## 5 Current Challenges and Future Perspectives  
The critical problem in the study of melanins is the obtaining of a pure macromolecule without mixture of any other cell components. Common isolation procedures imply an extraction with an alkaline solvent and further precipitation in acid media [13; 19; 110]. This protocol co-extracts a wide diversity of components, associated with the fungal cell wall. A different multi-step extraction procedure has been used by other authors seeking for the method of obtaining pure melanin. This procedure, applied to the extraction of _C. neoformans_ melanin, includes an enzymatic digestion of cell-wall components, followed by protein denaturation and subsequent degradation, chloroform extraction to remove lipids and acid hydrolysis [15; 101]. This melanin still contained some associated cell wall components, see Supplementary Figure S2. Therefore, a challenge is to get a suitable extraction procedure for the fungal melanins.  
A step further would be the use of synthetic melanin and the study of their pyrolysis products. Attempts to obtaining useful DHN melanin models (free DHN polymer or DHN-peptide polymer) failed because the pyrograms revealed a lot of impurities likely introduced during the chemical synthesis (protein from the enzymes used in the polymerization, alkanes, fatty acids, etc.) and the study of the pyrolysis products was inconclusive.  
Alternatively, chemical degradation of the _Ochroonis_ melanin versus other DOPA and DHN melanins seems to be needed in order to identify specific markers of each melanin-type and disclose the structural problem. However, as reported by [PERSON] et al. [103], \"due to the high heterogeneity of melanins, their analytical characterization can be a challenging task... Our thorough comparative evaluation of analytical data published so far on melanin analysis has proven to be a difficult task in terms of finding equivalent results, even when the same matrix was used\".  
Another approach is the study of _O. lascauxensis_ genome in search of genes involved in the synthesis of melanin or pyomelanin. Unfortunately the literature only reportedthe in silico genome analysis of _Ochroconis mirabilis_[111], an opportunistic pathogen. This analysis revealed that the fungus has the potential ability to synthesize melanin via the 1,8-dihydroxyroxynapthhalene (DHN)-melanin pathway and to produce trichothecene (mycotoxin). The possible synthesis of trichothecene is of interest because it has been suggested that the melanin of _Stachybotrys charaturn_ can be based on the polymerization of trichothecenes, as evidenced pyrolysis and \({}^{13}\)C NMR data [19; 37]. In fact, _S. charaturn_ synthesizes, in addition to phenols, a high number of mycotoxins, among which there are the sesquiterpenoid trichothecenes and the diterpeneoid atranones [11; 112].  
The \({}^{13}\)C NMR data of _S. charaturn_ melanin agree with the possible involvement of mycotoxins (trichothecenes and atranones, among others), which possess a terpenoid structure having methylene chains and carbonyl groups. It can be speculated that the reactive groups from mycotoxins and phenols can undergo polymerization reactions resulting in a complex macromolecule with a marked aliphatic character [19; 113]. In addition, the pyrolysis of this melanin produced compounds such as series of alkylcyclohexenes, alkylbenzes, and alkylnaphthalenes. These compounds are secondary thermal reaction products formed upon pyrolysis of aliphatic compounds through cyclization and aromatization reactions [24; 37].  
According to these data, another possible pathway for melanin formation is the polymerization of phenols and terpenoids. Recently, the formation of a pheomelanin-like polymer has been reported in the fungus _Plenodomus biglobosus_[114] which considerably complicates the situation, and leaves the chemical structure of fungal melanins even more open.