Alberto Jorge

Raman spectroscopy as a non-invasive technique for the quantification of melanins in feathers and hairs

The quantification of melanins is a complex task due to the chemical heterogeneity of the pigments and the difficulty of their isolation. The best accepted procedure currently consists in the chemical cleavage of melanins and the subsequent detection of degradation products by HPLC, which implies the destruction of samples. Here, we show that Raman spectroscopy is a non‐invasive technique that can be used to quantify melanins. We made parallel analyses of the characteristics of pheomelanin and eumelanin Raman spectra as measured by confocal Raman microscopy and of degradation products of pheomelanin (4‐amino‐3‐hydroxyphenylalanine, 4‐AHP) and eumelanin (pyrrole‐2,3,5‐tricarboxylic acid, PTCA) as measured by HPLC in feathers of red‐legged partridges and hairs of wild boars and humans. We found strong correlations between the spectral Raman characteristics and 4‐AHP and PTCA levels, which indicates that the Raman spectra of melanins can be used to determine their content.

Molecular characterization of a dual inhibitory and mutagenic activity of 5-fluorouridine triphosphate on viral RNA synthesis. Implications for lethal mutagenesis.

The basis for a dual inhibitory and mutagenic activity of 5-fluorouracil (5-FU) on foot-and-mouth disease virus (FMDV) RNA replication has been investigated with purified viral RNA-dependent RNA polymerase (3D) in vitro. 5-Fluorouridine triphosphate acted as a potent competitive inhibitor of VPg uridylylation, the initial step of viral replication. Peptide analysis by mass spectrometry has identified a VPg fragment containing 5-fluorouridine monophosphate (FUMP) covalently attached to Tyr3, the amino acid target of the uridylylation reaction. During RNA elongation, FUMP was incorporated in the place of UMP or CMP by FMDV 3D, using homopolymeric and heteropolymeric templates. Incorporation of FUMP did not prevent chain elongation, and, in some sequence contexts, it favored misincorporations at downstream positions. When present in the template, FUMP directed the incorporation of AMP and GMP, with ATP being a more effective substrate than GTP. The misincorporation of GMP was 17-fold faster opposite FU than opposite U in the template. These results in vitro are consistent with the mutational bias observed in the mutant spectra of 5-FU-treated FMDV populations. The dual mutagenic and inhibitory activity of 5-fluorouridine triphosphate may contribute to the effective extinction of FMDV by 5-FU through virus entry into error catastrophe.

Insects synthesize pheomelanin

1 Department of Evolutionary Ecology, Do~nana Biological Station—CSIC, Sevilla, Spain 2 National Museum of Natural Sciences—CSIC, Madrid, Spain 3 Department of Molecular Biology and Biochemical Engineering, Pablo de Olavide University, Sevilla, Spain 4 Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan Correspondence Ismael Galv!an, e-mail: galvan@ebd.csic.es

Dispersive Raman spectroscopy allows the identification and quantification of melanin types

Melanins are the most prevalent pigments in animals and are involved in visual communication by producing colored traits that often evolve as intraspecific signals of quality. Identifying and quantifying melanins are therefore essential to understand the function and evolution of melanin-based signals. However, the analysis of melanins is difficult due to their insolubility and the lack of simple methods that allow the identification of their chemical forms. We recently proposed the use of Raman spectroscopy as a simple, noninvasive technique that can be used to identify and quantify melanins in feathers and hairs. Contrarily, other authors later stated that melanins are characterized by a lack of defined Raman signals. Here, we use confocal Raman microscopy to confirm previous analyses showing that the two main chemical forms of melanins (eumelanin and pheomelanin) exhibit distinct Raman signal and compare different excitation wavelengths to analyze synthetic pheomelanin and natural melanins in feathers of different species of birds. Our analyses indicate that only laser excitation wavelengths below 1064 nm are useful for the analysis of melanins by Raman spectroscopy, and only 780-nm laser in the case of melanins in feathers. These findings show that the capacity of Raman spectroscopy to distinguish different chemical forms of melanins depends on laser power and integration time. As a consequence, Raman spectroscopy should be applied after preliminar analyses using a range of these parameters, especially in fragile biological tissues such as feathers.

Vibrational characterization of pheomelanin and trichochrome F by Raman spectroscopy

We characterize for the first time the vibrational state of natural pheomelanin using Raman spectroscopy and model pigment synthesized from 5-S-cysteinyldopa. The shape of the Raman spectrum was very different from that of eumelanin. Four Raman bands were visible in the 500-2000 cm(-1) wavenumber region about 500, 1150, 1490 and 2000 cm(-1), which we assigned to the out-of-plane deformation and the stretching vibration of the phenyl rings, to the stretching vibration of C-N bonds or the stretching and wagging vibration of CH2, and to overtone or combination bands. Interestingly, we also show that the Raman spectrum of synthetic trichochrome F, a pigment that may be produced along with pheomelanin during pheomelanogenesis, is different from that of pheomelanin and similar to the spectrum of eumelanin. We could detect Raman signal of both eumelanin and pheomelanin in feathers and hairs where both pigments simultaneously occur without the need of isolating the pigment. This indicates that Raman spectroscopy represents a non-invasive method to detect pheomelanin and distinguish it from other pigments. This may be especially relevant to detect pheomelanin in animal skin including humans, where it has been associated with animal appearance and classification, human phototypes, prevention of skin diseases and cancer risk.

Eumelanin and pheomelanin are predominant pigments in bumblebee (Apidae: Bombus) pubescence

Background Bumblebees (Hymenoptera: Apidae: Bombus) are well known for their important inter- and intra-specific variation in hair (or pubescence) color patterns, but the chemical nature of the pigments associated with these patterns is not fully understood. For example, though melanization is believed to provide darker colors, it still unknown which types of melanin are responsible for each color, and no conclusive data are available for the lighter colors, including white. Methods By using dispersive Raman spectroscopy analysis on 12 species/subspecies of bumblebees from seven subgenera, we tested the hypothesis that eumelanin and pheomelanin, the two main melanin types occurring in animals, are largely responsible for bumblebee pubescence coloration. Results Eumelanin and pheomelanin occur in bumblebee pubescence. Black pigmentation is due to prevalent eumelanin, with visible signals of additional pheomelanin, while the yellow, orange, red and brown hairs clearly include pheomelanin. On the other hand, white hairs reward very weak Raman signals, suggesting that they are depigmented. Additional non-melanic pigments in yellow hair cannot be excluded but need other techniques to be detected. Raman spectra were more similar across similarly colored hairs, with no apparent effect of phylogeny and both melanin types appeared to be already used at the beginning of bumblebee radiation. Discussion We suggest that the two main melanin forms, at variable amounts and/or vibrational states, are sufficient in giving almost the whole color range of bumblebee pubescence, allowing these insects to use a single precursor instead of synthesizing a variety of chemically different pigments. This would agree with commonly seen color interchanges between body segments across Bombus species.

The rusty plumage coloration of juvenile gyrfalcons is produced by pheomelanin and its expression is affected by an intracellular antioxidant

Abstract Juveniles of many diurnal raptors exhibit a characteristic rusty plumage coloration whose biochemical basis has never been determined. Using the Gyrfalcon (Falco rusticolus) as a model species, we analyzed feathers by Raman spectroscopy and showed that the rusty color is due to the presence of the pigment pheomelanin, which was also observed in the feathers of a juvenile Peregrine Falcon (Falco peregrinus). We experimentally modified the expression of the rusty plumage coloration by treating four developing Gyrfalcons with buthionine sulfoximine (BSO), a specific and nontoxic inhibitor of glutathione (GSH) synthesis. Because cysteine, one of the three constitutive amino acids of GSH, is required for pheomelanin synthesis and GSH is the most important intracellular antioxidant, these findings indicate that the expression of rusty plumage coloration can be affected by environmental oxidative stress. Our results suggest that the rusty plumage coloration of at least some diurnal raptors is pheomelanin-based, and the dependence on GSH levels opens the possibility that the evolution of this trait in some species and the age-related variation in its expression across species may be explained by interspecific and intraspecific variation in exposure to environmental factors that generate oxidative stress and by age-related variations in endogenous levels of oxidative stress. La Coloración Ferruginosa del Plumaje Juvenil de Falco rusticolus es Producida por Feomelanina y su Expresión Es Afectada por un Antioxidante Intracelular Los juveniles de numerosas especies de rapaces exhiben una coloración ferruginosa típica del plumaje, cuya base bioquímica nunca ha sido determinada. Usando a Falco rusticolus como una especie modelo, analizamos sus plumas mediante espectroscopia Raman y demostramos que el color ferruginoso se debe a la presencia del pigmento feomelanina, el cual también fue observado en las plumas de un individuo juvenil de Falco peregrinus. Modificamos experimentalmente la expresión de la coloración ferruginosa del plumaje tratando a cuatro individuos juveniles de F. rusticolus con butionina sulfoximina (BS), un inhibidor específico y no tóxico de la síntesis del glutatión (GSH). Debido a que se requiere cisteína (uno de los tres aminoácidos constituyentes del GSH) para la síntesis de feomelanina y el GSH es el antioxidante intracelular más importante, estos hallazgos indican que la expresión de la coloración ferruginosa del plumaje puede ser afectada por el estrés oxidativo ambiental. Nuestros resultados sugieren que la coloración ferruginosa del plumaje de al menos algunas especies de rapaces diurnas se debe a la feomelanina. Por otro lado, la dependencia en los niveles de GSH abre la posibilidad de que la evolución de este rasgo y la variación de su expresión relacionada con la edad en diferentes especies puede explicarse por la variación inter e intra-específica en la exposición a factores ambientales que generan un estrés oxidativo y por variaciones en los niveles endógenos de estrés oxidativo relacionados con la edad.

Raman Spectroscopy Reveals the Presence of Both Eumelanin and Pheomelanin in the Skin of Lacertids

Abstract Melanic pigments play a key role in the coloration of animals, but the type of melanin pigment in black, brown, and blue colored scales of Squamata has not been studied. Based on research on birds and mammals, we may expect that pheomelanin is the majority pigment in brownish colorations and eumelanin is the majority pigment in black and blue colorations of Squamata. To characterize the pigments that underlie the melanin-based colorations of lizards, we analyzed the skin of nine genera of lacertids using dispersive Raman spectroscopy. Our results suggest that no prediction can be made on the type of pigmentary melanin present in the skin of the lacertids based alone on the hue of the sample. Indeed, brownish patterns in the skin of Psammodromus, Gallotia, Acanthodactylus, and Algyroides lizards presented both chemical forms of melanin: eumelanin and pheomelanin. Therefore, pheomelanogenesis might be an ancient characteristic within Lacertidae, because it was detected in genera in the Lacertini, Eremini, and Gallotini. Raman spectra of melanic-based patterns of genus Zootoca and ultraviolet (UV)-blue patches of Podarcis, Iberolacerta, Lacerta, and Timon lizards suggested that eumelanin is the majority pigment in these patches. Raman spectroscopy is a suitable nondestructive technique useful to identify melanin forms in the skin of lizards, and it demonstrated that pheomelanin is synthesized by Squamata. Resumen Los pigmentos melánicos juegan un papel clave en la coloración de los animales. Sin embargo, en los Squamata se desconoce la identidad molecular de los pigmentos melánicos (i.e., eumelanina y feomelanina) responsables de las coloraciones negras, marrones y azules. Basado en estudios anteriores en aves y mamíferos, cabría esperar que la feomelanina fuera el pigmento mayoritario en coloraciones marrones y que la eumelanina sea el pigmento mayoritario en las coloraciones negras y azules de los Squamata. Para caracterizar los pigmentos responsables de la coloración melánica en lagartos hemos analizado la piel de nueve géneros de lacértidos usando espectroscopía Raman dispersiva. Nuestros resultados sugieren que no se puede predecir el tipo de melanina pigmentaria presente en la piel de los lácertidos basándonos solo en la tonalidad de la muestra. De hecho, en los patrones de color marrón de la piel de lagartos de los géneros Psammodromus, Gallotia, Acanthodactylus y Algyroides están presentes ambas formas químicas de la melanina pigmentaria, es decir, eumelanina y feomelanina. En los patrones melánicos del género Zootoca y en las manchas ultravioleta (UV)-azules de lagartos en los géneros Podarcis, Iberolacerta, Lacerta, y Timon el espectro Raman sugirió que la forma eumelánica debe de ser el pigmento mayoritario en estas manchas. La espectroscopía Raman es una técnica no destructiva que es aplicable para identificar las formas melánicas presentes en la piel de los lagartos y demostró que los Squamata pueden sintetizar feomelanina.

Solar and terrestrial radiations explain continental-scale variation in bird pigmentation

Animals living on the earth’s surface are protected from the damaging effects of solar ultraviolet (UV) radiation by melanin pigments that color their integument. UV levels that reach the earth’s surface vary spatially, but the role of UV exposure in shaping clinal variations in animal pigmentation has never been tested. Here, we show at a continental scale in Europe that golden eagles Aquila chrysaetos reared in territories with a high solar UV-B radiation exposure deposit lower amounts of the sulphurated form of melanin (pheomelanin) in feathers and consequently develop darker plumage phenotypes than eagles from territories with lower radiation exposure. This clinal variation in pigmentation is also explained by terrestrial γ radiation levels in the rearing territories by a similar effect on the pheomelanin content of feathers, unveiling natural radioactivity as a previously unsuspected factor shaping animal pigmentation. These findings show for the first time the potential of solar and terrestrial radiations to explain pigmentation phenotype diversity in animals, including humans, at large spatial scales.

Analyzing materials in the microscopes: From the Sorby thin sections up to the non-destructive large chambers

One hundred and sixty five years ago, Henry Clifton Sorby developed a revolutionary technique to prepare thin and polished sections of rocks and minerals to be observed by transmission and reflectance in the optical polarized light microscopes. Nowadays, Sorby’s methods are still employed for near all inorganic materials with scarce modifications. The optical microscopy subject is essential for rocks analysis; it is an irreplaceable technique for specific complex samples, such as twinned-exsolved feldspars and it must be preserved in the geosciences curriculum. However, for many valuable and common specimens we observe a strong growing of non-destructive techniques coupled to the large chambers of modern microscopes including new chemical, molecular, luminescent, metrical, imaging and structural probes. Suitable explanations for the thin sections decreasing in research laboratories could be: (i) the undesirable cutting of the specimen; (ii) the contamination of surfaces by abrasives and glues; (iii) the e...