Neuromelanins in brain aging and Parkinson's disease: synthesis, structure, neuroinflammatory, and neurodegenerative role.

Neuromelanins are compounds accumulating in neurons of human and animal brain during aging, with neurons of substantia nigra and locus coeruleus having the highest levels of neuromelanins. These compounds have melanic, lipid, peptide, and inorganic components and are contained inside special autolysosomes. Neuromelanins can participate in neuroprotective or toxic processes occurring in Parkinson's disease according to cellular environment. Their synthesis depends on the concentration of cytosolic catechols and is a protective process since it prevents the toxic accumulation of catechols-derived reactive compounds. Neuromelanins can be neuroprotective also by binding reactive/toxic metals to produce stable and non-toxic complexes. Extraneuronal neuromelanin released by dying dopamine neurons in Parkinson's disease activates microglia which generate reactive oxygen species, reactive nitrogen species, and proinflammatory molecules, thus producing still neuroinflammation and neuronal death. Synthetic neuromelanins have been prepared with melanic, protein structure, and metal content closely mimicking the natural brain pigment, and these models are also able to activate microglia. Neuromelanins have different structure, synthesis, cellular/subcellular distribution, and role than melanins of hair, skin, and other tissues. The main common aspect between brain neuromelanin and peripheral melanin is the presence of eumelanin and/or pheomelanin moieties in their structure.

A Model Eumelanin from 5,6-Dihydroxyindole-2-Carboxybutanamide Combining Remarkable Antioxidant and Photoprotective Properties with a Favourable Solubility Profile for Dermo-Cosmetic Applications.

The search for new synthetic melanin-related pigments that maintain the antioxidant and photoprotective properties of naturally occurring dark eumelanins, while overcoming their unfavorable solubility, and molecular heterogeneity is presently a very active issue for dermo-cosmetic purposes. In this work, we explored the potential of a melanin obtained from the carboxybutanamide of a major eumelanin biosynthetic precursor, 5,6-dihydroxyindole-2-carboxylic acid (DHICA), by aerobic oxidation under slightly alkaline conditions. Analysis of the pigment by EPR, ATR-FTIR and MALDI MS indicated a substantial structural similarity to DHICA melanin, while investigation of the early intermediates confirmed unchanged regiochemistry of the oxidative coupling. The pigment exhibited a UVA-visible absorption even more intense than that of DHICA melanin, and a noticeable solubility in polar solvents of dermo-cosmetic relevance. The hydrogen- and/or electron-donor ability, and the iron (III) reducing power as determined by conventional assays provided evidence for marked antioxidant properties not merely ascribable to the more favorable solubility profile, while the inhibitory action of the radical- or photosensitized solar light-induced lipid peroxidation was more marked compared to that of DHICA melanin. Overall, these results hint at this melanin, which remarkable properties are, in part, due to the electronic effects of the carboxyamide functionality as a promising functional ingredient for dermo-cosmetic formulations.

Multiphoton FLIM Analyses of Native and UVA-Modified Synthetic Melanins.

To better understand the impact of solar light exposure on human skin, the chemical characterization of native melanins and their structural photo-modifications is of central interest. As the methods used today are invasive, we investigated the possibility of using multiphoton fluorescence lifetime (FLIM) imaging, along with phasor and bi-exponential fitting analyses, as a non-invasive alternative method for the chemical analysis of native and UVA-exposed melanins. We demonstrated that multiphoton FLIM allows the discrimination between native DHI, DHICA, Dopa eumelanins, pheomelanin, and mixed eu-/pheo-melanin polymers. We exposed melanin samples to high UVA doses to maximize their structural modifications. The UVA-induced oxidative, photo-degradation, and crosslinking changes were evidenced via an increase in fluorescence lifetimes along with a decrease in their relative contributions. Moreover, we introduced a new phasor parameter of a relative fraction of a UVA-modified species and provided evidence for its sensitivity in assessing the UVA effects. Globally, the fluorescence lifetime properties were modulated in a melanin-dependent and UVA dose-dependent manner, with the strongest modifications being observed for DHICA eumelanin and the weakest for pheomelanin. Multiphoton FLIM phasor and bi-exponential analyses hold promising perspectives for in vivo human skin mixed melanins characterization under UVA or other sunlight exposure conditions.

The First Homozygote Mutation c.499G>T (Asp167Tyr) in the RPE65 Gene Encoding Retinoid Isomerohydrolase Causing Retinal Dystrophy

RPE65, an abundant membrane-associated protein present in the retinal pigment epithelium (RPE), is a vital retinoid isomerase necessary for regenerating 11-cis-retinaldehyde from all-trans retinol in the visual cycle. In patients with inherited retinal dystrophy (IRD), precise genetic diagnosis is an indispensable approach as it is required to establish eligibility for the genetic treatment of RPE65-associated IRDs. This case report aims to report the specific phenotype−genotype correlation of the first patient with a homozygous missense variant RPE65 c.499G>T, p. (Asp167Tyr). We report a case of a 66-year-old male who demonstrated a unique phenotype manifesting less severe functional vision deterioration in childhood and adolescence, and extensive nummular pigment clusters. The underlying causes of the differences in the typical bone spicule and atypical nummular pigment clumping are unknown, but suggest that the variant itself influenced the rate of photoreceptor death. Functional studies are needed to define whether the substitution of aspartate impairs the folding of the tertiary RPE65 structure only and does not lead to the complete abolishment of chromophore production, thus explaining the less severe phenotype in adolescence.

Melanins from the Lichens Lobaria pulmonaria and Lobaria retigera as Eco-Friendly Adsorbents of Synthetic Dyes.

Synthetic dyes are widely used in the industry; they are chemically stable, difficult to neutralize, and therefore they are a threat to the environment when released into wastewaters. The dyes have a significant impact on plant performance by impairing photosynthesis, inhibiting growth, and entering the food chain and may finally result in the toxicity, mutagenicity and carcinogenicity of food products. Implementation of the dark piment melanin for the adsorption of the synthetic dyes is a new ecologically friendly approach for bioremediation. The aim of the present work was to study the physico-chemical characteristics of melanins from the lichens Lobaria pulmonaria and Lobaria retigera, analyze their adsorption/desorption capacities towards synthetic dyes, and assess the capacity of melanins to mitigate toxicity of the dyes for a common soil bacterium Bacillus subtilis. Unique chelating properties of melanins determine the perspectives of the use of these high molecular weight polymers for detoxification of xenobiotics.

Gingival pigmentation: Concurrent assessment of distribution, intensity, and extent in a black population.

OBJECTIVE: To evaluate prevalence, distribution, intensity and extent of physiologic gingival melanin pigmentation (GMP) in black individuals. MATERIALS AND METHODS: For this cross-sectional study, GMP was evaluated on digital images by three calibrated examiners, according to de Krom (distribution), DOPI (intensity) and Melanin Index (extent) classifications. Descriptive statistics, Wilcoxon, Mann-Whitney, Kruskal-Wallis, Two-way ANOVA, chi square, and K-means cluster analysis were used. RESULTS: Seventy participants were recruited. The most prevalent GMP categories were: de Krom category 2 (34.3%), DOPI heavy intensity (57.2%), and Melanin index Degree IV (50%). Significant inter-group differences were found for age (p < 0.05) but not gender (p > 0.05). Significant correspondence/overlap was observed between classifications (p > 0.05). Three GMP clusters were identified: Cluster 1 (27%; n = 19) had mild asymmetric and interspersed pigmentation; Cluster 2 (46%; n = 32) had heavy pigmentation in one long continuous ribbon, with pink marginal gingiva; and Cluster 3 (27%; n = 19) had heavy pigmentation in one long continuous ribbon, symmetric, and uniform. CONCLUSIONS: There may be overlap among GMP classification systems. In black individuals, the predominant GMP presentation is one of a broad zone of heavily pigmented attached gingiva, in a continuous strip from central incisors to canines, symmetrical across the midline, and with pink free marginal gingiva. CLINICAL RELEVANCE: This is the first study to analyze distribution, intensity, and extent of gingival melanin pigmentation in the same population and to integrate the various classification systems through cluster analysis. The novel findings provide a foundation for patient assessment and counseling and for future studies.

A Straightforward Access to New Amides of the Melanin Precursor 5,6-Dihydroxyindole-2-carboxylic Acid and Characterization of the Properties of the Pigments Thereof.

We report herein an optimized procedure for preparation of carboxamides of 5,6-dihydroxyindole-2-carboxylic acid (DHICA), the main biosynthetic precursor of the skin photoprotective agents melanins, to get access to pigments with more favorable solubility properties with respect to the natural ones. The developed procedure was based on the use of a coupling agent (HATU/DIPEA) and required protection of the catechol function by easily removable acetyl groups. The O-acetylated compounds could be safely stored and taken to the reactive o-diphenol form just before use. Satisfactorily high yields (>85%) were obtained for all amides. The oxidative polymerization of the synthesized amides carried out in air in aqueous buffer at pH 9 afforded melanin-like pigmented materials that showed chromophores resembling those of DHICA-derived pigments, with a good covering of the UVA and the visible region, and additionally exhibited a good solubility in alcoholic solvents, a feature of great interest for the exploitation of these materials as ingredients of dermocosmetic formulations.

Genomic investigation of colour polymorphism and phylogeographic variation among populations of black-headed bulbul (Brachypodius atriceps) in insular southeast Asia.

Intraspecific polymorphism in birds, especially plumage colour polymorphism, and the mechanisms that control it are an area of active research in evolutionary biology. The black-headed bulbul (Brachypodius atriceps) is a polymorphic species with two distinct morphs, yellow and grey. This species inhabits the mainland and virtually all continental islands of Southeast Asia where yellow morphs predominate, but on two islands in the Sunda region, Bawean and Maratua, grey morphs are common or exclusive. Here, we generated a high-quality reference genome of a yellow individual and resequenced genomes of multiple individuals of both yellow and grey morphs to study the genetic basis of coloration and population history of the species. Using PCA and STRUCTURE analysis, we found the Maratua Island population (which is exclusively grey) to be distinct from all other B. atriceps populations, having been isolated c. 1.9 million years ago (Ma). In contrast, Bawean grey individuals (a subset of yellow and grey individuals on that island) are embedded within an almost panmictic Sundaic clade of yellow birds. Using FST and dxy to compare variable genomic segments between Maratua and yellow individuals, we located peaks of divergence and identified candidate loci involved in the colour polymorphism. Tests of selection among coding-proteins in high FST regions, however, did not indicate selection on the candidate genes. Overall, we report on some loci that are potentially responsible for the grey/yellow polymorphism in a species that otherwise shows little genetic diversification across most of its range.

Colourful traits in female birds relate to individual condition, reproductive performance and male-mate preferences: a meta-analytic approach.

Colourful traits in females are suggested to have evolved and be maintained by sexual selection. Although several studies have evaluated this idea, support is still equivocal. Evidence has been compiled in reviews, and a handful of quantitative syntheses has explored cumulative support for the link between condition and specific colour traits in males and females. However, understanding the potential function of females' colourful traits in sexual communication has not been the primary focus of any of those previous studies. Here, using a meta-analytic approach, we find that evidence from empirical studies in birds supports the idea that colourful female ornaments are positively associated with residual mass and immune response, clutch size and male-mate preferences. Hence, colourful traits in female birds likely evolved and are maintained by sexual selection as condition-dependent signals.

Impairment of mixed melanin-based pigmentation in parrots.

Parrots and allies (Order Psittaciformes) have evolved an exclusive capacity to synthesize polyene pigments called psittacofulvins at feather follicles, which allows them to produce a striking diversity of pigmentation phenotypes. Melanins are polymers constituting the most abundant pigments in animals, and the sulphurated form (pheomelanin) produces colors that are similar to those produced by psittacofulvins. However, the differential contribution of these pigments to psittaciform phenotypic diversity has not been investigated. Given the color redundancy, and physiological limitations associated to pheomelanin synthesis, we hypothesized that the latter would be avoided by psittaciform birds. Here we test this by using Raman spectroscopy to identify pigments in feathers exhibiting colors suspicious of being produced by pheomelanin (i.e., dull red, yellow and grey- and green-brownish) in 26 species from the three main lineages of Psittaciformes. We detected the non-sulphurated melanin form (eumelanin) in black, grey and brown plumage patches, and psittacofulvins in red, yellow and green patches, but no evidence of pheomelanin. As natural melanins are assumed to be composed of eumelanin and pheomelanin in varying ratios, our results represent the first report of impairment of mixed melanin-based pigmentation in animals. Given that psittaciforms also avoid the uptake of circulating carotenoid pigments, these birds seem to have evolved a capacity to avoid functional redundancy between pigments, likely by regulating follicular gene expression. Ours study provides the first vibrational characterization of different psittacofulvin-based colors and thus helps to determine the relative polyene chain length in these pigments, which is related to their antireductant protection activity.

Biological activities of melanin pigment extracted from Bombyx mori gut-associated yeast Cryptococcus rajasthanensis KY627764.

Melanin pigment has been produced and extracted from a wide variety of living forms ranging from microorganisms to higher organisms. Owing to the therapeutic nature of the pigment, various microbial populations have been explored for its production. Hence, we isolated a melanin producing yeast from the insect Bombyx mori gut microflora and identified it as Cryptococcus rajasthanensis based on the molecular characterization. The isolated yeast produced enhanced melanin pigment when cultured in the minimal L-tyrosine broth as compared to the Saboraud medium. The pigment was extracted and characterized as melanin based on UV-Visible spectroscopy, FTIR (Fourier-transform infrared) spectroscopy and 1H NMR (Nuclear magnetic resonance). The melanin pigment was evaluated as a potent bioactive molecule with bioactivity like antimicrobial, antioxidant, anti-inflammatory, and anticancer activity that describes the therapeutic nature of the extracted melanin pigment. Distinct from the biologically active role the melanin pigment isolated from the yeast, the Cryptococcus extract also exhibited killer toxin activity against the pathogenic yeast Candida albicans.

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.

Pigmented lesions of the oral mucosa: A cross-sectional study of 458 histopathological specimens.

OBJECTIVES: To evaluate clinical, demographic, and histopathological characteristics of pigmented lesions of the oral mucosa. MATERIALS AND METHODS: A cross-sectional study was conducted over a 64-year period. Information was collected from medical charts, and all archived histopathological specimens with diagnoses of any pigmented lesion were retrieved. Statistical analysis was performed using SPSS software. RESULTS: A total of 34,127 archived specimens were reviewed, revealing 458 (1.34%) pigmented lesions, of which 230 were melanocytic and 228 nonmelanocytic. Most patients were females (74.2%), white-skinned (49.1%), in the third and seventh decades of life (mean of 45 years). Most lesions were macular (59.8%), followed by plaques and nodules (4.8%), measuring 0-5 mm (41.9%). Cheek mucosa (21.0%), alveolar mucosa (16.6%), and gingiva (11.8%) were the most commonly affected sites. Amalgam tattoo was applied in 212 cases (46.3%), followed by melanotic macule (22.9%) and nevus (20.5%). Other diagnoses included racial pigmentation, exogenous pigmentation, melanotic neuroectodermal tumor of infancy, melanoma, melanoacanthoma, smoker's melanosis, and heavy metal pigmentation. CONCLUSIONS: Pigmented lesions represent an uncommon diagnosis in oral pathology routines. The most frequent entities are amalgam tattoo, melanotic macule, and nevus. Patients are usually middle-aged women presenting a small, long-lasting, macular lesion on the cheek mucosa.

The Late Stages of Melanogenesis: Exploring the Chemical Facets and the Application Opportunities.

In the last decade, the late stages of melanin biosynthesis involving the oxidative polymerization of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) have been extensively investigated. Most of the information derived from a biomimetic approach in which the oxidation of melanogenic indoles was carried out under conditions mimicking those occurring in the biological environment. Characterization of the early oligomers allowed for drawing a structural picture of DHI and DHICA melanins, providing also an interpretative basis for the different properties exhibited by these pigments, e.g., the chromophore and the antioxidant ability. The improved knowledge has opened new perspectives toward the exploitation of the unique chemistry of melanins and its precursors in cosmetic and health care applications. A noticeable example is the development of an innovative hair dyeing system that is based on the marked ease of DHI to give rise to black melanin on air oxidation under slightly alkaline conditions. The advantage of this method for a step-wise coverage of gray hair with a natural shade pigmentation on repeated treatment with a DHI-based formulation with respect to traditional dyes is presented. A variant of DHICA melanin combining solubility in water-miscible organic solvents, an intense chromophore in the UltraViolet-A UV-A region, and a marked antioxidant potency was evaluated as an ingredient for cosmetic formulations.

Transplantable Melanomas in Hamsters and Gerbils as Models for Human Melanoma. Sensitization in Melanoma Radiotherapy-From Animal Models to Clinical Trials.

The focus of the present review is to investigate the role of melanin in the radioprotection of melanoma and attempts to sensitize tumors to radiation by inhibiting melanogenesis. Early studies showed radical scavenging, oxygen consumption and adsorption as mechanisms of melanin radioprotection. Experimental models of melanoma in hamsters and in gerbils are described as well as their use in biochemical and radiobiological studies, including a spontaneously metastasizing ocular model. Some results from in vitro studies on the inhibition of melanogenesis are presented as well as radio-chelation therapy in experimental and clinical settings. In contrast to cutaneous melanoma, uveal melanoma is very successfully treated with radiation, both using photon and proton beams. We point out that the presence or lack of melanin pigmentation should be considered, when choosing therapeutic options, and that both the experimental and clinical data suggest that melanin could be a target for radiosensitizing melanoma cells to increase efficacy of radiotherapy against melanoma.

Modeling Fungal Melanin Buildup: Biomimetic Polymerization of 1,8-Dihydroxynaphthalene Mapped by Mass Spectrometry.

Due to the emerging biomedical relevance and technological potential of fungal melanins, and prompted by the virtual lack of information about their structural arrangement, an optimized synthetic protocol has been devised for a potential structural model of Ascomyces allomelanin through enzyme-catalyzed oxidative polymerization of 1,8-dihydroxynaphthalene (1,8-DHN). Electrospray ionization mass spectrometry (ESI-MS) measurements of freshly synthesized DHN-polymer recorded in the negative ion mode allowed detection of oligomers up to m/z 4000, separated by 158 Da, corresponding to the in-chain DHN-unit. The dominant peaks were assigned to singly-charged distribution, up to 23 repeating units, whereas a doubly charged polymer distribution was also detectable. Chemical derivatization, ultra-performance liquid chromatography (UPLC)-ESI MS, and MS/MS data confirmed that oxidative polymerization of 1,8-DHN proceeds through C-C coupling of the naphthalene rings. The new insights reported here into synthetic 1,8-DHN oligomers/polymers as a mimic of fungal melanins may guide novel interesting advances and applications in the field of biomimetic functional materials.

Melanization Affects the Content of Selected Elements in Parmelioid Lichens.

Lichens belonging to Parmeliaceae are highly diversified, but most of them share an extremely conserved morpho-chemical trait: the lower cortex is heavily melanized. The adaptive value of this character is still uncertain. Melanins are ubiquitous compounds found in most organisms since they fulfil several biological functions including defense against UV radiation, oxidizing agents, microbial stress, and metal complexation. This work aims to establish whether melanization can affect the elemental content of lichen thalli. The relative abundance of macro- (Ca, K and S) and micro- (Fe, Mn and Zn) nutrients in melanized and non-melanized pseudotissues of nine species was first evaluated by a non-destructive micro-X-ray fluorescence elemental analysis on either the upper and lower cortex, and on the internal medulla, which was artificially exposed to the mechanical removal of the lower cortex. Afterwards, the total concentration of the same elements was measured in composite samples by inductively coupled plasma atomic emission spectroscopy after acidic digestion. In order to verify whether Fe and Zn are chemically bound to the melanized pseudotissues, a sequential elution experiment was performed on two species: the two-side heavily melanized Melanelixia glabratula and the one-side lightly melanized Punctelia subrudecta. The content of Fe and Zn was higher in the melanized species than in the non-melanized ones. Species deprived of their melanized lower cortex showed a sharp decrease in Fe but not in Zn, suggesting that the melanized lower cortex is involved in Fe complexation, whereas Zn is homogeneously distributed throughout the thallus.

Testosterone activates sexual dimorphism including male-typical carotenoid but not melanin plumage pigmentation in a female bird.

In males it is frequently testosterone (T) that activates the expression of sexually selected morphological and behavioral displays, but the role of T in regulating similar traits in females is less clear. Here, we combine correlational data with results from T and gonadotropin-releasing hormone (GnRH) manipulations in both sexes to assess the role of T in mediating sexually dimorphic coloration and morphology in the red-backed fairy-wren (Malurus melanocephalus). We show that: (1) natural variation in female expression of ornamental traits (darkened bills and red back feathers) is positively associated with age and circulating androgen titres, (2) females have the capacity to express most male-typical traits in response to exogenous T, including carotenoid-pigmented body plumage, shorter feathers, darkened bill and enlarged cloacal protuberance, but (3) appear constrained in production of male-typical melanin-pigmented plumage, and (4) low androgen levels during the pre-nuptial molt, probably because of low ovarian capacity for steroid production (or luteinizing hormone sensitivity), prevent females from developing male-like ornamentation. Thus, females appear to retain molecular mechanisms for hormonally regulated male-typical ornamentation, although these are rarely activated because of insufficient production of the hormonal signal.

Bird Integumentary Melanins: Biosynthesis, Forms, Function and Evolution.

Melanins are the ubiquitous pigments distributed in nature. They are one of the main pigments responsible for colors in living cells. Birds are among the most diverse animals regarding melanin-based coloration, especially in the plumage, although they also pigment bare parts of the integument. This review is devoted to the main characteristics of bird melanins, including updated views of the formation and nature of melanin granules, whose interest has been raised in the last years for inferring the color of extinct birds and non-avian theropod dinosaurs using resistant fossil feathers. The molecular structure of the two main types of melanin, eumelanin and pheomelanin, and the environmental and genetic factors that regulate avian melanogenesis are also presented, establishing the main relationship between them. Finally, the special functions of melanin in bird feathers are also discussed, emphasizing the aspects more closely related to these animals, such as honest signaling, and the factors that may drive the evolution of pheomelanin and pheomelanin-based color traits, an issue for which birds have been pioneer study models.

"Fifty Shades" of Black and Red or How Carboxyl Groups Fine Tune Eumelanin and Pheomelanin Properties.

Recent advances in the chemistry of melanins have begun to disclose a number of important structure-property-function relationships of crucial relevance to the biological role of human pigments, including skin (photo) protection and UV-susceptibility. Even slight variations in the monomer composition of black eumelanins and red pheomelanins have been shown to determine significant differences in light absorption, antioxidant, paramagnetic and redox behavior, particle morphology, surface properties, metal chelation and resistance to photo-oxidative wear-and-tear. These variations are primarily governed by the extent of decarboxylation at critical branching points of the eumelanin and pheomelanin pathways, namely the rearrangement of dopachrome to 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and the rearrangement of 5-S-cysteinyldopa o-quinoneimine to 1,4-benzothiazine (BTZ) and its 3-carboxylic acid (BTZCA). In eumelanins, the DHICA-to-DHI ratio markedly affects the overall antioxidant and paramagnetic properties of the resulting pigments. In particular, a higher content in DHICA decreases visible light absorption and paramagnetic response relative to DHI-based melanins, but markedly enhances antioxidant properties. In pheomelanins, likewise, BTZCA-related units, prevalently formed in the presence of zinc ions, appear to confer pronounced visible and ultraviolet A (UVA) absorption features, accounting for light-dependent reactive oxygen species (ROS) production, whereas non-carboxylated benzothiazine intermediates seem to be more effective in inducing ROS production by redox cycling mechanisms in the dark. The possible biological and functional significance of carboxyl retention in the eumelanin and pheomelanin pathways is discussed.