Melanin-Binding Colorants: Updating Molecular Modeling, Staining and Labeling Mechanisms, and Biomedical Perspectives

Round 1

Reviewer 1 Report

Malignant melanoma is the most aggressive skin cancer. It is important to understand the biological characteristics of melanin prior to the development of specific drugs for treatment. This manuscript could represent a background for future studies in this field. However, before publication, a few questions/comments should be addressed or answered.

1. The authors should consider rewriting the lines of 64-67, 92-93, 102-103, 175-176, 490-491, 303-305, and 555-557.
2. In Section 6 (Binding of Colorants and Drugs to Melanin), the authors should mention the 18F-labeled picolinamide or nicotinamide derivatives for melanin targeting and their imaging applications.
3. Line 797, 10B is not a radioactive isotope, please correct it.
4. Line 799, the killing effect of 10B-mediated BNCT is mainly caused by alpha and lithium particles releasing high energy within a limited distance to induce DNA double strand breaks. Thus, the “γ photon” should be deleted.

Author Response

Author's Reply to Reviewer 1

              

Comments and Suggestions for Authors

Malignant melanoma is the most aggressive skin cancer. It is important to understand the biological characteristics of melanin prior to the development of specific drugs for treatment. This manuscript could represent a background for future studies in this field. However, before publication, a few questions/comments should be addressed or answered.

--We thank the positive comments of the reviewer.

 

The authors should consider rewriting the lines of 64-67, 92-93, 102-103, 175-176, 490-491, 303-305, and 555-557.

--lines 64-67: Rewriting of the sentences in lines 64-67, and 490-491 have been made.  Rewriting suggestion for line 555-557 has been performed in relation to a suggestion of reviewer 2. We consider that the content of lines 92-93, 102-103, 175-176, and 303-305 is correct and no changes are necessary to improve the corresponding sentences.

 

In Section 6 (Binding of Colorants and Drugs to Melanin), the authors should mention the 18F-labeled picolinamide or nicotinamide derivatives for melanin targeting and their imaging applications.

--The mention of 18F-labeled picolinamide or nicotinamide derivatives for melanin targeting and imaging applications has been made.

 

Line 797, 10B is not a radioactive isotope, please correct it.

--The reviewer is right; we have named it as isotope 10B.

 

Line 799, the killing effect of 10B-mediated BNCT is mainly caused by alpha and lithium particles releasing high energy within a limited distance to induce DNA double strand breaks. Thus, the “γ photon” should be deleted.

--Agreed. The most relevant biological action is due to the helium and lithium nuclei. We have rewritten the sentence to read “When the isotope ¹⁰B is used and then irradiated with slow neutrons, the products of the nuclear fission (⁷Li and ⁴He nuclei) due to neutron capture could damage specifically melanoma cells because of their very short braking distance in condensed matter.”

 

Author Response File: Author Response.pdf

Reviewer 2 Report

This review (?) is devoted to melanin, melanoma, and colorants in general. The first part of the review is appropriate for this Journal. There are so many published reviews on melanin, but this review contains some points of view that are not usually included. For instance, it contains a lot of information about the physical chemical and possible models on the molecular structure of melanin, and this is valuable. Other sections about metal chelating properties of melanin are also appropriate and rare in melanin reviews. However, the paper sometimes become long and reiterative since the extension. On the other hand, the final part, about some biomedical applications on melanoma, is not so suitable. A lot (too many?) aspects are considered. The introduction starts discussing some colorants and the application to antiparasitic diseases and the manuscript finished presenting some proposals on melanoma therapy. The recommendation is focusing and significant reduction to get a readable manuscript.

 

The following points would be considered. Some of them can be easily repaired by modification, but others need extensive re-writing  

Line 62: This is because melanin for staining is not appropriate. Melanin binds covalently or just by absorption thousands of chemicals, so that the specificity is null. Adding melanin to any biological structure is probably getting a dark material. I think that melanin cannot be used as a stain reagent.

Line 67: Assuming the respect to authors about the choice of the references, I suggest the reference http://dx.doi.org/10.1155/2014/498276 as the most appropriate for this issue, 

Line 95: “clear electron microscopic and X-ray crystallographic features”. Delete the term clear. These features are unclear, as melanin is an ill-defined structure.

Line 107: Despite the comment at line 102, the term eumelanin is used a few lines after. I suggest maintaining the term “melanin” instead of “eumelanin”. Concerning melanoma, pheomelanin is much more involved than eumelanin in melanomagenesis.

Line 112: Concerning Section 2. Molecular Structure of Eumelanin, the carboxylated unit DHICA is mentioned at line 119 but then it is not considered (see Figures 1 and 2), since the dopachrome tautomerase activity is not taken into account. This should be repaired, as the existence of carboxyl groups at the polymer greatly affects the structure, chelating capacity, color (from black to brown as the content of DHICA units at the polymer increases), and many properties of the polymer discussed throughout the manuscript. In addition, at Figure 2B, have the authors any probe about the existence of hydrated IQ species?. If so, please give the reference, otherwise, delete that possibility.

Line 188: “ortho-benzoquinones (eumelanin, allomelanin) “. Delete ortho, as some allomelanins (including pyomelanins) contains paraquinones derived from homogentisic acid.

Line 301. Concerning Raman, some reference about the use of this technique for melanin determination would be valuable (i.e. https://doi.org/10.1111/pcmr.12140)

Line 320: staining of pheomelanin or allomelanin seem rather overlooked. This is why these types of melanin are usually not formed inside melanosomes. On the other hand, do not forget that the staining of colorized and easily visible material seems to have a limited value. Authors might focused or re-focused this aspect.

Line 333: diammine, double mm?

Section about melanin-lanthanide complexes could also yield an enhanced hyperthermic effect in melanoma cells is original, but would be melt with other part at the final part of the manuscript. The manuscript should have coordination, and not different authors pasting individual contributions.

So far, the manuscript seemed to be a review, but then subsequent sections seem to contain original data. In general, the manuscript is evolving and the final part seems to be original results with theoretical schemes without methods description. This evolution should be cleared up. Journals do not allow the introduction of original results in reviews, as no methods and discussion are given.  If the manuscript is just a long review, the source of all figures should be given. Figure 9, 13, 14, 15, 16, 17, 18 and 19 are not. In turn, the permission of other reproduced figures should be available. The content should be split in two different contributions, and one of them is out of the scope of Colorants

Figure 15: The labeling of ml as melanosomes and nuclei in that picture of melanoma cells is doubtful. The introduction of unpublished material in a review by courtesy collaborators is not acceptable without experimental details, magnification, cell culturing conditions and so on..

Figures 16, 17, 18 and 19 have nothing to do with lysosomes and melanosomes. The manuscript at this stage derives to unacceptable. Too long and unfocused.

Line 450: All abbreviations should be defined. PDA should be polydopamine, but this should be detailed.

It is assumed that lipofuscin always contain melanin, and this is not correct. This should be cleared up.

Line 555: “As lysosomes and melanosomes share numerous properties, it seems difficult to distinguish between both organelles, and perhaps this feature makes it difficult to find specific vital probes for their recognition”.

Melanosomes contain melanin and all the melanin forming machinery, but this is not the case for lysosome. This sentence is not appropriate.  The paragraph should be re-written. The final question at line 565-566 is already answered, as this term is used in RPE. (Retinal pigment epithelium). I feel that authors are going too far away about cytological aspects, and I am afraid that they seem to have a limited knowledge about them.

Section 6 and paragraphs as that stating at line 631 are reiterative. All these colorants and the affinity to melanin have been described in previous sections, and the review become reiterative and verbose. Reduction is highly recommended. The difference among colorants and drugs is unclear. In addition, it contains clear flaws, i.e line 671: “line melanin-bearing tissues and organs (melanomas, uveal tract, hair follicles, thyroid and Langerhans’s islets)”. To state that thyroid and Langerhans’s islets contain melanin is unacceptable. The use of radioisotopes and the affinity to melanin in melanoma therapy is a different point, but this would be re-written in a completely different manner.

Section 7: It is correct that sonodynamic therapy (SDT), generates damaging ROS and radicals, but melanin behave as a ROS scavenger, and this is one of the main reason for the resistance of melanoma to radiotherapy. However, this is not discussed at all. Figure 20 is a theoretical proposal model (with some flaws, as hydrogen production), but the application proposed at lines 755-757 is not realistic and questionable. Moreover, the review has been converted in a hotchpotch manuscript.

Author Response

Author's Reply to Reviewer 2

 

Comments and Suggestions for Authors

This review (?) is devoted to melanin, melanoma, and colorants in general. The first part of the review is appropriate for this Journal. There are so many published reviews on melanin, but this review contains some points of view that are not usually included. For instance, it contains a lot of information about the physical chemical and possible models on the molecular structure of melanin, and this is valuable. Other sections about metal chelating properties of melanin are also appropriate and rare in melanin reviews. However, the paper sometimes become long and reiterative since the extension. On the other hand, the final part, about some biomedical applications on melanoma, is not so suitable. A lot (too many?) aspects are considered. The introduction starts discussing some colorants and the application to antiparasitic diseases and the manuscript finished presenting some proposals on melanoma therapy. The recommendation is focusing and significant reduction to get a readable manuscript.

--We thank the valuable comments and criticism of the reviewer, specially the following concepts:

“There are so many published reviews on melanin, but this review contains some points of view that are not usually included. For instance, it contains a lot of information about the physical chemical and possible models on the molecular structure of melanin, and this is valuable. Other sections about metal chelating properties of melanin are also appropriate and rare in melanin reviews.”

--We recognize that some Sections and/or paragraphs are long and reiterative, and accordingly, efforts have been made to achieve a shortened paper. Most Sections, and the last one on Biomedical Perspectives have been shortened and simplified.    

 

The following points would be considered. Some of them can be easily repaired by modification, but others need extensive re-writing.

--In order to shorten several Sections and paragraphs, some phrases have been slightly modified. Various paragraphs from several Sections have been rewritten.   

 

Line 62: This is because melanin for staining is not appropriate. Melanin binds covalently or just by absorption thousands of chemicals, so that the specificity is null. Adding melanin to any biological structure is probably getting a dark material. I think that melanin cannot be used as a stain reagent.

--The reviewer is right, but our sentence refers to the use of melanin as “substrate”, not “stain”.  And application of true colorants to stain melanin is not mainly used to see melanin better, but to analyze some structural features of the macromolecular structure of melanin as substrate.

 

Line 67: Assuming the respect to authors about the choice of the references, I suggest the reference http://dx.doi.org/10.1155/2014/498276 as the most appropriate for this issue,

--We agree with the suggestion, the reference:  Solano, F. Melanins: Skin Pigments and Much More—Types, Structural Models, Biological Functions, and Formation Routes. New Journal of Science, 2014, 1–28. doi:10.1155/2014/498276 has been added as indicated by the reviewer.

 

Line 95: “clear electron microscopic and X-ray crystallographic features”. Delete the term clear. These features are unclear, as melanin is an ill-defined structure.

--We agree, the word “clear” has been deleted.

 

Line 107: Despite the comment at line 102, the term eumelanin is used a few lines after. I suggest maintaining the term “melanin” instead of “eumelanin”. Concerning melanoma, pheomelanin is much more involved than eumelanin in melanomagenesis.

--We agree, the term “melanin” is here maintained, and also in other sites, when it is convenient.

 

Line 112: Concerning Section 2. Molecular Structure of Eumelanin, the carboxylated unit DHICA is mentioned at line 119 but then it is not considered (see Figures 1 and 2), since the dopachrome tautomerase activity is not taken into account. This should be repaired, as the existence of carboxyl groups at the polymer greatly affects the structure, chelating capacity, color (from black to brown as the content of DHICA units at the polymer increases), and many properties of the polymer discussed throughout the manuscript. In addition, at Figure 2B, have the authors any probe about the existence of hydrated IQ species? If so, please give the reference, otherwise, delete that possibility.

--We agree in that the precursor dihydroxy-indole-2-carboxylic acid (DHICA) can play a role in melanin biosynthesis, and that the existence of carboxyl groups affects the structure, chelating capacity, color, and other properties of melanin, but DHICA has not been considered in this work because of the limitation of using the C2 site for polymerization, and the paler color when compared to the DHI polymer. This explanation has been added in the text after the line 120.

-- Regarding Figure 2B, the point mentioned by the reviewer on the reference for the hydrated IQ, the following reference has been added in the legend of the Figure (line 152), as well as in the Reference List (line 1252):   

Bishop, C.A., Tong, L.K.J (1964). The reversible addition of hydroxide ion to quinones. Tetrahedron Lett. 1964, 41, 3043-3046. DOI 10.1016/S0040-4039(01)89438-7.

 

Line 188: “ortho-benzoquinones (eumelanin, allomelanin) “. Delete ortho, as some allomelanins (including pyomelanins) contains paraquinones derived from homogentisic acid.

--The reviewer is right, the word “ortho” has been deleted (now, in lines 191 and 197).

 

Line 301. Concerning Raman, some reference about the use of this technique for melanin determination would be valuable (i.e. https://doi.org/10.1111/pcmr.12140)

--We agree and thank to the reviewer. Therefore, the following phrase has been added in the line 305: “Interestingly, non-invasive recording of Raman spectra has been proposed for quantification of eu- and pheomelanin [209].” The corresponding reference has been also added: Galván, I.; Jorge, A., Ito, K., Tabuchi, K., Solano, F., & Wakamatsu, K. Raman spectroscopy as a non-invasive technique for the quantification of melanins in feathers and hairs. Pigm. Cell Melan. Res. 2013, 26, 917-923. DOI 10.1111/pcmr.12140

 

Line 320: staining of pheomelanin or allomelanin seem rather overlooked. This is why these types of melanin are usually not formed inside melanosomes. On the other hand, do not forget that the staining of colorized and easily visible material seems to have a limited value. Authors might focused or re-focused this aspect.

--Regarding …“staining of pheomelanin or allomelanin seem rather overlooked”,  a slight modification in the line 320 has been introduced. Anyway, we are aware of the fact that staining of colored substrates does not seem to be necessary to visualize them. However, the relevant feature in the case of melanin staining is mainly the possibility of gathering information about the substrate structure by studying dye binding consequences (e.g., as monomers or aggregated forms, through hydrophobic or electrostatic forces, etc.).    

 

Line 333: diammine, double mm?

--(now line 346). Yes, diammine is with double mm; accordingly, ammoniacal metal complexes are also named ammine complexes.

 

Section about melanin-lanthanide complexes could also yield an enhanced hyperthermic effect in melanoma cells is original, but would be melt with other part at the final part of the manuscript. The manuscript should have coordination, and not different authors pasting individual contributions.

--We think that the paragraph about “melanin-lanthanide complexes” (lines 360-391) are clearly described in relation with affinity for metal cation binding and photothermal effects, and that the place where these features should be mentioned is just this. We think that in spite of the contribution of three authors, coordination between structural and functional or applicative aspects is maintained in this and other points along the text. Anyway, the phrase from lines 386 and 387 has been deleted for clarity.

 

So far, the manuscript seemed to be a review, but then subsequent sections seem to contain original data. In general, the manuscript is evolving and the final part seems to be original results with theoretical schemes without methods description. This evolution should be cleared up. Journals do not allow the introduction of original results in reviews, as no methods and discussion are given.  If the manuscript is just a long review, the source of all figures should be given. Figure 9, 13, 14, 15, 16, 17, 18 and 19 are not. In turn, the permission of other reproduced figures should be available. The content should be split in two different contributions, and one of them is out of the scope of Colorants

--We do not agree with the reviewer. Although figures with some original results and theoretical schemes have been just introduced mainly with illustrative purposes, descriptions of the methods used allow to repeat and confirm the shown data. We think that the source of figures is clearly and sufficiently described. However, to improve understanding, additional methodological aspects and technical details have been added in the legend of almost all figures (1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, and 18).

--Regarding original results, to justify the introduction of some results and images in a review article, we believe that ideas on review articles are somewhat changing, and accordingly, in the last paragraph of introduction (line 110) we comment recent conceptions and reference about this thematic:

49. Cranford, S. The ABCs of review articles. Matter 2021, 4, 1-3. DOI 10.1016/j.matt.2020.12.013.

--The permission for reproduction of figures is not necessary when they are from Open Access journals, and only the formal information of the corresponding reference is required. In spite of this and to make clearer this point, the conditions for reproduction in the legend of figures have been enhanced: “(Reproduced from the Open Access reference [xx])”.

--We suppose the reviewer claims that the contribution out of the scope of Colorants corresponds to Sections mainly related to microscopic and/or mechanistic aspects of melanin-colorant interactions, which are illustrated in figures 9, 10, 11, 13, 14, 15, 16, 17, and 19. At present, it is widely accepted that molecular mechanics and molecular orbitals of colorants and substrates are highly relevant approaches to understand properties and binding processes, and therefore these approaches are pertinent and specifically considered in this review.    

 

Figure 15: The labeling of ml as melanosomes and nuclei in that picture of melanoma cells is doubtful. The introduction of unpublished material in a review by courtesy collaborators is not acceptable without experimental details, magnification, cell culturing conditions and so on.

--We do not agree in that the acridine orange (AO) labeling as “doubtful”. In the legend of Figure 15, more experimental details have been incorporated in the legend. Cell culture and labeling conditions are better specified in order to repeat melanocyte cultures. In all cases, magnification is reflected by scale bars. Abbreviations have been simplified, and Figure 15 has been changed for more clarity. As expected, AO labels massively marks the endosome-lysosome-melanosome component of all melanocytes, in agreement with the high AO probe concentration present. Moreover, after 10 min of AO labeling, no signals of cell damage or death are observed at all.

 

Figures 16, 17, 18 and 19 have nothing to do with lysosomes and melanosomes. The manuscript at this stage derives to unacceptable. Too long and unfocused.

--Unfortunately, we do not agree with the reviewer. Precisely, these figures illustrate the most relevant features of melanin-dye interactions, which are the central points in this review. These features correspond to molecular orbital analysis of colorants when bound to relevant models of eumelanin. The mentioned figures are not related to cell organelles, but is central to the “melanin-colorant binding issue”, described in molecular mechanistic and orbital scenarios.    

 

Line 450: All abbreviations should be defined. PDA should be polydopamine, but this should be detailed.

--The reviewer is right. Regarding abbreviations, PDA is polydopamine, and its abbreviation (PDA) is introduced at the first place where the word appears (now, line 129).

 

It is assumed that lipofuscin always contain melanin, and this is not correct. This should be cleared up.

--We did not find where the assumption that lipofuscin always contain melanin. The reviewer does not indicate in what Section or line this assumption appears in our MS. In any case, we agree with the reviewer in that this assumption is not correct.

 

Line 555: “As lysosomes and melanosomes share numerous properties, it seems difficult to distinguish between both organelles, and perhaps this feature makes it difficult to find specific vital probes for their recognition”.

Melanosomes contain melanin and all the melanin forming machinery, but this is not the case for lysosome. This sentence is not appropriate.  The paragraph should be re-written. The final question at line 565-566 is already answered, as this term is used in RPE. (Retinal pigment epithelium). I feel that authors are going too far away about cytological aspects, and I am afraid that they seem to have a limited knowledge about them.

--Well, the sentence (line 555) referred by the reviewer (now in line 580) seems to be not adequate, and therefore it has re-written and simplified. In comparison with melanosomes of skin and skin melanoma, neuromelanin [77] and melanin from RPE are very special cases, because they have a complex organization, with specific lipids, lipofuscin, and melanosomes encased in melano-lipofuscin granules [25]. The sentence has been incorporated in line 594.

 

Section 6 and paragraphs as that stating at line 631 are reiterative. All these colorants and the affinity to melanin have been described in previous sections, and the review become reiterative and verbose. Reduction is highly recommended. The difference among colorants and drugs is unclear. In addition, it contains clear flaws, i.e line 671: “line melanin-bearing tissues and organs (melanomas, uveal tract, hair follicles, thyroid and Langerhans’s islets)”. To state that thyroid and Langerhans’s islets contain melanin is unacceptable. The use of radioisotopes and the affinity to melanin in melanoma therapy is a different point, but this would be re-written in a completely different manner.

---The reviewer is right, the text is reiterative, and we have shortened it as far as possible. Redaction has been modified from line 660, where instead microscopical observations, the binding of melanin to different agents is based on biochemical analysis.

--The difference among colorants and drugs is simply related to category and presentation purposes. Unfortunately, re-written Sections or paragraphs cannot be performed at this stage, but considerable reduction and simplification of the text has been made.

--The flaw indicated by the reviewer has been corrected, and the words “thyroid and Langerhans’s islets” removed.

--We consider that radioisotope-containing melanin-binding ligands is adequately and clearly described in the paragraph. Physical characteristics of these ligands such as fluorescence, radioactivity, etc. make them very useful for microscopic visualization or therapy. Therefore, the most convenient site for description of these ligands is just the indicated.

 

Section 7: It is correct that sonodynamic therapy (SDT), generates damaging ROS and radicals, but melanin behave as a ROS scavenger, and this is one of the main reason for the resistance of melanoma to radiotherapy. However, this is not discussed at all. Figure 20 is a theoretical proposal model (with some flaws, as hydrogen production), but the application proposed at lines 755-757 is not realistic and questionable. Moreover, the review has been converted in a hotchpotch manuscript.

--We partially agree with the reviewer on this point. In relation with ROS and radicals, this is a rather polemic issue for melanin (production of, or protection from oxidative stress? both?), but we think that the thematic is not central to this review. Anyway, there are numerous examples of simultaneous ROS and radical production and scavenger action of photo-active compounds, e.g., curcumin, anthracene derivatives, etc. In addition, it is well known that under certain conditions, eumelanin and pheomelanin can either generate or remove ROS.

--In the Fig. 20, the source of some claimed flaws (hydrogen production) are the quoted references in the figure. However, we have modified Figure 20 and its legend to better suit our envisioning of melanin as a photo-/sono-active compound.

--We do not agree with the reviewer. Possible applications are only proposed, but they are not questionable or unrealistic. In our opinion, they are possible and sufficiently well-grounded proposals.   

--Considerable shortening has been made at the beginning of Section 7, also removing some references.

--We do not believe that the manuscript had become a hotchpotch MS either. In any case, the other two reviewers do not hold an opinion that way either. In the revised version, we have made a great effort to make the redaction shorter, simple, and congruent, although in a review it is almost impossible to maintain a strict order and congruency, due to the large number of data, subjects, and interpretations. We hope to have improved the review according to most of the reviewer 2 suggestions, thus achieving an acceptable manuscript for publication.

Author Response File: Author Response.pdf

Reviewer 3 Report

This is a very timely, expected review representing a new view on melanin and its applications. I have only a couple of technical corrections concerning this manuscript.

1. line 76 - the pathway of migration of neural crest cells concerns also other vertebrates, not only mammals.
2. line 77 - the melanocytes settle not only in the basal layer of epidermis - they settle down also in hair follicles in the case of mice it is even just the opposite, the precursors of melanocytes undergo apoptosis in the epithelium, except of follicles. Please correct.
3. line 80 - please cite also doi: 10.1152/physrev.00044.2003.
4. line 88 and 900 (citation 42) please supplement the full list of authors in the citation.
5. line 330-335 please consider supplementation of the citation list with doi: 10.1111/exd.13016, DOI: 10.1111/exd.13040, and DOI: 10.18388/abp.2013_1987

Author Response

Author's Reply to Reviewer 3

 

Comments and Suggestions for Authors

This is a very timely, expected review representing a new view on melanin and its applications. I have only a couple of technical corrections concerning this manuscript.

--We thank the reviewer for positive comments.

 

line 76 - the pathway of migration of neural crest cells concerns also other vertebrates, not only mammals.

--The reviewer is right, the word “mammal” has been replaced by “vertebrate”.

 

line 77 - the melanocytes settle not only in the basal layer of epidermis - they settle down also in hair follicles in the case of mice it is even just the opposite, the precursors of melanocytes undergo apoptosis in the epithelium, except of follicles. Please correct.

---We agree; the sentence has been corrected.

 

line 80 - please cite also doi: 10.1152/physrev.00044.2003.

--We agree, the suggested citation has been added in both Text and Reference List:

Slominski, A.; Tobin, D.J.; Shibahara, S.; Wortsman, J. Melanin pigmentation in mammalian skin and Its hormonal regulation. Physiol. Rev. 2004, 84, 1155-1228. DOI 10.1152/physrev.00044.2003

 

line 88 and 900 (citation 42) please supplement the full list of authors in the citation.

--In the line 88, there is only the number of citation, the list of authors appears in the line 900 as citation 42.

--We agree, in the citation 42, the full list of authors has been added.

 

line 330-335 please consider supplementation of the citation list with doi: 10.1111/exd.13016, DOI: 10.1111/exd.13040, and DOI: 10.18388/abp.2013_1987

--We agree, these are interesting references, and two of the suggested citations have been incorporated in the Text and the Reference List:

Joly-Tonetti, N.; Wibawa, J.I.D.; Bell, M.; Tobin, D. Melanin fate in the human epidermis: a reassessment of how best to detect and analyze histologically. Exp. Dermatol. 2016, 25, 501-504. DOI 10.1111/exd.13016

D’ Ischia, M.; Napolitano, A.; Michalczyk-Wetula, D.; Płonka, P.M. Melanin “dust” or “ghost”? Exp. Dermatol. 2016, 25, 505-506. DOI 10.1111/exd.13040

--The following phrase describing these silver techniques have also been added in the Text (now line 348):

“In addition to FM, Warthin-Starry (WS) silver stain is also used to reveal melanin [211,212]. In the WS stain, hydroquinone is added to the silver solution, increasing the sensitivity and specificity of melanin detection, possibly by pre-reduction of melanin or by reducing the silver bound to melanin [212].”

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Authors have submitted a complete reply letter addressing all points I raised in my previous review. I read it carefully, and it is an excellent reply. Some of the particular points have been satisfied by appropriate modifications throughout the manuscript. Importantly, some reiterative paragraphs have been reduced (not so much as I would recommend), and the permission to reproduce figures has been clarified. This latter point is for the editor of the Journal, but I thought that I should mention it. Authors argue about other points according to their opinion and assessment on particular aspects of the review. To summarize my second comments, obviously authors and I do not need to be in agreement concerning all points. My report tried to highline points for manuscript improvements, and I feel that this goal has been achieved. I hope authors agree with me concerning the improvement, as they wrote at the final paragraph of the reply letter. Consequently, I am not going further to argue about the points of disagreement (i.e., Figure 15 and the interpretation “about massive accumulation of metachromatic (red) in melanosome-containing regions (m) and melanin labeling at the end of narrow cytoplasmic prolongations (encircled)”. It is clear to me that melanin is inside the melanosomes as melanin granules to be secreted to the culture and they cannot be transferred to surrounding keratinocytes as in biological tissue. So, this interpretation of the staining is doubtful to me. Anyway, I already recognized that the review contains some aspects that are not described in other reviews, and I have learnt in some of them.