Glossogyne tenuifolia Essential Oil Prevents Forskolin-Induced Melanin Biosynthesis via Altering MITF Signaling Cascade

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a comprehensive study, with a wide range of biomedical research techniques applied. There remains an unmet clinical need for how best to efficaciously treat hyperpigmentation conditions. More dubious however is any motivation to reduce 'natural' pigmentation in the skin. 

I have a few significant concerns here, principal among these is the poor (in my opinion) choice of module cell to test this botanical. Murine dermis-derived melanoma cells (B16) in vitro do not behave similarly (or at least similarly enough) to human epidermis located melanocytes in situ. These cells are unpredictable in terms of level of baseline melanin produced at different confluence levels), and it is of note that this study was focused on only looking at Forskolin-induced stimulated melanin. I would have liked to have seen GTEO's effect on baseline melanin level or melanin after UVB stimulation. 

Also, much is made of the subcellular localization change on MITF under the influence of GTEO. It should be know that normal epidermal melanocytes usually show a nuclear localization of this transcription factor, under baseline conditions in human epidermis in situ and also in vitro. Thus I am not sure of the meaning of these data, especially from the mouse cell line? It is interesting to note that 100ug/ml GTEO stimulates MITF gene/protein expression higher than baseline?

More minor issues: 

1) Page 2: Line 53: UVR does not result in a stimulation of melanocyte numbers, but does lead to a stimulation of melanin production. 

2) Page 2: Line 62: Pheomelanogenesis occurs by a different biosynthetic process than Eumelanin. 

3) Can you comment on why 'essential oils have been identified for their strong in vitro anti-melanogenic effects' is limited to In vitro only? 

4) Figure 1C: Was there a reduction in cell numbers  with GTEO? Implication? 

5) It is not clear how the MTT data should be interpreted? Viability versus effect on cell proliferation? Did you do actual cell counts?

6) Page 6: Line 239: Comment on the baseline level of melanin in these mouse pigment cells?

7) I could not see any data on pigment cell dendricity in Figure 1C as stated? 

8)Figure 3B: Unclear what is the impact of TRP2 increasing in expression after GTEO? Please comment. Does this match your comment on Page 8 Line 292: " Whereas, GTEO failed to modulate TRP-1 and TRP-2 expression levels..."

9) Please provide full gels of all western blots, to help reader judge correctly this experiment. 

10) define 'GA' in Figure 3. 

11) I found Fig 4C data weak. 

12) Section 3.4 needs references 

Discussion: Despite many many studies, few if any actives have had a clinically-appreciated effect (see important editorial on this at: doi: 10.1111/exd.14350, which should be referenced This is important given the huge number of actives proposed that either inhibit tyrosinase activity or some other element of skin pigmentation. 

 

Comments on the Quality of English Language

Overall reasonable standard.

Author Response

Reviewer’s opinion: This is a comprehensive study, with a wide range of biomedical research techniques applied.

Response:  We would appreciate the reviewer, whom provided a positive and constructive comment on our work. The reviewer’s comments and our responses as follows.

 

Major comments

Comment. 1: There remains an unmet clinical need for how best to efficaciously treat hyperpigmentation conditions. More dubious however is any motivation to reduce 'natural' pigmentation in the skin. I have a few significant concerns here, principal among these is the poor (in my opinion) choice of module cell to test this botanical. Murine dermis-derived melanoma cells (B16) in vitro do not behave similarly (or at least similarly enough) to human epidermis located melanocytes in situ. These cells are unpredictable in terms of level of baseline melanin produced at different confluence levels), and it is of note that this study was focused on only looking at Forskolin-induced stimulated melanin. I would have liked to have seen GTEO's effect on baseline melanin level or melanin after UVB stimulation. Also, much is made of the subcellular localization change on MITF under the influence of GTEO. It should be know that normal epidermal melanocytes usually show a nuclear localization of this transcription factor, under baseline conditions in human epidermis in situ and also in vitro. Thus I am not sure of the meaning of these data, especially from the mouse cell line? It is interesting to note that 100ug/ml GTEO stimulates MITF gene/protein expression higher than baseline?.

.

Response: Dear Reviewer, thank you for your detailed feedback on our manuscript. We appreciate your insights and would like to address your concerns as follows:

Unmet Clinical Need and Motivation to Reduce 'Natural' Pigmentation: We agree that treating hyperpigmentation conditions remains a significant clinical challenge. Our study aims to explore new botanical treatments that may offer safer and more effective options for patients. While reducing 'natural' pigmentation is not our primary goal, understanding how botanical compounds like GTEO influence pigmentation pathways can contribute to broader dermatological applications, including hyperpigmentation disorders.

Choice of Model Cell Line: We acknowledge the limitations of using B16 murine melanoma cells as a model. However, B16 cells are a well-established model for studying melanogenesis due to their robust melanin production and responsiveness to melanogenic stimulators. Despite differences from human melanocytes, B16 cells provide valuable initial insights. Future studies will include human epidermal melanocytes to validate our findings and address the variability in melanin production.

Baseline Melanin Level and UVB Stimulation: We appreciate the suggestion to examine GTEO's effects on baseline melanin levels and after UVB stimulation. We observed that the baseline melanin production in B16 cells was 2.5 micrograms/mL, which significantly increased to 7.4 micrograms/mL following forskolin stimulation. As our manuscript focuses on identifying and developing skin-whitening agents for hyperpigmentary skin disorders, we intentionally used a melanin stimulator to assess GTEO's effectiveness in a hyperpigmented condition. Furthermore, there is limited evidence that melanocytes directly respond to UVB stimulation. Instead, UVB exposure triggers a series of events, beginning with p53-mediated POMC and alpha-MSH secretion in keratinocytes, which subsequently stimulates melanin production in melanocytes. Our ongoing research indicates that GTEO can inhibit 50 mJ/cm2 UVB-induced alpha-MSH secretion in keratinocytes, suggesting that GTEO may influence melanogenesis through keratinocyte signaling pathways.

Subcellular Localization of MITF: We understand that MITF typically shows nuclear localization in normal epidermal melanocytes. Our immunofluorescence analysis revealed baseline levels of MITF in the nucleus, with increased levels observed after forskolin stimulation, as indicated by the white arrows. The observed changes in MITF localization in B16 cells may reflect similar processes in human cells, though confirmation in human melanocytes is needed. We will conduct additional studies to clarify the significance of these findings.

GTEO Stimulation of MITF Gene/Protein Expression: We would like to clarify that the gene and protein expression levels of MITF in response to 100 micrograms/mL GTEO were compared to the forskolin-stimulated group, not the control group. As shown in Figure 4B, the control cells exhibit baseline levels of MITF protein, whereas forskolin stimulation increased MITF levels by 6-fold. Notably, treatment with 100 micrograms/mL GTEO significantly reduced MITF expression to 1.5-fold under forskolin-stimulated conditions. Further experiments will be conducted to understand the underlying mechanisms and to verify these effects in human melanocytes.

We hope these responses address your concerns. We are committed to expanding our research to include human melanocyte models and additional experimental conditions to provide a comprehensive understanding of GTEO's effects.

 

Minor comments

Comment. 1: Page 2: Line 53: UVR does not result in a stimulation of melanocyte numbers, but does lead to a stimulation of melanin production. 

Response: Thank you for your observation. We agree with your correction that UVR does not stimulate an increase in melanocyte numbers but rather leads to an increase in melanin production. We revised the text on Page 2, Line 52-53 to accurately reflect that UVR stimulates melanin production rather than melanocyte proliferation.

 

Comment. 2: Page 2: Line 62: Pheomelanogenesis occurs by a different biosynthetic process than Eumelanin. 

Response: Thank you for pointing this out. We acknowledge that pheomelanogenesis occurs through a different biosynthetic process than eumelanin production. We revised this phrase as “It is via this sequence of processes that eumelanin is synthesized” (Page 2, Line 61-62).

 

Comment. 3: Can you comment on why 'essential oils have been identified for their strong in vitro anti-melanogenic effects' is limited to In vitro only? 

Response: Thank you for your comment. The statement that "essential oils have been identified for their strong in vitro anti-melanogenic effects" is limited to in vitro studies because most of the current research on the anti-melanogenic properties of essential oils has been conducted in controlled laboratory environments using cell cultures. These studies provide initial insights into the potential mechanisms and efficacy of essential oils in inhibiting melanin production. However, further research, including in vivo studies and clinical trials, is necessary to validate these effects in living organisms and determine their safety and effectiveness in human applications.

 

Comment. 4: Figure 1C: Was there a reduction in cell numbers with GTEO? Implication? 

Response: Thank you for your query. There is no reduction in cell number; all cells are healthy and viable at all concentrations. The photomicrographs were taken in areas with fewer cells to clearly show cell morphology and the subcellular localization of melanin.

 

Comment. 5: It is not clear how the MTT data should be interpreted? Viability versus effect on cell proliferation? Did you do actual cell counts?

Response: Thank you for your question regarding the interpretation of the MTT data. The MTT assay in our study was used primarily to assess cell viability rather than cell proliferation. This assay measures the metabolic activity of cells, which correlates with the number of viable cells. However, it does not distinguish between cell viability and proliferation directly. We also performed actual cell counts using a hemocytometer to confirm that there was no significant change in cell number across the different treatment groups. Our results showed that cell numbers remained consistent, indicating that the treatments did not affect cell proliferation. These findings support our conclusion that the observed changes in the MTT assay reflect changes in cell viability.

 

Comment. 6: Page 6: Line 239: Comment on the baseline level of melanin in these mouse pigment cells?

Response: As per your suggestion, we have included the details of baseline melanin levels in B16 cells and the changes in melanin levels after treatment with forskolin and GTEO (Line 236-242).

 

Comment. 7: I could not see any data on pigment cell dendricity in Figure 1C as stated? 

Response: Thank you for your comment. In Figure 1C (panel 2), forskolin-treated cells exhibit pigment cell dendricity. During the conversion of the manuscript from Microsoft Word to PDF, the photomicrograph lost its quality, making the pigment dendricity poorly visible.

 

Comment. 8: Figure 3B: Unclear what is the impact of TRP2 increasing in expression after GTEO? Please comment. Does this match your comment on Page 8 Line 292: " Whereas, GTEO failed to modulate TRP-1 and TRP-2 expression levels..."

Response: Thank you for query. The differential effects of the drug on tyrosinase, TRP-1, and TRP-2 likely result from the distinct roles and regulatory mechanisms of these proteins in melanin biosynthesis. Tyrosinase is the key enzyme catalyzing initial steps, while TRP-1 and TRP-2 function in later stages. Inhibiting tyrosinase might lead to compensatory mechanisms altering downstream proteins' expression, such as an increase in TRP-2 to manage DOPAchrome levels. The drug's high specificity for tyrosinase, due to structural compatibility or binding affinity, might not extend to TRP-1, leaving it unaltered. Cellular context, including cofactors and post-translational modifications, could influence these effects, selectively upregulating TRP-2. Compensatory upregulation of TRP-2 might occur to maintain balance in melanin intermediates. Additionally, different cell types may exhibit varying responses due to distinct gene expression profiles and regulatory networks. The drug might also affect transcription factors, microRNAs, or other regulatory molecules that specifically regulate TRP-2 but not TRP-1. Thus, the observed increase in TRP-2 expression, despite the inhibition of tyrosinase and unaltered TRP-1, suggests complex, multi-layered regulatory mechanisms. Further investigation into these pathways will provide deeper insights into these differential effects. In addition, we updated the results in the revised manuscript (Line 291-298).

 

Comment. 9: Please provide full gels of all western blots, to help reader judge correctly this experiment. 

Response: Thank you for your suggestion. In MDPI journals, authors are required to provide the original immunoblots at the time of final production. We guarantee that the original blots will be provided.

 

Comment. 10: define 'GA' in Figure 3. 

Response: Thank you for pointing this out. We described the effect of GA on FRK-induced tyrosinase, TRP-1 and TRP-2 mRNA expression levels in B16 cells (Line 298-299)

 

Comment. 11: I found Fig 4C data weak. 

Response: Thank you for your comment. We try our best to explain this data. Please review the response to major comment 1.

 

Comment. 12: Section 3.4 needs references 

Response: As per your suggestion, we included a reference for this statement (Line 312; 576-577).

 

Comment. 13: Discussion: Despite many many studies, few if any actives have had a clinically-appreciated effect (see important editorial on this at: doi: 10.1111/exd.14350, which should be referenced. This is important given the huge number of actives proposed that either inhibit tyrosinase activity or some other element of skin pigmentation. 

Response: Thank you for sharing this crucial information, which enhances our understanding of the significance of skin pigmentary studies. We will refer to this in all our future work.

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Glossogyne tenuifolia Essential Oil Prevents Forskolin-Induced Melanin Biosynthesis via Altering MITF Signaling Cascade

This is an in vitro study, carried out in mice, which reveals the power of Glossogyne tenuifolia essential oil to reduce melanogenesis and, therefore, its potential use in cosmetics as a depigmentant. This research, still in the initial phase, is framed in the use of plants used in traditional medicine, which contain many biomolecules that can be incorporated into conventional therapies, in this case, the skin.

Based on previous literature, the authors have designed an experiment, in my correct opinion, which I repeat, should continue, for example, carrying out toxicity tests since everything natural is not safe for health, and the skin is a very important organ in the organism. In this sense, it is important that molecular tests have been carried out and biochemical routes responsible for the action of the essential oil have been proposed.

 

To be published, authors must make the following changes:

In the title, put behind it in parentheses the botanical family to which this plant belongs.

The correct name of the plant is Glossogyne tenuifolia is G.tenuifolia (Labill.) Cass. ex Cass. Enter this name the first time you name yourself in the study (Page 76).

Paragraph 387-392 should move to the introduction.

In material and methods, in point 3.1 the origin of the essential oil used, nor its terpenoids, does not appear.

Figures 1, 2 and 3 appear before they are named in the text, they must be moved after their comments in the text.

The $$$ icons, won't they be ***?

Skip paragraph 359-362 to the beginning of the discussion.

Finally, it should be emphasized that although the majority components of the essential oil are D-Limonene and a-pinene, it cannot be forgotten that other secondary metabolites present in a smaller proportion can act as adjuvants to the main metabolites.

Author Response

Reviewer’s opinion: Glossogyne tenuifolia Essential Oil Prevents Forskolin-Induced Melanin Biosynthesis via Altering MITF Signaling Cascade. This is an in vitro study, carried out in mice, which reveals the power of Glossogyne tenuifolia essential oil to reduce melanogenesis and, therefore, its potential use in cosmetics as a depigmentant. This research, still in the initial phase, is framed in the use of plants used in traditional medicine, which contain many biomolecules that can be incorporated into conventional therapies, in this case, the skin. Based on previous literature, the authors have designed an experiment, in my correct opinion, which I repeat, should continue, for example, carrying out toxicity tests since everything natural is not safe for health, and the skin is a very important organ in the organism. In this sense, it is important that molecular tests have been carried out and biochemical routes responsible for the action of the essential oil have been proposed.

Response:  We would appreciate the reviewer, whom provided a positive and constructive comment on our work. The reviewer’s comments and our responses as follows.

 

Comment. 1: In the title, put behind it in parentheses the botanical family to which this plant belongs.

Response: Thank you for your suggestion. We accept your recommendation. However, we believe it is more appropriate to include the family name in the abstract rather than the title. Therefore, we have included this information in the abstract (Line 23). This addition will provide more context and clarity for readers.

 

Comment. 2: The correct name of the plant is Glossogyne tenuifolia is G.tenuifolia (Labill.) Cass. ex Cass. Enter this name the first time you name yourself in the study (Page 76).

Response: Thank you for your suggestion. We accept your recommendation. We included this information in the revised manuscript (Line 75). This addition will provide more context and clarity for readers.

 

Comment. 3: Paragraph 387-392 should move to the introduction.

Response: Thank you for your suggestion. Another reviewer recommended removing this information from the manuscript altogether. Therefore, we have removed it from the main text.

 

Comment. 4: In material and methods, in point 3.1 the origin of the essential oil used, nor its terpenoids, does not appear.

Response: Thank you for your suggestion. As per your recommendation, we included information regarding the source of essential oils and their major compounds in the introduction section (Line 84-91).

 

Comment. 5: Figures 1, 2 and 3 appear before they are named in the text, they must be moved after their comments in the text.

Response: Thank you for your suggestion. As per your recommendation, we have moved Figures 1, 2, and 3 to appear after they are mentioned in the main text.

 

Comment. 6: The $$$ icons, won't they be ***?

Response: Thank you for your query. We use the $ symbol to indicate the difference between the control and FRK alone treatment group, while the * symbol indicates the difference between FRK and sample treatment groups.

 

Comment. 7: Skip paragraph 359-362 to the beginning of the discussion.

Response: As per your suggestion, we have removed this information from the results section and it was stated in the discussion (Line 463-468).

 

Comment. 8: Finally, it should be emphasized that although the majority components of the essential oil are D-Limonene and a-pinene, it cannot be forgotten that other secondary metabolites present in a smaller proportion can act as adjuvants to the main metabolites.

Response: Thank you for your insightful comment. We agree that while D-Limonene and α-pinene are the major components of the essential oil, other secondary metabolites present in smaller proportions may indeed act as adjuvants to the main metabolites. We have now included this consideration in the revised manuscript to provide a more comprehensive understanding of the potential interactions and synergistic effects within the essential oil (Line 495-496).

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors

It is a well organized work with potential dermato-pharmaceutical and cosmetic application

Since ascorbic acid is not a traditional tyrosinase inhibitor (neither of Tyrosine to L-Dopa or L-Dopa to Dopaquinone), I wonder why you  did not use traditional and direct tyrosinase inhibitors (Figure 2A and B) like hydroquinone -although is used only by medical perscription and under strictly defined conditions, arbutin-widely used in cosmetics... Please answer

Author Response

Reviewer’s opinion: Dear Authors, it is a well-organized work with potential dermato-pharmaceutical and cosmetic application.

Response:  We would appreciate the reviewer, whom provided a positive and constructive comment on our work. The reviewer’s comments and our responses as follows.

 

Comment. 1: Since ascorbic acid is not a traditional tyrosinase inhibitor (neither of Tyrosine to L-Dopa or L-Dopa to Dopaquinone), I wonder why you did not use traditional and direct tyrosinase inhibitors (Figure 2A and B) like hydroquinone -although is used only by medical perscription and under strictly defined conditions, arbutin-widely used in cosmetics... Please answer.

Response: Thank you for your insightful comment. We acknowledge that ascorbic acid is not a traditional tyrosinase inhibitor. However, ascorbic acid was chosen for this study due to its well-documented antioxidant properties and its role in inhibiting melanin synthesis through reduction of dopaquinone back to L-DOPA, thereby preventing melanin polymerization. Additionally, ascorbic acid is widely used in cosmetic formulations for its skin-brightening effects.

Regarding the use of traditional and direct tyrosinase inhibitors such as hydroquinone and arbutin, we considered the safety profile and regulatory status of these compounds. Hydroquinone, although effective, is restricted in many regions due to potential side effects and the need for medical supervision. Arbutin, while safer, also has regulatory limitations in certain markets.

Our study aimed to explore alternatives that are both effective and have a favorable safety profile for broader application. Future studies could include comparisons with traditional tyrosinase inhibitors to provide a comprehensive understanding of the efficacy and safety of our proposed treatment.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors responses have been helpful, as have the revisions.

However the following issues still remain to be (more fully) addressed, in my opinion. 

I could not see in the figure the data to support: “However, while GTEO treatment reduced TRP-1 expression, the changes were 296 not statistically significant. In contrast, GTEO treatment increased TRP-2 expression, 297 which is also not statistically significant. Indeed, the positive drug control galic acid (GA) 298 significantly inhibited tyrosinase, TRP-1 and TRP-2 genes in FRK-stimulated cells”

How is it possible to have a reduction more than 100%: “The reduction was observed at concentrations of 5.22 µg/mL (172%), 240 3.84 µg/mL (165%), and 2.56 µg/mL (82%) with GTEO doses of 25 µg/mL, 50 µg/mL, and 241 100 µg/mL, respectively”

Was melanin level really measured in ug/ml? What does this mean? Per number of cells?  Nano/picograms of melanin?

Nuclear MITF is not visible in Fig 4C ‘control’  - at least not with my eyes.

Cell Model: weaknesses of this mouse dermal melanoma line in current study are underplayed.

Some comment of the increase in the perhaps counter-intuitive TRP-2/DCT needs to be added to the paper, in the absence of evidence for a ‘Compensatory upregulation of TRP-2‘.

I could not see doi: 10.1111/exd.14350. added to the revised manuscript. 

Author Response

Response to reviewer

Reviewer’s opinion: The authors responses have been helpful, as have the revisions. However, the following issues still remain to be (more fully) addressed, in my opinion.

Response:  We would appreciate the reviewer, whom again provided a positive and constructive comment on our work. The reviewer’s comments and our responses as follows.

 

Comment. 1: I could not see in the figure the data to support: “However, while GTEO treatment reduced TRP-1 expression, the changes were 296 not statistically significant. In contrast, GTEO treatment increased TRP-2 expression, 297 which is also not statistically significant. Indeed, the positive drug control galic acid (GA) 298 significantly inhibited tyrosinase, TRP-1 and TRP-2 genes in FRK-stimulated cells.

Response: Dear Reviewer, Thank you for your feedback. We acknowledge that the data on TRP-1 and TRP-2 are inconsistent with our hypothesis and may be challenging for a general audience to interpret. As a result, we have decided to remove the TRP-1 and TRP-2 data from our manuscript.

 

Comment. 2: How is it possible to have a reduction more than 100%: “The reduction was observed at concentrations of 5.22 µg/mL (172%), 240 3.84 µg/mL (165%), and 2.56 µg/mL (82%) with GTEO doses of 25 µg/mL, 50 µg/mL, and 241 100 µg/mL, respectively”. 

Response: Thank you for your query. The baseline melanin content was set at 3.1 µg/mL, which we defined as 100%. Following treatment with FRK, the melanin content increased to 8.54 µg/mL, representing a 256% increase compared to the baseline. Co-treatment with GTEO reduced the FRK-induced melanin production to 5.22 µg/mL (172% of baseline), 3.84 µg/mL (165% of baseline), and 2.56 µg/mL (82% of baseline) at GTEO doses of 25 µg/mL, 50 µg/mL, and 100 µg/mL, respectively. Notably, the 100 µg/mL GTEO treatment reduced melanin content to 2.56 µg/mL, which is below the baseline level of 3.1 µg/mL, hence the reduction percentage appears below 100%.  

 

Comment. 3: Was melanin level really measured in ug/ml? What does this mean? Per number of cells?  Nano/picograms of melanin? 

Response: Thank you for your query. Yes, the melanin content was measured in µg/mL. This measurement refers to the concentration of melanin in 1 mL of cell lysates.

 

Comment. 4: Nuclear MITF is not visible in Fig 4C ‘control’ - at least not with my eyes.

Response: Thank you for your comment. To address the issue, we have replaced the photomicrographs in the control group. Please review the updated Figure 4C.

 

Comment. 5: Cell Model: weaknesses of this mouse dermal melanoma line in current study are underplayed.

Response: Thank you for high lightening this point. We acknowledge the limitations of the mouse dermal melanoma cell line used in our study. Specifically, the B16-F10 melanoma cell line, were widely used for studying the skin lightening effects of natural or synthetic compounds (Choi et al., 2022). However, based on your advice, in our future study, we will use human dermal melanocytes rather than melanoma cells.

Reference:

Choi, M.R.; Lee, H.; Kim, H.K.; Han, J.; Seol, J.E.; Vasileva, E.A.; Mishchenko, N.P.; Fedoreyev, S.A.; Stonik, V.A.; Ju, W.S.; et al. Echinochrome A Inhibits Melanogenesis in B16F10 Cells by Downregulating CREB Signaling. Mar. Drugs 2022, 20, 555. https://doi.org/10.3390/md20090555

 

Comment. 6: Some comment of the increase in the perhaps counter-intuitive TRP-2/DCT needs to be added to the paper, in the absence of evidence for a ‘Compensatory upregulation of TRP-2‘.

Response: Thank you for your comment. We acknowledge the counter-intuitive nature of the increase in TRP-2/DCT. Given the lack of evidence for a compensatory upregulation of TRP-2 and the controversial nature of the TRP-1 and TRP-2 results, we have decided to remove this data from our manuscript.

 

Comment. 7: I could not see doi: 10.1111/exd.14350. added to the revised manuscript.

Response: Thank you pointing this out. As per your suggestion, we included this reference in the revised manuscript (Reference # 32). 

 

 

Author Response File: Author Response.pdf