Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques.

Choroidal melanocytes (HCMs) are melanin-producing cells in the vascular uvea of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment, and display cellular functions and extracellular interactions that are biologically distinct to skin melanocytes. HCMs (and melanins) are important in normal human eye physiology with roles including photoprotection, regulation of oxidative damage and immune responses. To extend knowledge of cytoplasmic melanins and melanosomes in label-free HCMs, a non-invasive 'fit-free' approach, combining 2-photon excitation fluorescence lifetimes and emission spectral imaging with phasor plot segmentation was applied. Intracellular melanin-mapped FLIM phasors showed a linear distribution indicating that HCM melanins are a ratio of two fluorophores, eumelanin and pheomelanin. A quantitative histogram of HCM melanins was generated by identifying the image pixel fraction contributed by phasor clusters mapped to varying eumelanin/pheomelanin ratio. Eumelanin-enriched dark HCM regions mapped to phasors with shorter lifetimes and longer spectral emission (580-625 nm) and pheomelanin-enriched lighter pigmented HCM regions mapped to phasors with longer lifetimes and shorter spectral emission (550-585 nm). Overall, we demonstrated that these methods can identify and quantitatively profile the heterogeneous eumelanins/pheomelanins within in situ HCMs, and visualize melanosome spatial distributions, not previously reported for these cells

Differential antifungal activity of human and cryptococcal melanins with structural discrepancies

Indexación: Scopus.Melanin is a pigment found in all biological kingdoms, and plays a key role in protection against ultraviolet radiation, oxidizing agents, and ionizing radiation damage. Melanin exerts an antimicrobial activity against bacteria, fungi, and parasites. We demonstrated an antifungal activity of synthetic and human melanin against Candida sp. The members of the Cryptococcus neoformans and C. gattii species complexes are capsulated yeasts, which cause cryptococcosis. For both species melanin is an important virulence factor. To evaluate if cryptococcal and human melanins have antifungal activity against Cryptococcus species they both were assayed for their antifungal properties and physico-chemical characters. Melanin extracts from human hair and different strains of C. neoformans (n = 4) and C. gattii (n = 4) were investigated. The following minimum inhibitory concentrations were found for different melanins against C. neoformans and C. gattii were (average/range): 13.7/(7.8-15.6) and 19.5/(15.6-31.2) μg/mL, respectively, for human melanin; 273.4/(125- > 500) and 367.2/(125.5- > 500) μg/mL for C. neoformans melanin and 125/(62.5-250) and 156.2/(62-250) μg/mL for C. gattii melanin. Using Scanning Electron Microscopy we observed that human melanin showed a compact conformation and cryptococcal melanins exposed an amorphous conformation. Infrared spectroscopy (FTIR) showed some differences in the signals related to C-C bonds of the aromatic ring of the melanin monomers. High Performance Liquid Chromatography established differences in the chromatograms of fungal melanins extracts in comparison with human and synthetic melanin, particularly in the retention time of the main compound of fungal melanin extracts and also in the presence of minor unknown compounds. On the other hand, MALDI-TOF-MS analysis showed slight differences in the spectra, specifically the presence of a minor intensity ion in synthetic and human melanin, as well as in some fungal melanin extracts. We conclude that human melanin is more active than the two fungal melanins against Cryptococcus. Although some physico-chemical differences were found, they do not explain the differences in the antifungal activity against Cryptococcus of human and cryptococcal melanins. More detailed studies on the structure should be considered to associate structure and antifungal activity.https://www.frontiersin.org/articles/10.3389/fmicb.2017.01292/ful

5,6-dihydroxyindole-2-carboxylic acid (DHICA): a First Principles Density-Functional Study

We report first principles density functional calculations for 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and several reduced forms. DHICA and 5,6-dihydroxyindole (DHI) are believed to be the basic building blocks of the eumelanins. Our results show that carboxylation has a significant effect on the physical properties of the molecules. In particular, the relative stabilities and the HOMO-LUMO gaps (calculated with the $\Delta$SCF method) of the various redox forms are strongly affected. We predict that, in contrast to DHI, the density of unpaired electrons, and hence the ESR signal, in DHICA is negligibly small.Comment: 5 pages, 2 figure

Towards Structure-Property-Function Relationships for Eumelanin

We discuss recent progress towards the establishment of important structure-property-function relationships in eumelanins - key functional bio-macromolecular systems responsible for photo-protection and immune response in humans, and implicated in the development of melanoma skin cancer. We focus on the link between eumelanin's secondary structure and optical properties such as broad band UV-visible absorption and strong non-radiative relaxation; both key features of the photo-protective function. We emphasise the insights gained through a holistic approach combining optical spectroscopy with first principles quantum chemical calculations, and advance the hypothesis that the robust functionality characteristic of eumelanin is related to extreme chemical and structural disorder at the secondary level. This inherent disorder is a low cost natural resource, and it is interesting to speculate as to whether it may play a role in other functional bio-macromolecular systems.Comment: 19 pages, 8 figures, Invited highlight article for Soft Matte

Quantitative scattering of melanin solutions

The optical scattering coefficient of a dilute, well solubilised eumelanin solution has been accurately measured as a function of incident wavelength, and found to contribute less than 6% of the total optical attenuation between 210 and 325nm. At longer wavelengths (325nm to 800nm) the scattering was less than the minimum sensitivity of our instrument. This indicates that UV and visible optical density spectra can be interpreted as true absorption with a high degree of confidence. The scattering coefficient vs wavelength was found to be consistent with Rayleigh Theory for a particle radius of 38+-1nm.Comment: 23 pages, 5 figure

Quantitative photoluminescence of broad band absorbing melanins: A procedure to correct for inner filter and re-absorption effects

We report methods for correcting the photoluminescence emission and excitation spectra of highly absorbing samples for re-absorption and inner filter effects. We derive the general form of the correction, and investigate various methods for determining the parameters. Additionally, the correction methods are tested with highly absorbing fluorescein and melanin (broadband absorption) solutions; the expected linear relationships between absorption and emission are recovered upon application of the correction, indicating that the methods are valid. These procedures allow accurate quantitative analysis of the emission of low quantum yield samples (such as melanin) at concentrations where absorption is significant.Comment: 20 pages, 13 figure

A case of partial leucism in the American Barn Owl (Tyto furcata) (Temminck, 1827), from Buenos Aires province, Argentina

The diverse colorations of a birds’ plumage are due to either structural colors or pigments that are synthesized in specialized cells or incorporated through the diet. However, plumage color aberrations can occur; several cases of albinism and leucism have been reported for different bird species and some examples correspond to Strigiformes. A specimen of the American barn owl (Tyto furcata) with partial leucism was found in Carhué, Buenos Aires province. The plumage of its facial disc, tail and the ventral region was completely white; furthermore, the neck, and the primary and secondary remiges were pigmented although in a significantly lighter fashion than in normal-phenotype specimens. The rest of the body presented a normal pattern for the species.Fil: Chiale, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Departamento Científico Zoología Vertebrados; ArgentinaFil: Pagano Luis Gerardo. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Departamento Científico Zoología Vertebrados; Argentina. Grupo Falco; Argentin