy metals (Cooper and Midling 2006;Cooper et al. 2011;Ooi et al. 2019). It is shown that melanin, normally found in melanised or pigmented tissues in, for example, muscles, eyes and feathers, is usually enriched with elements such as Zn, Cu, Mg, Ca or Fe (Bowness et al. 1952;Chatelain et al. 2014;Cooper and Midling 2006;Edwards et al. 2016;Hong and Simon 2007;McGraw 2003;Niecke et al. 1999;Wogelius et al. 2011) as it can function as a reservoir or sink for heavy metals and other toxic substances (Chatelain et al. 2014;Cooper and Midling 2006;Edwards et al. 2016;Hong and Simon 2007;McGraw 2003). As a result, melanin pigments in the melanised regions of muscle in sand flathead was suggested to be a sink for sequestering excessive heavy metals in the fish, especially Zn (Ooi et al. 2019). ...
... There was a significant difference in mean Zn levels when comparing the different types Presence of muscle melanisation in fish has been associated with elevated levels of heavy metals (Cooper and Midling 2006;Cooper et al. 2011;Ooi et al. 2019). It is shown that melanin, normally found in melanised or pigmented tissues in, for example, muscles, eyes and feathers, is usually enriched with elements such as Zn, Cu, Mg, Ca or Fe (Bowness et al. 1952;Chatelain et al. 2014;Cooper and Midling 2006;Edwards et al. 2016;Hong and Simon 2007;McGraw 2003;Niecke et al. 1999;Wogelius et al. 2011) as it can function as a reservoir or sink for heavy metals and other toxic substances (Chatelain et al. 2014;Cooper and Midling 2006;Edwards et al. 2016;Hong and Simon 2007;McGraw 2003). As a result, melanin pigments in the melanised regions of muscle in sand flathead was suggested to be a sink for sequestering excessive heavy metals in the fish, especially Zn (Ooi et al. 2019). ...
Muscle melanisation in sand flathead is defined as the presence of abnormal black spots in the fish's flesh and the phenomenon has been associated with Zn pollution in the environment. In this study, Zn levels in the fish muscle and crabs were analysed to investigate the role of Zn in causing muscle melanisation and the fish's uptake of Zn via its diet, crabs. An improved technique for extracting melanised fish muscle from fillets with black spots was developed. It enabled Zn levels in melanised muscle to be more accurately determined when compared to previous study and showed Zn levels were 2.1–3.5 times higher in melanised regions of muscle than non-melanised regions of muscle from fish with melanisation and fish unaffected by melanisation. This indicated elevated levels of Zn were localised in the melanised muscle. Muscle melanisation was potentially caused by Zn ions binding to the active site of tyrosinase, an enzyme involved in melanin production, likely facilitated by the presence of higher Zn than Cu in the polluted estuaries. A digestion reagent mixture suitable for microwave assisted acid digestion and analysis of Zn in the whole crab was developed using a mixture of HNO3 and H2O2. Zn levels in the whole crab were 1.5 times higher in 50% of the crab species studied from a polluted estuary compared to the same species from an unpolluted estuary. As sand flathead primarily feed on crabs, the fish's diet was a likely source of elevated Zn for the fish. Overall, this study has described new techniques useful for the study of melanised fish and heavy metal levels in the whole crab. The findings from this study also provided insights that will inform and guide the direction of future research on muscle melanisation in sand flathead.
... Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, X-ray spectroscopy, and secondary-ion mass spectroscopy (SIMS), have been employed to track the molecular, elemental and isotopic information. 1,5,6,[8][9][10][11][12][13][15][16][17][18] Unfortunately, previous studies all focused on non-destructive chemical imaging of small-sized specimens (e.g. Archaeopteryx, Confuciusornis), limited by the analytical instruments 6,8,11,12 or the chemical analyses of small samples destructively taken from fossils. ...
... 12,35,36 Some elements, such as Ca, Zn, Mn, Cu and Ni, have been associated with melanin pigmentation in modern and fossil feathers. 1,6,11,18,30,32,33 In the fossils of Jianianhualong, Anchiornis, Archaeopteryx, Gansus, and other extinct and living birds, the Cu level usually varies with different feather samples of an individual and is generally higher in the feathers than in the bones. 6,12,30,32 Though in DLXH 1218, the distributions of Ca, Cu, Mn and Ni match the plumage shapes, no obvious enrichment of Zn was observed with either the feathers or the bones, which is also different from the Thermopolis Archaeopteryx, Confuciusornis and the fossil coliiformes bird (AMNH FARB 30806) where the feathers and the bone materials have elevated Zn levels. ...
Jianianhualong tengi is a key taxon for understanding the evolution of pennaceous feathers as well as of troodontid theropods. It is known by only the holotype, which was recovered from the Lower Cretaceous Yixian Formation of western Liaoning, China. In this study, we carried out a large-area micro-X-Ray fluorescence (micro-XRF) analysis of the holotype of Jianianhualong tengi via a Brucker M6 Jetstream mobile XRF scanner. The elemental distribution measurements of the specimen show an enrichment of typical bone-associated elements, such as S, P and Ca, which allows to visualize the fossil structure. Additionally, the bones are enriched with several heavier elements, such as Sr, Th, Y and Ce relative to the surrounding rocks. The enrichment is most likely associated to secondary mineralization and the phosphates from the bones. Interestingly, the plumage shape correlates with an enrichment in elements, such as Cu, Ni and Ti, consistent with the findings of a previous study 1 on Archaeopteryx using synchrotron imaging. Elemental variations among the skeleton, the unguis and the sheath blade further indicate their possible compositional or ultrastructural differences, providing new biological and taphonomic information on the fossilized keratinous structures. An in-situ and nondestructive micro-XRF analysis is currently the most ideal way to map the chemistry of meter-sized fossils and has so far been mainly restricted to small samples. Micro-spatial chemical analysis of larger samples usually required a synchrotron facility. Our study demonstrated that a laboratory-based large-area micro-XRF scanner can provide a practical tool for the study of large specimens, thus allowing to collect full chemical data in order to obtain a better understanding of evolutionary and taphonomic processes.
... 1,6,9,10,[12][13][14] Various chemical imaging techniques, e.g., Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, X-ray spectroscopy, and secondary-ion mass spectroscopy (SIMS), have been employed to track the molecular, elemental and isotopic information. 1,5,6,[8][9][10][11][12][13][15][16][17][18] Unfortunately, previous studies all focused on non-destructive chemical imaging on small-sized specimens (e.g., Archaeopteryx, Confuciusornis) as limited by the analyzing instruments, 6,8,11,12 or chemical analyses on small samples destructively taken from fossils. 9,10,13,16,17 So far systematic non-destructive chemical imaging on meter-sized specimens of non-avialan dinosaurs have not been done. ...
... 12,35,36 Some elements such as Ca, Zn, Mn, Cu and Ni have been noticed to be associated with melanin pigmentation in modern and fossil feathers. 1,6,11,18,30,32,33 In the fossils of Jianianhualong, Anchiornis, Archaeopteryx, Gansus, and other extinct and living birds, the Cu level usually varies on different feather samples of an individual and is generally higher in the feathers than in the bones. 6,12,30,32 Though in DLXH 1218, the distributions of Ca, Cu, Mn and Ni match the plumage shapes, no obvious enrichment of Zn has been observed with either feathers or bones, which is also different from the Thermopolis Archaeopteryx, Confuciusornis and the fossil coliiformes bird (AMNH FARB 30806) where the feathers and bone materials have elevated Zn level. ...
... Furthermore, the European Institute for the Non-Destructive Photonics Analysis of Ancient Materials (IPANEMA) has been developing methodologies to support the study of ancient materials (e.g. archaeological, palaeontological and cultural heritage) using the beamlines of synchrotron SOLEIL (Bertrand et al., 2011Edwards et al., 2016). ...
... According to their valence states, these elements could be incorporated into melanin sheets of porphyrin-like oligomers, as theoretically suggested (Kim et al., 2016). A subsequent study using equal analytical approach further supported this interpretation, demonstrating that in several feathers of modern birds, elements and their valences states, are spatially correlated with pigmented areas (Edwards et al., 2016). More recently, using XRF and XAS, Manning et al. (2019) were able to identify evidence for Fig. 3. Workflow chart of the way synchrotron radiation might be applied in palaeontology. ...
Synchrotron radiation (SR) is an electromagnetic radiation produced when electrons are forced to assume a curved trajectory resulting in an emission of a bright beam of high energy photons. This phenomenon is carried out in large particle acceleration facilities called synchrotrons, which is equipped with experimental stations at the end of each spot where the beam is emitted. In an applied sense, SR allows the investigation of materials of different nature, from synthetic to biological, from macro to the nano-scale. Since fossils are typified as environmental samples (i.e. a mixture of different type of compounds), SR has become an important approach to modern palaeontology. Due to the selective nature of the fossil record, bias can occur in both, morphology and geochemistry. Therefore, palaeontologists are frequently susceptible to equivocal interpretations. To diminish this problem, the employment of analytical methods became paramount in many areas of palaeontology. However, most benchtop equipments are limited in resolution, they also require particular experimental conditions (e.g. vacuum), and complex sample preparations (e.g. extraction from rocks). In this scenario, SR is advantageous as it allows non-destructive experiments to be performed in situ in qualitative or quantitative modes, in larger areas of the samples, still maintaining high spatial resolution and low detection limits. Nonetheless, in contrast to worldwide palaeontology where SR is widely applied, this analytical approach remains still poorly explored in South America. In Brazil, only a few studies used SR to resolve palaeontological problems. This panorama contrasts with the fact that in Brazil had a 2nd generation synchrotron, the UV and Soft X-Ray Light Source (UVX), which operated for 22 years. Nevertheless, this machine was substituted by a new 4th generation synchrotron, Sirius, which has been in operation since early 2020. Among its 13 beamlines, some will be best suitable to analyse palaeontological material, both through spectroscopy and imaging at a nanoscale. Therefore, it is expected that fossils will be routinely analysed in Sirius, being able to push forward the current knowledge in palaeontology. Here, we review the application of SR to the investigation of fossilization and other challenging topics in palaeontology. In this context, a fossil beetle from the Cretaceous Crato Formation (NE Brazil) was analysed using synchrotron radiation micro-X-Ray fluorescence (SR-μXRF). Results revealed which elements are involved in the process of fossilization and diagenesis, providing additional evidence for the preservational model of insects in this unit. Due to the high resolution and non-destructive properties, the SR-techniques have provided invaluable information on rare and delicate fossils. Considering all these aspects, it is now clear that this analytical approach has a high potential of expanding not only the frontiers of palaeontology, but also of the South American Earth sciences as a whole.
... 3 spectroscopy, X-ray spectroscopy, and secondary-ion mass spectroscopy (SIMS)), have been employed to track the molecular, elemental and isotopic information [1,5,6,[8][9][10][11][12][13][15][16][17][18]. ...
... Some elements such as Ca, Zn, Mn, Cu and Ni have been noticed to be associated with melanin pigmentation in modern and fossil feathers [1,6,11,18,29,31,32]. In the fossils of Jianianhualong, Anchiornis, Archaeopteryx, Gansus, and other extinct and living birds, the Cu level usually varies on different feather samples of an individual and is generally higher in the feathers than in the bones [6,12,29,31]. ...
Jianianhualong tengi is a key taxon for understanding the evolution of pennaceous feathers as well as troodontid theropods, and it is known by only the holotype, which was recovered from the Lower Cretaceous Yixian Formation of western Liaoning, China. Here, we carried out a large-area micro-X-Ray fluorescence (micro-XRF) analysis on the holotypic specimen of Jianianhualong tengi via a Brucker M6 Jetstream mobile XRF scanner. The elemental distribution measurements of the specimen show an enrichment of typical bones couponing elements such as S, P and Ca allowing to visualize the fossil structure. Additionally, to this, the bones are enriched in several heavier elements such as Sr, Th, Y and Ce over the surrounding rocks. The enrichment is most likely associated to secondary mineralization and the phosphates from the bones. Interestingly the plumage shape correlates with an enrichment in elements such as Cu, Ni and Ti, consistent with a previous study [1] on Archaeopteryx using synchrotron imaging. The analysis presented here provide new biological and taphonomic information of this fossil. An in-situ and nondestructive micro-XRF analysis is currently the most ideal way to map the chemistry of fossils, so far this is manly restricted to small samples. Larger samples usually required a synchrotron facility for analysis. Our study demonstrated that laboratory-based large-area micro-XRF scanner can provides a practical tool for the study of large large-sized specimens allowing collect full chemical data for a better understanding of evolutionary and taphonomic processes.
... Novel state-of-the-art, non-destructive, chemical imaging and spectroscopy techniques, e.g. Synchrotron Rapid Scanning -X-Ray Fluorescence (SRS-XRF) imaging combined with X-ray absorption spectroscopy (XAS); has provided new information concerning the presence and distribution of eumelanin residue in fossil material over relatively large areas (dm 2 ), even after millions of years of potential degradation (Barden et al., 2015;Edwards et al., 2014;2016;Manning et al., 2013;Wogelius et al., 2011). Eumelanin produces dark black/brown colours in vertebrates and invertebrates but is only one of the critical pigments governing color in ancient and modern lifeforms. ...
... This in contrast to eumelanin that has no sulfur groups that trace metals can bind to. Indeed, recent detailed SRS-XRF and XAS analyses of extant pheomelanin-rich feathers indicated a distinct chemical signature for zinc and organic sulfur, with a significant portion of the zinc inventory bound to sulfur (forming organosulfur-zinc complexes), almost certainly through the sulfur contained within the pheomelanin molecule (Edwards et al. 2016). In light of these new results, the aim of this study was to test whether, using similar SRS-XRF and XAS analyses, pheomelanin residue can now be resolved and mapped in extinct fossil material. ...
... 6 and 7) is forging a new paradigm in paleobiology, allowing inferences of the adaptive coloration and behavior of extinct taxa (8,9) (although these interpretations are not without limitations) (5)(6)(7). In addition to studies of coloration sensu stricto [e.g., of melanin-based patterning (10), structural coloration, and melanosome diversity (9,11,12) in fossil feathers and of countershading in ancient vertebrates (8,(13)(14)(15)(16)(17)], recent studies have highlighted the chemical taphonomy of melanin (2,15,18,19), the melanin metallome (20,21), and the evolution of integumentary melanosomes (22). Recent evidence for nonintegumentary melanin (extracutaneous) (5) (i.e., from internal tissues, excluding the eyes) in extant vertebrates and fossil amphibians (23,24) supports hypotheses that melanosomes may have broader functions beyond integumentary coloration (25). ...
... Here, we confirm that this issue applies to vertebrates more broadly and that nonintegumentary melanosome sources should be considered before inferring original melaninbased coloration in diverse fossil vertebrates. Moreover, our results do not show a correlation between geometry and melanin chemistry (eumelanin vs. pheomelanin) of nonintegumentary melanosomes, unlike feathers from extant birds (21). ...
Significance
Recent reports of nonintegumentary melanosomes in fossils hint at functions for melanin beyond color production, but the biology and evolution of internal melanins are poorly understood. Our results show that internal melanosomes are widespread in diverse fossil and modern vertebrates and have tissue-specific geometries and metal chemistries. Tissue-specific chemical signatures can persist in fossils despite some diagenetic overprint, allowing the reconstruction of internal soft-tissue anatomy in fossil vertebrates, and suggest that links between melanin and metal regulation have deep evolutionary origins in vertebrates.
... In collagen, given that the disulfide bondcontaining regions are biologically removed during fibril formation, the dominant S species is likely derived from its methionine residues, which has a lower excitation energy than the inorganic S phase found in the bone bioapatite, sulfate (2473.3 vs. 2482.07 eV; Edwards et al., 2014Edwards et al., , 2016. It has been shown that methionine can be preserved in archaeological samples, suggesting that such preservation might persist further into the archaeological and paleontological records (Richards et al., 2001). ...
... Beamline I18 at the Diamond Light Source is a microfocus undulator end station with a flux of 2 × 10 12 ph/s at 10 keV. XRF imaging was performed using the experimental setup of (Anné et al., , 2017Edwards et al., 2014Edwards et al., , 2016 using a beam size of 5.5 μm via a system of Kirkpatrick-Baez focusing mirrors with a 4-element Vortex Si drift detector. The specimens were mounted at 45°to the incident beam and the detector mounted at a 90°scattering angle. ...
... Melanization is the pigmentation process wherein precursors (catecholamine) are converted into pigment molecules that are incorporated into the cuticle. There are a number of studies showing the incorporation of metals within the melanosomes in other organisms 22 . However, there is no information about the distribution of trace metal associated with melanin present in wing cuticle. ...
... Since melanin contains substantial amounts of Cu and Zn 31,32 , the higher metal readings could be indicative of higher melanin concentrations in the male damselfly wing. Thus, our results support the key role of melanin in the visual, stronger pigmentation of the male damselfly wings (e.g. with high eumelanin contents) 22,31,32 . ...
Insects represent the majority of known animal species and exploit a variety of fascinating nanotechnological concepts. We investigated the wings of the damselfly Calopteryx haemorrhoidalis, whose males have dark pigmented wings and females have slightly pigmented wings. We used scanning electron microscopy (SEM) and nanoscale synchrotron X-ray fluorescence (XRF) microscopy analysis for characterizing the nanostructure and the elemental distribution of the wings, respectively. The spatially resolved distribution of the organic constituents was examined by synchrotron Fourier transform infrared (s-FTIR) microspectroscopy and subsequently analyzed using hierarchical cluster analysis. The chemical distribution across the wing was rather uniform with no evidence of melanin in female wings, but with a high content of melanin in male wings. Our data revealed a fiber-like structure of the hairs and confirmed the presence of voids close to its base connecting the hairs to the damselfly wings. Within these voids, all detected elements were found to be locally depleted. Structure and elemental contents varied between wing membranes, hairs and veins. The elemental distribution across the membrane was rather uniform, with higher Ca, Cu and Zn levels in the male damselfly wing membranes.
... 70 Alternatively, chemical differences among fossils (as reected in the distribution of specimens in Raman parameter chemospace) may reect shis in the position and shape of the D and G bands due to differences in the melanin metallome in different tissues and/or species. 66,67 Fossilized melanosomes 17,71,72 and leaves 73 are associated with various metals that may be biological 17,74 and/or diagenetic [75][76][77] in origin. Future studies will assess the extent to which diagenetically incorporated metals can impact the Raman signal of thermally matured organic materials. ...
Ancient biomolecules provide a unique perspective on the past but are underutilized in paleontology because of challenges in interpreting the chemistry of fossils. Most organically preserved soft tissues in fossils have been altered by thermal maturation during the fossilization process, obscuring original chemistry. Here, we use a comprehensive program of thermal maturation experiments on soft tissues from diverse extant organisms to systematically test whether thermally altered biosignatures can be discriminated using Raman spectroscopy. All experimentally matured samples show chemical signatures that are superficially similar. Comparative analysis of Raman spectra following peak deconvolution, however, reveals strong tissue-specific signals. Application of this approach to fossils from the Bolca (49 Ma) and Libros (10 Ma) Konservat-Lagerstätten successfully discriminates fossil vertebrate soft tissue from that of fossil plants. Critically, our data confirm that a robust interrogation of Raman spectra coupled with multivariate analysis is a powerful tool to shed light on the taxonomic origins of thermally matured fossil soft tissues.
... Synchrotron XRF and XAS has been used to examine and interpret the biochemistry of fossils for over 10 years, in specimens recovered from a wide range of geologic ages (~400 mya to recent), tissue types (non-biomineralized (e.g., skin) and mineralized (e.g., bones, teeth) and taxa (invertebrates, vertebrates and plants) [4,8,[10][11][12]15,16,[31][32][33][34]. These studies revealed biological structures that cannot be observed in visible light, as well as the fractionation of elements within discrete biological structures that can be compared with similar tissues in living organisms. ...
Simple Summary
Our understanding of what can preserve in the fossil record, and for how long, is constantly evolving with the use of new scientific techniques and exceptional fossil discoveries. In this study, we examine the state of preservation of a Tyrannosaurus rex that died about 66 million years ago. This specimen has previously been studied using a number of advanced methods, all of which have indicated preservation of original soft tissues and bone biomolecules. Here, we use synchrotron—a type of particle accelerator—analyses to generate data identifying and quantifying elements that constitute this fossil bone. We show that trace elements incorporated by the living animal during bone deposition and remodeling, such as zinc, are preserved in the fossil bone in a pattern similar to what is seen in modern bird bones. This pattern is not observed in a microscopically well preserved, but molecularly more degraded dinosaur, a herbivorous Tenontosaurus. These data further support the preservation of original biological material in this T. rex, suggesting new possibilities for deciphering extinct species life histories. This study also highlights that preservation of original biochemistry in fossils is specimen-specific and cannot be determined by pristine appearance alone.
Abstract
Biomolecules preserved in deep time have potential to shed light on major evolutionary questions, driving the search for new and more rigorous methods to detect them. Despite the increasing body of evidence from a wide variety of new, high resolution/high sensitivity analytical techniques, this research is commonly met with skepticism, as the long standing dogma persists that such preservation in very deep time (>1 Ma) is unlikely. The Late Cretaceous dinosaur Tyrannosaurus rex (MOR 1125) has been shown, through multiple biochemical studies, to preserve original bone chemistry. Here, we provide additional, independent support that deep time bimolecular preservation is possible. We use synchrotron X-ray fluorescence imaging (XRF) and X-ray absorption spectroscopy (XAS) to investigate a section from the femur of this dinosaur, and demonstrate preservation of elements (S, Ca, and Zn) associated with bone remodeling and redeposition. We then compare these data to the bone of an extant dinosaur (bird), as well as a second non-avian dinosaur, Tenontosaurus tilletti (OMNH 34784) that did not preserve any sign of original biochemistry. Our data indicate that MOR 1125 bone cortices have similar bone elemental distributions to that of an extant bird, which supports preservation of original endogenous chemistry in this specimen.
... The F-test and dimensional Hamilton test was performed to justify each path included in the fit [33]. Finally, shells were chosen that gave the best fit, e.g., split oxygen shell, and that were chemically reasonable based on the chemistry of each system (see Supplementary Material SS1) [28][29][30][34][35][36][37][38][39]. ...
In order to provide important details concerning the adsorption reactions of Sr, batch reactions and a set of both ex situ and in situ Grazing Incidence X-ray Absorption Fine Structure (GIXAFS) adsorption experiments were completed on powdered TiO2 and on rutile(110), both reacted with either SrCl2 or SrCO3 solutions. TiO2 sorption capacity for strontium (Sr) ranges from 550 ppm (SrCl2 solutions, second order kinetics) to 1400 ppm (SrCO3 solutions, first order kinetics), respectively, and is rapid. Sr adsorption decreased as a function of chloride concentration but significantly increased as carbonate concentrations increased. In the presence of carbonate, the ability of TiO2 to remove Sr from the solution increases by a factor of ~4 due to rapid epitaxial surface precipitation of an SrCO3 thin film, which registers itself on the rutile(110) surface as a strontianite-like phase (d-spacing 2.8 Å). Extended X-ray Absorption Fine Structure (EXAFS) results suggest the initial attachment is via tetradental inner-sphere Sr adsorption. Moreover, adsorbates from concentrated SrCl2 solutions contain carbonate and hydroxyl species, which results in both inner- and outer-sphere adsorbates and explains the reduced Sr adsorption in these systems. These results not only provide new insights into Sr kinetics and adsorption on TiO2 but also provide valuable information concerning potential improvements in effluent water treatment models and are pertinent in developing treatment methods for rutile-coated structural materials within nuclear power plants.
... Melanosomes typically show tissue-specific metal associations in extant (Edwards et al., 2016;Rossi et al., 2019) and some fossil vertebrates (Wogelius et al., 2011;Manning et al., 2019;Rossi et al., 2019). Other fossil vertebrates, however, show widespread Cu enrichment in melanosomes from all tissues (Rossi et al., 2020). ...
Fossil melanosomes are a major focus of paleobiological research because they can inform on the original coloration, phylogenetic affinities, and internal anatomy of ancient animals. Recent studies of vertebrate melanosomes revealed tissue-specific trends in melanosome-metal associations that can persist in fossils. In some fossil vertebrates, however, melanosomes from all body regions are enriched only in Cu, suggesting diagenetic overprinting of original chemistry. We tested this hypothesis using laboratory experiments on melanosomes from skin and liver of the African clawed frog Xenopus laevis. After maturation in Cu-rich media, the metal chemistry of melanosomes from these tissues converged toward a common composition, and original differences in Cu oxidation state were lost. Elevated Cu concentrations and a pervasive Cu(II) signal are likely indicators of diagenetically altered melanosomes. These results provide a robust experimental basis for interpretating the chemistry of fossil melanosomes.
... Turacoverdin Green [36] Coproporphyrin III Red Brown [46] Turacin Red [47] carotenoids [28,29]. On the one hand, melanin-based coloration switches from brown to black due to the presence of phaeomelanin or eumelanin, respectively, or the number and distribution of the melanosomes [30,31]. On the other hand, carotenoids-based colorations vary from yellow to red as previously mentioned. ...
Some seabirds or coastal birds such as flamingos or pelicans display elegant pink
or reddish colours. These colours are due to pigments that birds cannot synthesize
de novo. Thus, this coloration is mainly originated from carotenoids ingested
trough carotenoid rich food sources like microalgae (Dunaliella) or small shrimps
(Artemia), which are microorganisms inhabiting the salty environments where the
mentioned birds live. New advances in this field of knowledge have revealed that
extreme microorganisms belonging to the haloarchaea group (Archaea Domain)
may contribute significantly to the characteristic pink- red colour of flamingos’
feathers for instance. Alive haloarchaea cells have been found on the surface of the
feathers. Besides, the major carotenoid produced by haloarchaea (bacterioruberin)
has also been identify within the feathers structure. This work summarizes the main
contributions recently reported about this topic as well as general aspects regarding
bacterioruberin as a powerful colour carotenoid. Discussions about potential role of
these microorganisms in the life of seaside birds are also included.
... New analyses of melanosome-associated metals highlight melanin functions in metal regulation [7]. Vertebrate integumentary melanosomes are usually enriched in Ca and Zn (plus Fe and Cu in feathers [59]). Melanosomes from the heart, lungs, spleen, liver, and kidneys are usually enriched in Fe (plus Cu in the liver). ...
Melanins are widespread pigments in vertebrates, with important roles in visual signaling, UV protection, and homeostasis. Fossil evidence of melanin and melanin-bearing organelles-melanosomes-in ancient vertebrates may illuminate the evolution of melanin and its functions, but macroevolutionary trends are poorly resolved. Here, we integrate fossil data with current understanding of melanin function, biochemistry, and genetics. Mapping key genes onto phenotypic attributes of fossil vertebrates identifies potential genomic controls on melanin evolution. Taxonomic trends in the anatomical location, geometry, and chemistry of vertebrate melanosomes are linked to the evolution of endo-thermy. These shifts in melanin biology suggest fundamental links between melanization and vertebrate ecology. Tissue-specific and taxonomic trends in melanin chemistry support evidence for evolutionary tradeoffs between function and cytotoxicity. Melanin in Vertebrates Melanins (see Glossary) are dark to rufous pigments that are widespread in vertebrates and underpin critical functions in physiology and behavior [1]. Fossil evidence of melanin extending to over 300 million years ago has triggered a paradigm shift in paleobiology, prompting remarkable reconstructions of the coloration and behavior of extinct vertebrates [2-6]. New discoveries of internal melanins in vertebrate fossils have broadened our understanding of the functional diversity of ancient melanins [7-9] and invite a re-evaluation of the macroevolu-tionary history of melanin and its functions. Here, we synthesize trends in the fossil record of melanin and explore fossil evidence for the evolution of melanin function and the genetic basis of melanization. This highlights the value of the fossil record as a resource for tracking melanin evolution through deep time.
... Synchrotron X-ray imaging allows for scanning larger specimens , Manning et al. 2013, and X-ray absorption near edge structure can detect aspects of coordination chemistry. It has been argued that the detection of chelated copper and zinc may be diagnostic of eumelanin ) and phaeomelanin (Edwards et al. 2016, Manning et al. 2019. It is unclear, however, if diagenetic alterations would affect original metal distributions. ...
Melanin and other pigments are now well known to be important in exceptional preservation of soft tissues in vertebrates and other animals. Because pigments confer coloration and even structural colors, they have opened a new field of paleocolor reconstruction. Since its inception about a decade ago, reconstruction of color patterns has been performed on several vertebrates, including feathered and scale-clad dinosaurs. Iridescence and other types of structural color can also be identified through melanosome shape and arrangement. How pigments and melanosomes fossilize and are altered has become an important research subject. Ancient color patterns that may range from crypsis to brilliant displays have revealed insights into the evolution and escalation of visual systems, the nature of ancient animal interactions, and how several unique characteristics of birds already arose among dinosaurs. ▪ Melanin and other pigments preserve in exceptional fossils; this opens paths for reconstructing coloration of extinct organisms, such as dinosaurs. ▪ The most abundant pigment is melanin, which can be identified chemically and through preserved melanosome microbodies. ▪ Melanosome shape reveals clues to original hue ranging from reddish brown and black to gray and structural coloration. ▪ Other pigments may preserve, such as porphyrin pigments in theropod dinosaur eggshells. ▪ Fossil color patterns contribute new insights into the evolution of visual systems, predator-prey interactions, and key innovations.
... Therefore, the complex nature and the subtle differences in the rhizosphere make it difficult to apply traditional wet-chemistry speciation, which is prone to artefacts and biases (Prietzel et al., 2003;Prietzel et al., 2007b). The use of X-ray absorption near-edge structure (XANES) in combination with synchrotron X-ray fluorescence (SR-XRF) mapping have been proven to be a powerful nondestructive technique for direct speciation of various elements in biological and geological samples (Egerton et al., 2015;Edwards et al., 2016). ...
Rhizosphere soil has distinct physical and chemical properties from bulk soil. However, besides root‐induced physical changes, chemical changes have not been extensively measured in situ on the pore scale.
In this study, we couple structural information, previously obtained using synchrotron X‐ray computed tomography (XCT), with synchrotron X‐ray fluorescence microscopy (XRF) and X‐ray absorption near‐edge structure (XANES) to unravel chemical changes induced by plant roots.
Our results suggest that iron (Fe) and sulfur (S) increase notably in the direct vicinity of the root via solubilization and microbial activity. XANES further shows that Fe is slightly reduced, S is increasingly transformed into sulfate (SO4²⁻) and phosphorus (P) is increasingly adsorbed to humic substances in this enrichment zone. In addition, the ferrihydrite fraction decreases drastically, suggesting the preferential dissolution and the formation of more stable Fe oxides. Additionally, the increased transformation of organic S to sulfate indicates that the microbial activity in this zone is increased. These changes in soil chemistry correspond to the soil compaction zone as previously measured via XCT.
The fact that these changes are colocated near the root and the compaction zone suggests that decreased permeability as a result of soil structural changes acts as a barrier creating a zone with increased rhizosphere chemical interactions via surface‐mediated processes, microbial activity and acidification.
... High deposition rates observed for essential elements as Cu and Zn are most probably due to their structural role in the keratinous composition of the feather (Howell et al., 2017). Due to the lack of pigmentation in the sixth rectrix of KP, deposition rates of Zn and Cu cannot be related with the occurrence of eumelanin or pheomelanin, known to serve as metal reservoir also in feathers (Edwards et al., 2016). Low deposition rate observed for Co indicates negligible blood concentration during feather growth and thus a limited availability of Co through the food web. ...
A non-invasive study of trace element accumulation in tail feathers of the Kentish plover (Charadrius alexandrinus) was performed along the coastline of the northern littoral strip of the Venice Lagoon, with the aim to verify whether contamination may be a factor affecting conservation status of Kentish plover populations. Body burdens in feathers of 11 trace elements including toxic metals/metalloids and essential elements (As, Cd, Co, Cr, Cu, Hg, Ni, Pb, Se, V, Zn) were quantified by ICP-MS, then concentrations were normalized to feather's age calculated using ptilochronology in order to obtain daily deposition rates. Mercury emerged as a major threat to the conservation of the species, since average feather concentration was clearly above the adverse-effect threshold associated with impairment in the reproductive success in a number of bird species. Also Cd and Se occurred at levels that may impact on the conservation status of the studied species at local scale, even if to a lesser extent than Hg. Gender-related differences in trace element accumulation emerged only for As, although for this element the risks associated to environmental exposure seem to be negligible.
... This study therefore, has tried to establish a link between metal concentration and melanin based colouration in birds. Likewise, the melanin pigment formation within the feathers of birds influenced by accumulation of Cu has been reported (Edwards et al. 2016). In case of bacterial cells, there are few reports available suggesting the synthesis of melanin by bacteria under heavy metal stress as a protective mechanism and its role in metal ion chelation. ...
Heavy metal pollution destruct soil microbial compositions and functions, plant’s performance and subsequently human health. Culturable microbes among many metal abatement strategies are considered inexpensive, viable and environmentally safe. In this study, nitrogen fixing bacterial strain CAZ3 recovered from chilli rhizosphere tolerated 100, 1000 and 1200 µg mL⁻¹ of cadmium, chromium and nickel, respectively and was identified as Azotobacter chroococcum by 16S rDNA sequence analysis. Under metal stress, cellular morphology of A. chroococcum observed under SEM was found distorted and shrinkage of cells was noticed when grown with 50 µg mL⁻¹ of Cd (cell size 1.7 µm) and 100 of µg mL⁻¹ Ni (cell size 1.3 µm) compared to untreated control (cell size 1.8 µm). In the presence of 100 µg mL⁻¹ of Cr, cells became elongated and measured 1.9 µm in size. Location of metals inside the cells was revealed by EDX. A dose dependent growth arrest and consequently the death of A. chroococcum cells was revealed under CLSM. A. chroococcum CAZ3 secreted 320, 353 and 133 µg EPS mL⁻¹ when grown with 100 µg mL⁻¹ each of Cd, Cr and Ni, respectively. The EDX revealed the presence of 0.4, 0.07 and 0.24% of Cd, Cr and Ni, respectively within EPS extracted from metal treated cells. Moreover, a dark brown pigment (melanin) secreted by A. chroococcum cells under metal pressure displayed tremendous metal chelating activity. The EDX spectra of melanin extracted from metal treated cells of A. chroococcum CAZ3 displayed 0.53, 0.22 and 0.12% accumulation of Cd, Cr and Ni, respectively. The FT-IR spectra of EPS and melanin demonstrated stretching vibrations and variations in surface functional groups of bacterial cells. The C-H stretching of CH3 in fatty acids and CH2 groups, stretching of N-H bond of proteins and O-H bond of hydroxyl groups caused the shifting of peaks in the EPS spectra. Similar stretching vibrations were recorded in metal treated melanin which involved CHO, alkyl, carboxylate and alkene groups resulting in significant peak shifts. Nuclear magnetic resonance (NMR) spectrum of EPS extracted from A. chroococcum CAZ3 revealed apparent peak signals at 4.717, 9.497, 9.369 and 9.242 ppm. However, ¹H NMR peaks were poorly resolved due largely to the impurity/viscosity of the EPS. The entrapment of metals by EPS and melanin was confirmed by EDX. Also, the induction and excretion of variable amounts of metallothioneins (MTs) by A. chroococcum under metal pressure was interesting. Conclusively, the present findings establish- (i) cellular damage due to Cd, Cr and Ni and (ii) role of EPS, melanin and MTs in adsorption/complexation and concurrently the removal of heavy metals. Considering these, A. chroococcum can be promoted as a promising candidate for supplying N efficiently to plants and protecting plants from metal toxicity while growing under metal stressed environment.
... The amount of ROS is controlled by the self-defense systems that are antioxidant-mediated in their normal state, such as antioxidants. These include vitamin C, vitamin E, or glutathione [4], which scavenge free radicals to prevent cellular damage. However, they disrupt the balance of the cellular oxidation state which results in too many ROS. ...
N
-Hydroxycinnamoylphenalkylamides (36H) exhibited both antioxidation and antityrosinase abilities. The compound was studied for its antioxidative properties, using a 1,1-diphenyl-2-picrylhydrazul- (DPPH-) scavenging test, a ferric ion-reducing antioxidant power assay (FRAP) assessment, and a metal-chelating power assay. The results showed that 36H had antioxidative capabilities in the DPPH-scavenging and ferric-reducing power examinations but the chelating power assay did not demonstrate antioxidative capability. 36H was also measured for tyrosinase inhibitory activity applying various species platforms, including in vitro mushroom, B16F10 mouse melanoma, and human melanocyte cells. In terms of in vitro mushroom tyrosinase suppression, 36H restrained the melanogenesis processes. It is assumed that 36H blocked the tyrosinase active site as a competitive inhibitor for mushroom tyrosinase, hence not decreasing the human normal melanocyte cellular viability. A quantitative real-time polymerase chain reaction (qRT-PCR) and western blot discovered that 36H downregulated melanogenesis-related RNA and proteins, including pigment production (MITF, tyrosinase, TRP-1, and TRP-2), melanosome maturation (Rab27a), and melanosome transportation (Myo5a, MLPH and Mreg). Overall, 36H displayed the biofunctions of antioxidation and melanin suppression, so there was a possibility for its application as a food additive or a skin-whitening agent.
... However, chemical specific XRF imaging is only possible for chemical species that are well resolved in the XANES spectrum. It is for instance possible to map selenium chemical species [21], Cr(III) vs Cr(VI) oxidation states [22], arsenic chemical species [23,24], and sulfur chemical species [25]. A third alternative for microscopic speciation analysis is micro-XANES imaging that consists in recording a full XANES spectrum for each pixel of the image. ...
Synchrotron-radiation X-ray absorption spectroscopy (XAS) is a direct method for speciation analysis with atomic resolution, providing information about the local chemical environment of the probed element. This article gives an overview of the basic principles of XAS and its application to element speciation in biomedical research. The basic principle and experimental modalities of XAS are introduced, followed by a discussion of both its limitations, such as beam damage or detection limits, and practical advices to improve experiments. An updated review of biomedical studies involving XAS published over the last 5 years is then provided, paying special attention to metal-based drug biotransformation, metal and nanoparticle toxicology, and element speciation in cancer, neurological, and general pathophysiology. Finally, trends and future developments such as hyphenated methods, in situ correlative imaging and speciation, in vivo X-ray Absorption Near Edge Spectroscopy (XANES), full-field XANES, and X-ray Free Electron Laser (XFEL) XAS are presented.
... Following acid digestion of the samples, several methods are commonly used to carry out analysis of the metal content in the feathers, such as FAAS, ETAAS, ICP-OES, and ICP-MS [31][32][33]. Alternatively, the elemental composition of the feather samples can be determined by methods based on the direct analysis of solids such as laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) [31], energydispersive X-ray fluorescence spectrometry (EDXRF) or Synchrotron Rapid Scanning X-ray fluorescence (SRS-XRF) [34,35]. These techniques have the potential to provide images of the elemental distribution in the feathers in comparison with other techniques after digestion. ...
The laser ablation-ICP-MS method was used for a quantitative assessment of the distribution of elements Al, Ba, Ca, Cu, Mg, Mn, Pb, Sr, and Zn in feathers. We analysed the rectrices of first-year red-breasted flycatchers characterized by a black-and-white colouration, which gave the opportunity to examine the content of elements in two aspects: (i) quantitative distribution of elements depending on the analysed part of the feathers; and (ii) direct imaging of elements in the context of variation in feather colouration (black – eumelanin pigment and white – free of pigmentation). The results show that distributions of Ca, Cu, Mg, and Zn in the feather shaft were related to melanin pigmentation – concentrations of these elements were significantly higher within black parts. Furthermore, the highest concentrations of Al, Ba, Mn, Pb and Sr were recorded at the end of a shaft – the oldest part of the feathers analysed – which may be connected with the time and dynamic of nestling plumage development.
... Evidence for original colouration (rather than simply patterning), both structural and chemical, have been found in fossils (e.g. Colleary et al., 2015;Glass et al., 2012;Vitek et al., 2013;Vinther, 2015;Edwards et al., 2016). So, pigmentation and/or structural colours could be discovered in fossil spiders using these techniques. ...
Spiders are the most diverse and important terrestrial predators in modern ecosystems. Therefore, fossil spiders are fundamental to understanding past terrestrial ecosystems, especially coevolution with their principal prey, the insects. Being generally soft bodied, spiders have a poor fossil record, but where they do occur, it is in the exceptional circumstance of a Fossil-Lagerstätte. By far the greatest number of fossil spider specimens are found in amber (fossilized tree resin), but earlier than the Cretaceous and outside of amber forest areas, rock-matrix preservation is essential for the spider fossil record. Every taphonomic situation requires a special technique for study. Here, we review imaging techniques in the study of fossil spiders. The earliest depictions of fossil spiders are drawings made with the unaided eye. Light microscopy enabled detailed drawings to be produced and, later, photographs of fossil spiders appeared in the literature. In the 21st century, digital photography has revolutionized image capture and reproduction, and scanning electron microscopy has been applied recently to fossil spiders. The most exciting modern technique, X-ray (including synchrotron source) CT scanning, is now producing extraordinary images of three-dimensional fossil spiders embedded in amber, and the method is also applicable to rock-matrix preservation. We expect to see considerable refinement of these techniques in the future, as well as the possibility of novel ones. Thanks to the excellent preservation of some fossil spiders, and modern techniques available to provide exquisite images of fine morphological details, fossil spiders can now be considered taxonomically (sub)equal to modern forms. Hence, the usual excuse for excluding fossil spiders in phylogenetic and other studies no longer carries as much weight as it once did, and we encourage neontologists to consider the fossil record whenever possible.
Pelage coloration, which serves numerous functions, is crucial to the evolution of behavior, physiology, and habitat preferences of mammals. However, little is known about the coloration of Mesozoic mammaliaforms that coevolved with dinosaurs. In this study, we used a dataset of melanosome (melanin-containing organelle) morphology and quantitatively measured hair colors from 116 extant mammals to reliably reconstruct the coloration of six Mesozoic mammaliaforms, including a previously undescribed euharamiyidan. Unlike the highly diverse melanosomes discovered in feathered dinosaurs, hairs in six mammaliaforms of different lineages and diverse ecomorphotypes showed uniform melanosome geometry, corresponding to dark-brown coloration consistent with crypsis and nocturnality. Our results suggest that the melanosome variation and color expansion seen in extant mammals may have occurred during their rapid radiation and diversification after the Cretaceous-Paleogene extinction.
Melanin, a widely distributed pigment found in various organisms, possesses distinct structures that can be classified into five main types: eumelanin (found in animals and plants), pheomelanin (found in animals and plants), allomelanin (found in plants), neuromelanin (found in animals), and pyomelanin (found in fungi and bacteria). In this review, we present an overview of the structure and composition of melanin, as well as the various spectroscopic identification methods that can be used, such as Fourier transform infrared (FTIR) spectroscopy, electron spin resonance (ESR) spectroscopy, and thermogravimetric analysis (TGA). We also provide a summary of the extraction methods of melanin and its diverse biological activities, including antibacterial properties, anti-radiation effects, and photothermal effects. The current state of research on natural melanin and its potential for further development is discussed. In particular, the review provides a comprehensive summary of the analysis methods used to determine melanin species, offering valuable insights and references for future research. Overall, this review aims to provide a thorough understanding of the concept and classification of melanin, its structure, physicochemical properties, and structural identification methods, as well as its various applications in the field of biology.
The book writes on birds as challenges and opportunities for business, conservation and research. The main chapter explains how the new genetic research has and will change the taxonomy and number of bird species in the world.
Zinc is an essential cofactor for many cellular processes including gene transcription, insulin secretion and retinal function. Excessive free Zn ²⁺ is highly toxic and consequently intracellular zinc is tightly controlled by a system of transporters, metallothioneins (MTs) and storage vesicles. Here we describe the developmental consequences of a missense allele of zinc efflux transporter slc30a8 ( znt8) in zebrafish. Homozygous slc30a8 hu1798 larvae are virtually blind and develop very little or no bone mineral. We show that zinc is stored in pigmented cells (melanophores) of healthy larvae but in slc30a8 hu1798 mutants it instead accumulates in the bone and brain. Supporting a role for pigment cells in zinc homeostasis, nacre zebrafish, which lack melanophores, also show disrupted zinc homeostasis. The photoreceptors of slc30a8 hu1798 fish are severely depleted while those of nacre fish are enriched with zinc. We propose that developing zebrafish utilise pigmented cells as a zinc storage organ, and that Slc30a8 is required for transport of zinc into these cells and into photoreceptors.
A panel of geochemical techniques is used here to investigate the taphonomy of fossil feathers preserved in association with the skeleton of the Jurassic theropod Anchiornis huxleyi . Extant feathers were analysed in parallel to test whether the soft tissues morphologically preserved in the fossil also exhibit a high degree of chemical preservation. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) indicate that clays and iron oxide pseudomorphs occur in the surrounding sediment and also reveal the preservation of melanosome‐like microbodies in the fossil. Carbon gradient along a depth profile and co‐occurrence of carbon and sulphur are shown in the fossil by elastic backscattering (EBS) and particle‐induced x‐ray emission (PIXE), which are promising techniques for the elemental analysis of fossil soft tissues. The molecular composition of modern and fossil soft tissues was assessed from micro‐attenuated total reflectance fourier transform infrared spectroscopy (micro‐ATR FTIR), solid‐state ¹³C nuclear magnetic resonance (CP‐MAS ¹³C NMR) and pyrolysis gas chromatography mass spectrometry in the presence of TMAH (TMAH‐Py‐GC‐MS). Results indicate that the proteinaceous material that comprises the modern feathers is not present in the fossil feathers. The fossil feathers and the embedding sediment exhibit a highly aliphatic character. However, substantial differences exist between these samples, revealing that the organic matter of the fossil feathers is, at least partially, derived from original constituents of the feathers. Our results suggest that, despite the morphological preservation of Anchiornis feathers, original proteins, that is keratin, were probably not preserved in the 160‐myr‐old feathers.
Feathers can be used to investigate exposure to pollution in birds because they are a secondary route for the excretion of trace elements. Evidence based on analytical imaging and spectroscopy suggests that the spatial distribution of the essential trace element zinc within feathers is related to melanin pigmentation. However, our understanding of how trace elements are deposited into growing feathers is poor and has been hampered by a lack of analytical tools to examine the localization of trace elements within a feather. Here, synchrotron micro X‐ray fluorescence spectroscopy was used to map zinc directly within the barb and barbules of lesser scaup (Aythya affinis) feathers grown after experimental increases in dietary zinc. The results showed distinct spatial variation in zinc within barbs and barbules, with higher levels observed in the latter. Furthermore, increases in dietary zinc were found to increase the relative levels of zinc throughout the barbules from the base to the tip of the feather. Finally, analysis of feather cross sections revealed that regions of the feather barb and barbules with higher melanosome density also contained higher levels of zinc. These results provide a more detailed understanding of zinc and melanosome arrangement within the feather barb and barbules. Moreover, these results provide further support for the use of feathers as a noninvasive tool to study exposure to trace elements and highlight the utility of X‐ray spectroscopy in studies investigating impacts of a rapidly changing environment on wild bird health.
Feathers have long been regarded as the innovation that drove the success of birds. However, feathers have been reported from close dinosaurian relatives of birds, and now from ornithischian dinosaurs and pterosaurs, the cousins of dinosaurs. Incomplete preservation makes these reports controversial. If true, these findings shift the origin of feathers back 80 million years before the origin of birds. Gene regulatory networks show the deep homology of scales, feathers, and hairs. Hair and feathers likely evolved in the Early Triassic ancestors of mammals and birds, at a time when synapsids and archosaurs show independent evidence of higher metabolic rates (erect gait and endothermy), as part of a major resetting of terrestrial ecosystems following the devastating end-Permian mass extinction.
This paper describes a new large-range rapid-scan X-ray fluorescence (XRF) imaging station at beamline 6-2 at the Stanford Synchrotron Radiation Lightsource at SLAC National Accelerator Laboratory. This station uses a continuous rapid-scan system with a scan range of 1000 × 600 mm and a load capacity of up to 25 kg, capable of 25–100 µm resolution elemental XRF mapping and X-ray absorption spectroscopy (XAS) of a wide range of objects. XRF is measured using a four-element Hitachi Vortex ME4 silicon drift detector coupled to a Quantum Detectors Xspress3 multi-channel analyzer system. A custom system allows the X-ray spot size to be changed quickly and easily via pinholes ranging from 25 to 100 µm, and the use of a poly-capillary or axially symmetric achromatic optic may achieve a <10 µm resolution in the future. The instrument is located at wiggler beamline 6-2 which has an energy range of 2.1–17 keV, creating K emission for elements up to strontium, and L or M emission for all other elements. XAS can also be performed at selected sample positions within the same experiment, allowing for a more detailed chemical characterization of the elements of interest. Furthermore, sparse excitation energy XRF imaging can be performed over a wide range of incident X-ray energies. User friendliness has been emphasized in all stages of the experiment, including versatile sample mounts, He purged chambers for low-Z analyses, and intuitive visualization hardware and software. The station provides analysis capabilities for a wide range of materials and research fields including biological, chemical, environmental and materials science, paleontology, geology and cultural heritage.
In situ characterization of the chemical and structural properties of black and white sheep hair was performed with a spatial resolution of 25 nm using infrared nano-spectroscopy. Comparing data sets from two types of hair allowed us to isolate the keratin FTIR fingerprint and so mark off chemical properties of the hair's melanosomes. From a polarization sensitive analysis of the nano-FTIR spectra, we showed that keratin intermediate filaments (IFs) present anisotropic molecular ordering. In stark contrast with white hair which does not contain melanosomes, in black hair, we spatially resolved single melanosomes and achieved unprecedented assignment of the vibrational modes of pheomelanin and eumelanin. The in situ experiment presented here avoids harsh chemical extractive methods used in previous studies. Our findings offer a basis for a better understanding of the keratin chemical and structural packing in different hair phenotypes as well as the involvement of melanosomes in hair color and biological functionality.
During the past decade, melanins and melanogenesis have attracted growing interest for a broad range of biomedical and technological applications. The burst of polydopamine-based multifunctional coatings in materials science is just one example, and the list may be expanded to include melanin thin films for organic electronics and bioelectronics, drug delivery systems, functional nanoparticles and biointerfaces, sunscreens, environmental remediation devices. Despite considerable advances, applied research on melanins and melanogenesis is still far from being mature. A closer intersectoral interaction between research centers is essential to raise the interests and increase the awareness of the biomedical, biomaterials science and hi-tech sectors of the manifold opportunities offered by pigment cells and related metabolic pathways. Starting from a survey of biological roles and functions, the present review aims at providing an interdisciplinary perspective of melanin pigments and related pathway with a view to showing how it is possible to translate current knowledge about physical and chemical properties and control mechanisms into new bioinspired solutions for biomedical, dermocosmetic, and technological applications.
Current understanding of bone healing and remodelling strategies in vertebrates has traditionally relied on morphological observations through the histological analysis of thin sections. However, chemical analysis may also be used in such interpretations, as different elements are known to be absorbed and used by bone for different physiological purposes such as growth and healing. These chemical signatures are beyond the detection limit of most laboratory-based analytical techniques (e.g. scanning electron microscopy). However, synchrotron rapid scanning-X-ray fluorescence (SRS-XRF) is an elemental mapping technique that uniquely combines high sensitivity (ppm), excellent sample resolution (20-100 µm) and the ability to scan large specimens (decimetre scale) approximately 3000 times faster than other mapping techniques. Here, we use SRS-XRF combined with microfocus elemental mapping (2-20 µm) to determine the distribution and concentration of trace elements within pathological and normal bone of both extant and extinct archosaurs (Cathartes aura and Allosaurus fragilis). Results reveal discrete chemical inventories within different bone tissue types and preservation modes. Chemical inventories also revealed detail of histological features not observable in thin section, including fine structures within the interface between pathological and normal bone as well as woven texture within pathological tissue.
Large-scale Synchrotron Rapid Scanning X-ray Fluorescence (SRS-XRF) elemental mapping and X-ray absorption spectroscopy are applied here to fossil leaf material from the 50 Mya Green River Formation (USA) in order to improve our understanding of the chemistry of fossilized plant remains. SRS-XRF of fossilized animals has previously shown that bioaccumulated trace metals and sulfur compounds may be preserved in their original distributions and these elements can also act as biomarkers for specific biosynthetic pathways. Similar spatially resolved chemical data for fossilized plants is sparsely represented in the literature despite the multitude of other chemical studies performed. Here, synchrotron data from multiple specimens consistently show that fossil leaves possess chemical inventories consisting of organometallic and organosulfur compounds that: (1) map discretely within the fossils, (2) resolve fine scale biological structures, and (3) are distinct from embedding sedimentary matrices. Additionally, the chemical distributions in fossil leaves are directly comparable to those of extant leaves. This evidence strongly suggests that a significant fraction of the chemical inventory of the examined fossil leaf material is derived from the living organisms and that original bioaccumulated elements have been preserved in situ for 50 million years. Chemical information of this kind has so far been unknown for fossilized plants and could for the first time allow the metallome of extinct flora to be studied.
Trace metals produced by anthropogenic activities are of major importance in urban areas and might constitute a new evolutionary force selecting for the ability to cope with their deleterious effects. Interestingly, melanin pigments are known to bind metal ions, thereby potentially sequestering them in inert body parts such as coat and feathers, and facilitating body detoxification. Thus, a more melanic plumage or coat coloration could bring a selective advantage for animals living in polluted areas. We tested this hypothesis by investigating the link between melanin-based coloration and zinc and lead concentrations in feathers of urban feral pigeons, both at capture time and after one year of captivity in standardized conditions. Results show that differently coloured pigeons had similar metal concentrations at capture time. Metal concentrations strongly decreased after one year in standardized conditions, and more melanic pigeons had higher concentrations of zinc (but not lead) in their feathers. This suggests that more melanic pigeons have a higher ability to store some metals in their feathers compared with their paler counterparts, which could explain their higher success in urbanized areas. Overall, this work suggests that trace metal pollution may exert new selective forces favouring more melanic phenotypes in polluted environments.
Thiophenic compounds are major constituents of fossil fuels and pose problems for fuel refinement. The quantification and speciation of these compounds is of great interest in different areas such as biology, fossil fuels studies, geology, and archaeology. Sulfur 1s Near-Edge X-ray Absorption Fine Structure (NEXAFS) spectroscopy has emerged as a qualitative and quantitative method for sulfur speciation. A firm understanding of the sulfur 1s NEXAFS spectra of organosulfur species is required for these analytical studies. To support this development, the sulfur 1s NEXAFS spectra of simple thiols and thioethers were previously examined, and are now extended to studies of thiophenic and aromatic thioether compounds, in the gas and condensed phases. High-resolution spectra have been further analyzed with the aid of Improved Virtual Orbital (IVO) and Δ(self-consistent field) ab initio calculations. Experimental sulfur 1s NEXAFS spectra show fine features predicted by calculation, and the combination of experiment and calculation has been used to improve the assignment of spectroscopic features important for the speciation and quantification of sulfur compounds. Systematic differences between gas and condensed phases are also explored; these differences suggest a significant role for conformational effects in the NEXAFS spectra of condensed species.
A general problem when fitting EXAFS data is determining whether particular parameters are statistically significant. The F‐test is an excellent way of determining relevancy in EXAFS because it only relies on the ratio of the fit residual of two possible models, and therefore the data errors approximately cancel. Although this test is widely used in crystallography (there, it is often called a “Hamilton test”) and has been properly applied to EXAFS data in the past, it is very rarely applied in EXAFS
analysis. We have implemented a variation of the F‐test adapted for EXAFS
data analysis in the RSXAP analysis package, and demonstrate its applicability with a few examples, including determining whether a particular scattering shell is warranted, and differentiating between two possible species or two possible structures in a given shell.
Extended X-ray absorption fine structure (EXAFS) spectroscopy at the Zn - K edge is applied for the study of the bonding geometry of Zn in human nails. The studied nail clippings belong to healthy donors and donors who suffer from lung diseases. Fitting of the first nearest neighboring shell of Zn reveals that it is bonded with N and S, at distances that take values in the ranges 2.00–2.04 Å and 2.23–2.28Å, respectively. Zn is four - fold coordinated and the ratio of the number of sulfur and nitrogen atoms (NS/NN) in the first coordination shell ranges from 0.52 to 1. The sample that belongs to the donor who suffers from lung fibrosis, a condition that is related to keratinization of the lung tissue, is characterized by the highest number of NS/NN. Simulation, using the FEFF8 code, of the Zn - K edge EXAFS spectra with models of tetrahedrally coordinated Zn with 1 (or 2) cysteine and 3 (or 2) histidines is satisfactory.
UV radiation of the skin triggers keratinocytes to secrete endothelin-1 (ET-1) that binds to endothelin receptors on neighboring melanocytes. Melanocytes respond with a prolonged increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), which is necessary for proliferation and melanogenesis. A major fraction of the Ca(2+) signal is caused by entry through Ca(2+)-permeable channels of unknown identity in the plasma membrane. ORAI Ca(2+) channels are molecular determinants of Ca(2+) release-activated Ca(2+) (CRAC) channels and are expressed in many tissues. Here, we show that ORAI1-3 and their activating partners stromal interaction molecules 1 and 2 (STIM1 and STIM2) are expressed in human melanocytes. Although ORAI1 is the predominant ORAI isoform, STIM2 mRNA expression exceeds STIM1. Inhibition of ORAI1 by 2-aminoethoxydiphenyl borate (2-APB) or downregulation of ORAI1 by small interfering RNA (siRNA) reduced Ca(2+) entry and CRAC current amplitudes in activated melanocytes. In addition, suppression of ORAI1 caused reduction in the ET-1-induced cellular viability, melanin synthesis, and tyrosinase activity. Our results imply a role for ORAI1 channels in skin pigmentation and their potential involvement in UV-induced stress responses of the human skin.
The speciation and quantification of sulfur species based on sulfur K-edge x-ray absorption spectroscopy is of wide interest, particularly for biological and petroleum science. These tasks require a firm understanding of the sulfur 1s near-edge x-ray absorption fine structure (NEXAFS) spectra of relevant species. To this end, we have examined the gas phase sulfur 1s NEXAFS spectra of a group of simple thiol and thioether compounds. These high-resolution gas phase spectra are free of solid-state broadening, charging, and saturation effects common in the NEXAFS spectra of solids. These experimental data have been further analyzed with the aid of improved virtual orbital Hartree-Fock ab initio calculations. The experimental sulfur 1s NEXAFS spectra show fine features predicted by calculation, and the combination of experiment and calculation has been used to improve assignment of spectroscopic features relevant for the speciation and quantification of the sulfur compounds.
Well-preserved fossils of pivotal early bird and nonavian theropod species have provided unequivocal evidence for feathers
and/or downlike integuments. Recent studies have reconstructed color on the basis of melanosome structure; however, the chemistry
of these proposed melanosomes has remained unknown. We applied synchrotron x-ray techniques to several fossil and extant organisms,
including Confuciusornis sanctus, in order to map and characterize possible chemical residues of melanin pigments. Results show that trace metals, such as
copper, are present in fossils as organometallic compounds most likely derived from original eumelanin. The distribution of
these compounds provides a long-lived biomarker of melanin presence and density within a range of fossilized organisms. Metal
zoning patterns may be preserved long after melanosome structures have been destroyed.
Non-destructive Fourier Transform InfraRed (FTIR) mapping of Eocene aged fossil reptile skin shows that biological control on the distribution of endogenous organic components within fossilized soft tissue can be resolved. Mapped organic functional units within this approximately 50 Myr old specimen from the Green River Formation (USA) include amide and sulphur compounds. These compounds are most probably derived from the original beta keratin present in the skin because fossil leaf- and other non-skin-derived organic matter from the same geological formation do not show intense amide or thiol absorption bands. Maps and spectra from the fossil are directly comparable to extant reptile skin. Furthermore, infrared results are corroborated by several additional quantitative methods including Synchrotron Rapid Scanning X-Ray Fluorescence (SRS-XRF) and Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS). All results combine to clearly show that the organic compound inventory of the fossil skin is different from the embedding sedimentary matrix and fossil plant material. A new taphonomic model involving ternary complexation between keratin-derived organic molecules, divalent trace metals and silicate surfaces is presented to explain the survival of the observed compounds. X-ray diffraction shows that suitable minerals for complex formation are present. Previously, this study would only have been possible with major destructive sampling. Non-destructive FTIR imaging methods are thus shown to be a valuable tool for understanding the taphonomy of high-fidelity preservation, and furthermore, may provide insight into the biochemistry of extinct organisms.
Visible pigmentation in mammals results from the synthesis and distribution of melanin in the skin, hair bulbs, and eyes. The melanins are produced in melanocytes and can be of two basic types: eumelanins, which are brown or black, and phaseomelanins, which are red or yellow. In mammals typically there are mixtures of both types. The most essential enzyme in this melanin biosynthetic pathway is tyrosinase and it is the only enzyme absolutely required for melanin production. However, recent studies have shown that mammalian melanogenesis is not regulated solely by tyrosinase at the enzymatic level, and have identified additional melanogenic factors that can modulate pigmentation in either a positive or negative fashion. In addition, other pigment-specific genes that are related to tyrosinase have been cloned which encode proteins that apparently work together at the catalytic level to specify the quantity and quality of the melanins synthesized. Future research should provide a greater understanding of the enzymatic interactions, processing, and tissue specificity that are important to pigmentation in mammals.
A software package for the analysis of X-ray absorption spectroscopy (XAS) data is presented. This package is based on the IFEFFIT library of numerical and XAS algorithms and is written in the Perl programming language using the Perl/Tk graphics toolkit. The programs described here are: (i) ATHENA, a program for XAS data processing, (ii) ARTEMIS, a program for EXAFS data analysis using theoretical standards from FEFF and (iii) HEPHAESTUS, a collection of beamline utilities based on tables of atomic absorption data. These programs enable high-quality data analysis that is accessible to novices while still powerful enough to meet the demands of an expert practitioner. The programs run on all major computer platforms and are freely available under the terms of a free software license.
The function of the dark polymer pigment neuromelanin found in catecholaminergic neurons of the human brain is not understood, especially as most published data are based upon a synthetic model melanin which differs structurally to the native pigment. Nevertheless human neuromelanin has been shown to efficiently bind transition metals such as iron, as well as other potentially toxic molecules. The pigment may have a protective function in the healthy brain by, for example, contributing to iron homeostasis within pigmented nuclei. We have demonstrated that synthetic dopamine melanin stimulates cell damage in both cell lines and primary cells in vitro, an effect associated with increased hydroxyl radical production and apoptosis. In contrast, at low iron concentrations native neuromelanin does not induce cell damage but rather protects cells in culture from oxidative stress. This protective function appears to be lost at high iron concentrations where neuromelanin saturated with iron functions as a source of oxidative load, rather than an iron chelator. Changes to neuromelanin and tissue iron load in Parkinson's disease may decrease the protective potential of the pigment, thus increase the potential for cell damage in this disorder.
Melanin is a ubiquitous pigment in living organisms with multiple important functions, yet its structure is not well understood. We propose a structural model for eumelanin protomolecules, consisting of 4 or 5 of the basic molecular units (hydroquinone, indolequinone, and its tautomers), in arrangements that contain an inner porphyrin ring. We use time-dependent density functional theory to calculate the optical absorption spectrum of the structural model, which reproduces convincingly the main features of the experimental spectrum of eumelanin. Our model also reproduces accurately other important properties of eumelanin, including x-ray scattering data, its ability to capture and release metal ions, and the characteristic size of the protomolecules.
A non-synonymous single nucleotide polymorphism in the human SLC24A5 gene is associated with natural human skin color variation. Multiple sequence alignments predict that this gene encodes a
member of the potassium-dependent sodium-calcium exchanger family denoted NCKX5. In cultured human epidermal melanocytes we
show using affinity-purified antisera that native human NCKX5 runs as a triplet of ∼43 kDa on SDS-PAGE and is partially localized
to the trans-Golgi network. Removal of the NCKX5 protein through small interfering RNA-mediated knockdown disrupts melanogenesis in human
and murine melanocytes, causing a significant reduction in melanin pigment production. Using a heterologous expression system,
we confirm for the first time that NCKX5 possesses the predicted exchanger activity. Site-directed mutagenesis of NCKX5 and
NCKX2 in this system reveals that the non-synonymous single nucleotide polymorphism in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 activity. We suggest that NCKX5 directly regulates human epidermal
melanogenesis and natural skin color through its intracellular potassium-dependent exchanger activity.
An introduction to melanin research melanin producing cells tyrosinase natural and synthetic melanins eumelanins neuromelanin pheomelanins and trichochromes pigment cell metabolism - enzymatic and chemical control genetic and hormonal regulation of melanogenesis photobiology and photochemistry of melanogenesis.
Melanin within melanosomes exists as eumelanin or pheomelanin. Distributions of these melanins have been studied extensively within tissues, but less often within individual melanosomes. Here, we apply X-ray fluorescence analysis with synchrotron radiation to survey the nanoscale distribution of metals within purified melanosomes of mice. The study allows a discovery-based characterization of melanosomal metals, and, because Cu is specifically associated with eumelanin, a hypothesis-based test of the "casing model" predicting that melanosomes contain a pheomelanin core surrounded by a eumelanin shell. Analysis of Cu, Ca, and Zn shows variable concentrations and distributions, with Ca/Zn highly correlated, and at least three discrete patterns for the distribution of Cu vs. Ca/Zn in different melanosomes - including one with a Cu-rich shell surrounding a Ca/Zn-rich core. Thus, the results support predictions of the casing model, but also suggest that in at least some tissues and genetic contexts, other arrangements of melanin may co-exist. This article is protected by copyright. All rights reserved.
Many animals display large patches of black or brown color in their integument, which contain melanin pigments and function as important sexually selected indicators of mate quality. To date, however, the particular means by which melanin-based color ornaments are costly to produce and serve as honest advertisements have remained elusive. Here, I propose a novel biochemical mechanism by which melanin-based coloration may serve as a reliable indicator of quality, involving the interplay of metal ions. In animals and other biochemical systems, a series of scarce macro- and microminerals (e.g. Ca, Zn, Cu, Fe) obtained from the diet act as critical regulatory factors in the biosynthesis of eumelanin and phacomelanin pigments, but can also be toxic to the body when accumulated in high concentrations. As large polymers with many functional (carboxyl) groups, melanin granules also bind these metals and store them in pigmented cells. Thus, by sequestering large deposits of melanin, often in dead tissue (as in hair or feathers), animals may directly reveal dietary access to these rare elements and the physiological protection they have afforded themselves from initially beneficial, but eventually damaging, high mineral concentrations.
Despite considerable advances in the past decade, melanin research still suffers from the lack of universally accepted and shared nomenclature, methodologies and structural models. This paper stems from the joint efforts of chemists, biochemists, physicists, biologists and physicians with recognized and consolidated expertise in the field of melanins and melanogenesis, who critically reviewed and experimentally revisited methods, standards and protocols to provide for the first time a consensus set of recommended procedures to be adopted and shared by researchers involved in pigment cell research. Aim of the paper is the definition of an unprecedented frame of reference built on cutting-edge knowledge and state-of-the-art methodology, to enable reliable comparison of results among laboratories and new progress in the field based on standardized methods and shared information. This article is protected by copyright. All rights reserved.
Inductively coupled plasma mass spectrometry (ICP-MS) was used to quantify the total amount of trace elements in retina from adult male Sprague-Dawley rats (n = 6). Concentration of trace elements within individual retinal areas in frozen sections of the fellow eye was established with the use of two methodologies: (1) particle-induced X-ray emission (PIXE) in combination with 3D depth profiling with Rutherford backscattering spectrometry (RBS) and (2) synchrotron X-ray fluorescence (SXRF) microscopy. The most abundant metal in the retina was zinc, followed by iron and copper. Nickel, manganese, chromium, cobalt, selenium and cadmium were present in very small amounts. The PIXE and SXRF analysis yielded a non-homogenous pattern distribution of metals in the retina. Relatively high levels of zinc were found in the inner part of the photoreceptor inner segments (RIS)/outer limiting membrane (OLM), inner nuclear layer and plexiform layers. Iron was found to accumulate in the retinal pigment epithelium/choroid layer and RIS/OLM. Copper in turn, was localised primarily in the RIS/OLM and plexiform layers. The trace elements iron, copper, and zinc exist in different amounts and locations in the rat retina.
The significance of our understanding of the chemistry of melanin and melanogenesis is reviewed. Melanogenesis begins with the production of dopaquinone, a highly reactive o-quinone. Pulse radiolysis is a powerful tool to study the fates of such highly reactive melanin precursors. Based on pulse radiolysis data reported by Land et al. (J Photochem Photobiol B: Biol 2001;64:123) and our biochemical studies, a pathway for mixed melanogenesis is proposed. Melanogenesis proceeds in three distinctive steps. The initial step is the production of cysteinyldopas by the rapid addition of cysteine to dopaquinone, which continues as long as cysteine is present (1 μM). The second step is the oxidation of cysteinyldopas to give pheomelanin, which continues as long as cysteinyldopas are present (10 μM). The last step is the production of eumelanin, which begins only after most cysteinyldopas are depleted. It thus appears that eumelanin is deposited on the preformed pheomelanin and that the ratio of eu- to pheomelanin is determined by the tyrosinase activity and cysteine concentration. In eumelanogenesis, dopachrome is a rather stable molecule and spontaneously decomposes to give mostly 5,6-dihydroxyindole. Dopachrome tautomerase (Dct) catalyses the tautomerization of dopachrome to give mostly 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Our study confirmed that the role of Dct is to increase the ratio of DHICA in eumelanin and to increase the production of eumelanin. In addition, the cytotoxicity of o-quinone melanin precursors was found to correlate with binding to proteins through the cysteine residues. Finally, it is still unknown how the availability of cysteine is controlled within the melanosome.
The element concentration of moult feathers of White-tailed Eagles was investigated. Using the 2- MeV Hamburg proton microprobe we tried to differentiate between elements incorporated into the feather via the food chain and those which are deposited externally onto the feather vane. Regarding incorporated elements, special attention has been given to a possible correlation between element concentration and feather pigmentation. Concerning the elements detected in this work (S, K, Ca, Ti, Mn, Fe, Cu, Zn, Hg, Pb), calcium, manganese and zinc show a considerable enhancement within the pigmented feather as compared with pigment-free feather sections. On the other hand, no differences were found in concentrations for sulfur, titanium, iron, copper, mercury and lead. Our findings therefore imply a special enrichment of Ca, Mn and Zn within melanin, the source of the feather's pigmentation. The possible role of these elements with regard to melanin formation is discussed.
A user friendly program for X-ray fluorescence analysis has been developed at the European Synchrotron Radiation Facility. The program allows interactive as well as batch processing of large data sets and it is particularly well suited for X-ray imaging. Its implementation of a complete description of the M shell is particularly helpful for analysis of data collected at low energies. The code is platform independent (Linux, Windows, MacOS X, Solaris …) and it is freely available for non-commercial use. Description of the algorithms used and practical examples are presented.
Understanding the interaction of metal ions with melanin structures is essential to know some intricate functions of this kind of compounds in the living systems. The coordination chemistry of vanadyl oxycation, VO(II), and copper, Cu(II), in a synthetic l-dopa melanin (SM) was investigated using electron paramagnetic resonance (EPR), Fourier transform infrared (FT IR) and ultraviolet–visible (UV–Vis) spectroscopy. The EPR and FT IR spectra indicated axially coordinated VO(II) ions bound to catecholate functional groups, and a fraction of the Cu(II) ions in a square planar structure partially coordinated to carboxylate functional groups. The resolved EPR perpendicular super hyperfine splitting constant, AN⊥, and the Cu(II) complex EPR parallel parameters g‖ and A‖ values indicated two magnetically equivalent nitrogen atoms in a plane with the coordination sphere, CuN2O2. The ultraviolet–visible (UV–Vis) spectra of solid samples indicate supramolecular structure constitution for the synthesized VO(II)SM and Cu(II)SM complexes.
Human hair fibers are primarily composed of keratin protein, characterized by a very high content of cysteine, a sulfur-containing amino acid, which ordinarily forms cystine via a disulfide bond. It is known that some cystine residues are converted to cysteic acid during permanent waving or hair coloring, although details of their distribution and extent are still unclear. In this study, by using difference in XANES profiles of cystine and cysteic acid at the S-K absorption edge, the formation of cysteic acid was confirmed for homogenized samples of permed or bleached hair. Furthermore chemical mapping of cysteic acid was performed on hair-section samples with X-ray contact microscopy. The peripheral region, cuticle, in bleached hair showed the highest content of cysteic acid compared with the other parts, while permed hair showed relatively uniform distribution. This finding suggests that perming and bleaching damage hair by different mechanisms.
XANES spectra of biomacromolecules such as histone, hemoglobin or bovine serum albumin (BSA) were measured in transmission at the S-K absorption edge for comparison with those of sulfur containing low-molecular weight biomolecules with special reference to the dependence on the chemical environment of sulfur. The spectra of dry histone and hemoglobin exhibited a prominent peak at the same energy as that of cysteine and glutathione (GSH), while the BSA spectrum showed an additional peak at a lower energy, which coincided with that of cystine and glutathione disulfide (GSSG). XANES peaks were found at the same energy even in a mammalian cell dry pellet. Spectra in the hydrated state exhibited similar profiles except for a very slight shift of resonance peaks to the lower energy. These results indicate that XANES profiles could be applicable to the mapping of S-C and S-S bonds in dry/hydrated biological systems using a spectromicroscopic technique. In addition, mass absorption coefficients of GSH and GSSG were determined.
Eumelanin and pheomelanin in tissue samples can be specifically measured as the markers pyrrole-2,3,5-tricarboxylic acid (PTCA) and 4-amino-3-hydroxyphenylalanine after acidic permanganate oxidation and hydroiodic acid hydrolysis, respectively. Those degradation methods, although widely applied, are not easily performed in most laboratories. To overcome this difficulty, we developed alkaline H(2)O(2) oxidation in 1 M K(2)CO(3) that produces, in addition to the eumelanin marker PTCA, thiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA) as markers for pheomelanin and pyrrole-2,3-dicarboxylic acid (PDCA) as a marker for 5,6-dihydroxyindole-derived eumelanin. Those four degradation products can be easily separated by HPLC and analyzed with ultraviolet detection. The alkaline H(2)O(2) oxidation method is simple, reproducible and applicable to all pigmented tissues. Its application to characterize eumelanin and pheomelanin in human hair shows that PTCA and TTCA serve as specific markers for eumelanin and pheomelanin, respectively, although some caution is needed regarding the artificial production of TTCA from eumelanic tissue proteins.
Pheomelanogenesis is a complex pathway that starts with the oxidation of tyrosine (or DOPA, 3,4-dihydroxyphenylalanine) by tyrosinase in the presence of cysteine, which results in the production of 5-S-cysteinyldopa and its isomers. Beyond that step, relatively little has been clarified except for a possible intermediate produced, dihydro-1,4-benzothiazine-3-carboxylic acid (DHBTCA). We therefore carried out a detailed study on the course of pheomelanogenesis using DOPA and cysteine and the physiological enzyme tyrosinase. To elucidate the later stages of pheomelanogenesis, chemical degradative methods of reductive hydrolysis with hydroiodic acid and alkaline peroxide oxidation were applied. The results show that: (1) DHBTCA accumulates after the disappearance of the cysteinyldopa isomers, (2) DHBTCA is then oxidized by a redox exchange with dopaquinone to form ortho-quinonimine, which leads to the production of pheomelanin with a benzothiazine moiety, and (3) the benzothiazine moiety gradually degrades to form a benzothiazole moiety. This latter process is consistent with the much higher ratio of benzothiazole-derived units in human red hair than in mouse yellow hair. These findings may be relevant to the (photo)toxic effects of pheomelanin.
The enzyme activity of tyrosinase, catalase, and peroxidase and the number of metal ions in melanogenic subcellular compartments in pigment cells were measured. Coated vesicles were richest in tyrosinase and catalase, whereas premelanosomes had the highest amount of peroxidase. Among metals ions examined, copper, zinc, and iron were more concentrated in pre-melanosomes than in coated vesicles. A quantitative analysis revealed that peroxidase served to enhance eumelanin polymer formation from monomers in the presence of hydrogen peroxide and metal ions, especially copper ions, which had the greatest enhancing effect on the conversion of monomers to polymers.
Melanin is an irregular light-absorbing polymer containing indoles and other intermediate products derived from the oxidation of tyrosine. Melanin is widely dispersed in the animal and plant kingdoms. It is the major pigment present in the surface structures of vertebrates. The critical step in melanin biogenesis is the oxidation of tyrosine by the enzyme tyrosinase. In vertebrates this enzyme is active only in specialized organelles in retinal pigment epithelium and melanocytes. In mammals melanin is formed as intracellular granules. Melanin granules are transferred from melanocytes to epithelial cells and form the predominant pigment of hair and epidermis. Melanin has many biological functions. Reactive quinone intermediates in the melanin biosynthetic pathway exhibit antibiotic properties and the polymer is an important strengthening element of plant cell walls and insect cuticle. Light absorption by melanin has several biological functions, including photoreceptor shielding, thermoregulation, photoprotection, camouflage and display. Melanin is a powerful cation chelator and may act as a free radical sink. Melanin is used commercially as a component of photoprotective creams, although mainly for its free radical scavenging rather than its light absorption properties. The pigment is also a potential target for anti-melanoma therapy.
Melanins are difficult to characterize because of their intractable chemical properties, the heterogeneity in their structural features, and the lack of methods to split melanin polymers into monomer units. To overcome this difficulty, we developed a rapid and sensitive method for quantitatively analyzing eumelanin and pheomelanin in biological samples that is based on the formation of pyrrole-2,3,5-tricarboxylic acid and/or aminohydroxyphenylalanine followed by HPLC determination. The method has been applied to the study of melanogenesis. The results summarized in this review are: 1) Biochemical studies show that in the process of mixed melanogenesis, cysteinyldopas are produced first, which are then oxidized to give pheomelanin; following cysteine depletion, eumelanin is then deposited on the preformed pheomelanin. 2) In vitro and in vivo studies show that tyrosinase activity is the most important factor that regulates the switch of melanogenesis, with lower tyrosinase activities favoring pheomelanogenesis; further suppression of melanogenesis results in a lack of pigment production. 3) In cultured melanocytes, the concentrations of tyrosine and cysteine, and their ratio in the medium, are important in determining the concentrations of eumelanin and pheomelanin produced and their ratio in the cells. In conclusion, our HPLC microanalytical method for characterizing eumelanin and pheomelanin has become a useful tool for the study of melanogenesis.
Many sulfide-oxidizing organisms, including the photosynthetic sulfur bacteria, store sulfur in "sulfur globules" that are readily detected microscopically. The chemical form of sulfur in these globules is currently the focus of a debate, because they have been described as "liquid" by some observers, although no known allotrope of sulfur is liquid at physiological temperatures. In the present work we have used sulfur K-edge X-ray absorption spectroscopy to identify and quantify the chemical forms of sulfur in a variety of bacterial cells, including photosynthetic sulfur bacteria. We have also taken advantage of X-ray fluorescence self-absorption to derive estimates of the size and density of the sulfur globules in photosynthetic bacteria. We find that the form of sulfur that most resembles the globule sulfur is simply solid S(8), rather than more exotic forms previously proposed.
The color of hair, skin, and eyes in animals mainly depends on the quantity, quality, and distribution of the pigment melanin, which occurs in two types: black to brown eumelanin and yellow to reddish pheomelanin. Microanalytical methods to quantify the amounts of eumelanin and pheomelanin in biological materials were developed in 1985. The methods are based on the chemical degradation of eumelanin to pyrrole-2,3,5-tricarboxylic acid and of pheomelanin to aminohydroxyphenylalanine isomers, which can be analyzed and quantitated by high performance liquid chromatography. This review summarizes and compares eumelanin and pheomelanin contents in various pigmented tissues obtained from humans, mice, and other animals. These methods have become valuable tools to study the functions of melanin, the control of melanogenesis, and the actions and interactions of pigmentation genes. The methods have also found applications in many clinical studies. High levels of pheomelanin are found only in yellow to red hairs of mammals and in red feathers of birds. It remains an intriguing question why lower vertebrates such as fishes do not synthesize pheomelanin. Detectable levels of pheomelanin are detected in human skin regardless of race, color, and skin type. However, eumelanin is always the major constituent of epidermal melanin, and the skin color appears to be determined by the quantity of melanin produced but not by the quality.
Sepia eumelanin is associated with many metal ions, yet little is known about its metal binding capacity and the chemical nature of the binding site(s). Herein, the natural concentrations of metal ions are presented and the ability to remove metals by exposure of the melanin granules to EDTA is quantified. The results reveal that the binding constants of melanin at pH 5.8 for Mg(II), Ca(II), Sr(II) and Cu(II) are, respectively, 5, 4, 14 and 34 times greater than the corresponding binding constants of these ions with EDTA. By exposing Sepia eumelanin to aqueous solutions of FeCl(3), the content of bound Fe(III) can be increased from a natural concentration of approximately 180 ppm to a saturation limit of approximately 80 000 ppm or 1.43 mmol/g of melanin. Similar saturation limits are found for Mg(II) and Ca(II). Exposure of Sepia melanin granules to aqueous solutions containing Ca(II) results in the stoichiometric replacement of the initially bound Mg(II), arguing that these two ions occupy the same binding site(s) in the pigment. The pH-dependent binding of Mg(II) and Ca(II) suggests coordination of these ions to carboxylic acid groups in the pigment. Mg(II) and Ca(II) can be added to a Fe(III)-saturated melanin sample without affecting the amount of Fe(III) pre-adsorbed, clearly establishing Fe(III) and Mg(II)/Ca(II) occupy different binding sites. Taking recent Raman spectroscopic data into account, the binding of Fe(III) is concluded to involve coordination to o-dihydroxyl groups. The effects of metal ion content on the surface morphology were analyzed. No significant changes were found over the full range of Fe(III) concentration studied, which is supported by the Brunauer-Emmett-Teller surface area analysis. These observations imply the existence of channels within the melanin granules that can serve to transport metal ions.
The structural organization of melanin granules isolated from ink sacs of Sepia officinalis was examined as a function of metal ion content by scanning electron microscopy and atomic force microscopy. Exposing Sepia melanin granules to ethelenediaminetetraacetic acid (EDTA) solution or to metal salt solutions changed the metal content in the melanin, but did not alter granular morphology. Thus ionic forces between the organic components and metal ions in melanin are not required to sustain the natural morphology once the granule is assembled. However, when aqueous suspensions of Sepia melanin granules of varying metal content are ultra-sonicated, EDTA-washed and Fe-saturated melanin samples lose material to the solution more readily than the corresponding Ca(II) and Mg(II)-loaded samples. The solubilized components are found to be 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-rich constituents. Associated with different metal ions, Na(I), Ca(II) and Mg(II) or Fe(III), these DHICA-rich entities form distinct two-dimensional aggregation structures when dried on the flat surface of mica. The data suggest multiply-charged ions play an important role in assisting or templating the assembly of the metal-free organic components to form the three-dimensional substructure distributed along the protein scaffold within the granule.
C57BL/6 a/a mice have been widely used to study melanogenesis, including in electron paramagnetic resonance (EPR) studies. Zinc cations modulate melanogenesis, but the net effect of Zn2+ in vivo is unclear, as the reported effects of Zn2+ on melanogenesis are ambiguous: zinc inhibits tyrosinase and glutathione reductase in vitro, but also enhances the activity of dopachrome tautomerase (tyrosinase-related protein-2) and has agonistic effects on melanocortin receptor signalling.
To determine in a C57BL/6 a/a murine pilot study whether excess zinc ions inhibit, enhance or in any other way alter hair follicle melanogenesis in vivo, and to test the usefulness of EPR for this study.
ZnSO(4).7H2O was continuously administered orally to C57BL/6 a/a mice during spontaneous and depilation-induced hair follicle cycling (20 mg mL-1; in drinking water; mean+/-SD daily dose 1.2+/-0.53 mL), and hair pigmentation was examined macroscopically, by routine histology and by EPR.
Oral zinc cations induced a bright brown lightening of new hair shafts produced during anagen, but without inducing an EPR-detectable switch from eumelanogenesis to phaeomelanogenesis. The total content of melanin in the skin and hair shafts during the subsequent telogen phase, i.e. after completion of a full hair cycle, was significantly reduced in Zn-treated mice (P=0.0005). Compared with controls, melanin granules in precortical hair matrix keratinocytes, hair bulb melanocytes and hair shafts of zinc-treated animals were reduced and poorly pigmented. Over the course of several hair cycles, lasting hair shaft depigmentation was seen during long-term exposure to high-dose oral Zn2+.
High-dose oral Zn2+ is a potent downregulator of eumelanin content in murine hair shafts in vivo. The C57BL/6 mouse model offers an excellent tool for further dissecting the as yet unclear underlying molecular basis of this phenomenon, while EPR technology is well suited for the rapid, qualitative and quantitative monitoring of hair pigmentation changes.
A new cysteinyldopa model ligand Cydo {3-[(2-aminoethyl)sulfanyl]-4,6-di-tert-butylbenzene-1,2-diol} was prepared and its reactivity with Cu(II) explored. Under anaerobic conditions, tetranuclear [Cu4(Cydo)4] is isolated, but in the presence of O2, a benzothiazine intermediate accumulates that is trapped as the Cu(II) complex [Cu(zine)2]. Under slightly different reaction conditions, the benzothiazine further oxidizes to benzothiazole (zole). All three compounds were characterized by X-ray crystallography, and the reactions were monitored spectrophotometrically.
In this article, we review the current state of knowledge concerning the physical and chemical properties of the eumelanin pigment. We examine properties related to its photoprotective functionality, and draw the crucial link between fundamental molecular structure and observable macroscopic behaviour. Where necessary, we also briefly review certain aspects of the pheomelanin literature to draw relevant comparison. A full understanding of melanin function, and indeed its role in retarding or promoting the disease state, can only be obtained through a full mapping of key structure-property relationships in the main pigment types. We are engaged in such an endeavor for the case of eumelanin.
Metal chelation is often invoked as one of the main biological functions of melanin. In order to understand the interaction between metals and melanin, extensive studies have been carried out to determine the nature of the metal binding sites, binding capacity, and affinity. These data are central to efforts aimed at elucidating the role metal binding plays in determining the physical, structural, biological, and photochemical properties of melanin. This article examines the current state of understanding of this field.
Cysteinyldopas are naturally occurring conjugates of cysteine and dopa (3,4-dihydroxy-l-phenylalanine) that are precursors to red pheomelanin pigments. Metal ions are known to influence pheomelanogenesis in vitro and may be regulatory factors in vivo. Cydo (3-[(2-amino-ethyl)sulfanyl]-4,6-di-tert-butylbenzene-1,2-diol) and CarboxyCydo (2-amino-3-(4,6-di-tert-butyl-2,3-dihydroxyphenylsulfanyl)-propionic acid) are model compounds of cysteinyldopa that retain its metal-binding functionalities but cannot polymerize due to the presence of blocking tert-butyl groups. Cydo reacts readily with zinc(II) acetate or nickel(II) acetate to form a cyclized 1,4-benzothiazine (zine) intermediate that undergoes ring contraction to form benzothiazole (zole) unless it is stabilized by coordination to a metal ion. The crystal structure of [Ni(zine)2] is reported. The acetate counteranion is required for the zinc-promoted reactivity, as neither zinc(II) sulfate nor zinc(II) chloride alone promotes the transformation. The counterion is less important for redox-active copper and iron, which both readily promote the oxidation of Cydo to zine and zole species; Cu(II) complexes of both zine and zole have been characterized by X-ray crystallography. In the case of CarboxyCydo, a 3-carboxy-1,4-benzothiazine intermediate decarboxylates to form [Cu(zine)2] under basic conditions, but in the absence of base forms a mixture of products that includes the carboxylated dimer 2,2'-bibenzothiazine (bi-zine). These products are consistent with species implicated in the pheomelanogenesis biosynthetic pathway and emphasize how metal ions, their counteranions, and reaction conditions can alter pheomelanin product distribution.
The control mechanisms and information content of melanin-based color signals in birds have generated much recent interest and controversy among evolutionary biologists. Initial experimental studies on this topic manipulated coarse metrics of an individual's condition (i.e. food intake, disease state) and failed to detect significant condition-dependence of melanin ornament expression. However, three new lines of research appear profitable and target specific factors associated with the production of melanin pigments. These include the role of (i) metals, (ii) amino acids, and (iii) testosterone and social interactions in shaping the extent and intensity of melanin-colored plumage patches. Here, I review recent studies of and evidence for these honesty-reinforcing mechanisms.
- P A Riley
- Melanin
Riley, P. A. Melanin. Int. J. Biochem. Cell Biol. 29, 1235-1239 (1997).
Unravelling the melanocyte
- V J Hearing
Hearing, V. J. Unravelling the melanocyte. Am. J. Hum. Genet 52, 1-7 (1993).