Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review by Fedele and Cardinali focuses on the physiological and clinical effects of circadian alterations in shift workers and discusses strategies to dampen its impact. The text moves linearly from SCN anatomy to molecular mechanisms, to PRCs, through to clinical applications in shift work. The figures (especially 3, 6, and 10) are effective as synthesis tools. Regarding the clinical relevance, the section on shift workers, long-haul drivers, and practical recommendations (orange glasses, light timing, 3-6 mg melatonin, fast clockwise rotation) has concrete applied value, useful for a clinical/occupational health audience. References 78-80 (studies on drivers in the Buenos Aires metropolitan area) represent an original empirical contribution from the group itself, not just a review of third-party work — this is probably the element of greatest real added value.

Some concerns are raised about originality of the review. In particular, the paper suffers of:

• heavy dependence on a single source for key figures: Figures 5, 6, 7, 9, and 10 — that is, almost the entire visual/conceptual framework of the paper — are "adapted with permission from [28]," which is a book by Cardinali himself (Ma Vie En Noir, 2016). This means the visual/conceptual backbone of the review is not new but recycled from a previous work by the same author. A reviewer should ask: what does this review add compared to the 2016 book/chapter from which the main figures are drawn?
• No new quantitative data, no meta-analysis, no systematic review with PRISMA-type criteria or similar: this is a classic narrative review, which isn't a flaw in itself, but it lowers the bar for "contribution" — the value has to come from critical synthesis, not mere exposition.
• Lack of critical/debate elements: the text presents almost everything in an assertive, consistent tone (e.g., the melatonin-cancer hypothesis, light/melatonin PRCs) without sufficiently discussing controversies, methodological limitations of the cited studies, heterogeneity across shift-worker populations, or conflicting findings in the literature. A good review that "justifies publication" usually includes an explicit gaps/limitations section that's more developed than the current brief concluding paragraph.
• Overlap with well-established existing literature: concepts on SCN core/shell, the CLOCK-BMAL1-PER-CRY molecular loop, light and melatonin PRCs, ALAN and shift workers are already covered in numerous highly cited reviews (Golombek & Rosenstein 2010, already cited as ref. 4; Mohawk et al. 2012, ref. 36). An expert reader in the field won't find a conceptually new perspective here.

On the basis of the above considerations, as a didactic/clinical update review, the work is solid, well-written, and useful for a non-specialist audience or occupational medicine physicians — this type of contribution is typically suited for publishing synthesis reviews without requiring original data.

But if the question is whether the work provides conceptual originality that justifies publication in the strict sense (a new critical synthesis, a new framework, substantial unpublished data), the answer is: partially. The real added value lies almost entirely in the section on original data from Argentine drivers and in the practical synthesis of recommendations for shift workers — the rest is a competent but not particularly innovative compilation, with a conspicuous reuse of graphic material from a previous publication by the same senior author.

With the aim to improve the manuscript I would suggest the authors to: (a) more clearly articulate what distinguishes this review from the 2016 book chapter the figures come from, (b) add a critical discussion of limitations/controversies in the literature, (c) potentially expand the section on their own original data, which is the manuscript's real strong point.

Author Response

Comments: The review by Fedele and Cardinali focuses on the physiological and clinical effects of circadian alterations in shift workers and discusses strategies to dampen its impact. The text moves linearly from SCN anatomy to molecular mechanisms, to PRCs, through to clinical applications in shift work. The figures (especially 3, 6, and 10) are effective as synthesis tools. Regarding the clinical relevance, the section on shift workers, long-haul drivers, and practical recommendations (orange glasses, light timing, 3-6 mg melatonin, fast clockwise rotation) has concrete applied value, useful for a clinical/occupational health audience. References 78-80 (studies on drivers in the Buenos Aires metropolitan area) represent an original empirical contribution from the group itself, not just a review of third-party work — this is probably the element of greatest real added value.

Some concerns are raised about originality of the review. In particular, the paper suffers of:

• heavy dependence on a single source for key figures: Figures 5, 6, 7, 9, and 10 — that is, almost the entire visual/conceptual framework of the paper — are "adapted with permission from [28]," which is a book by Cardinali himself (Ma Vie En Noir, 2016). This means the visual/conceptual backbone of the review is not new but recycled from a previous work by the same author. A reviewer should ask: what does this review add compared to the 2016 book/chapter from which the main figures are drawn?
• No new quantitative data, no meta-analysis, no systematic review with PRISMA-type criteria or similar: this is a classic narrative review, which isn't a flaw in itself, but it lowers the bar for "contribution" — the value has to come from critical synthesis, not mere exposition.
• Lack of critical/debate elements: the text presents almost everything in an assertive, consistent tone (e.g., the melatonin-cancer hypothesis, light/melatonin PRCs) without sufficiently discussing controversies, methodological limitations of the cited studies, heterogeneity across shift-worker populations, or conflicting findings in the literature. A good review that "justifies publication" usually includes an explicit gaps/limitations section that's more developed than the current brief concluding paragraph.
• Overlap with well-established existing literature: concepts on SCN core/shell, the CLOCK-BMAL1-PER-CRY molecular loop, light and melatonin PRCs, ALAN and shift workers are already covered in numerous highly cited reviews (Golombek & Rosenstein 2010, already cited as ref. 4; Mohawk et al. 2012, ref. 36). An expert reader in the field won't find a conceptually new perspective here.

On the basis of the above considerations, as a didactic/clinical update review, the work is solid, well-written, and useful for a non-specialist audience or occupational medicine physicians — this type of contribution is typically suited for publishing synthesis reviews without requiring original data.

But if the question is whether the work provides conceptual originality that justifies publication in the strict sense (a new critical synthesis, a new framework, substantial unpublished data), the answer is: partially. The real added value lies almost entirely in the section on original data from Argentine drivers and in the practical synthesis of recommendations for shift workers — the rest is a competent but not particularly innovative compilation, with a conspicuous reuse of graphic material from a previous publication by the same senior author.

With the aim to improve the manuscript I would suggest the authors to: (a) more clearly articulate what distinguishes this review from the 2016 book chapter the figures come from, (b) add a critical discussion of limitations/controversies in the literature, (c) potentially expand the section on their own original data, which is the manuscript's real strong point.

Response:

a) We thank the reviewer for these valuable comments. We would like to clarify that Figures 5, 6, 7, and 9 are not direct reproductions of the figures published in the 2016 book. Rather, they were adapted, redesigned, and substantially modified to improve their clarity and to better illustrate the concepts discussed in the present review. More importantly, although several figures were adapted from a previous publication by one of the authors to illustrate well-established concepts, the scope and objectives of the present review differ substantially from those of the 2016 book Ma Vie en Noir. The book primarily explores the history of melatonin and pineal gland research, whereas the present review focuses on the current understanding of circadian disruption associated with modern 24/7 society, and evidence-based strategies to mitigate its physiological and clinical consequences, particularly in shift workers. In addition, since the publication of the book, considerable advances have been made on this topic. As we mentioned in the abstract, this review integrates recent evidence published over the past decade and provides an updated synthesis linking physiological and clinical effects of circadian disruption with clinically relevant applications in occupational health.

We also agree that concepts such as SCN core/shell organization, the CLOCK-BMAL1-PER-CRY molecular loop, light and melatonin PRCs, ALAN, and shift work have been comprehensively described in previous reviews. However, because the aim of the present article is to provide an integrated overview linking fundamental circadian biology with its clinical applications, we considered a concise description of these mechanisms necessary to provide the appropriate background for readers from clinical and occupational health disciplines.

Therefore, we believe that the novelty of the present review lies not in revisiting these well-established concepts individually, but in integrating recent advances in circadian biology with current evidence on circadian disruption, chronotherapeutic interventions, and their practical implications for occupational health.

b) We thank the reviewer for this thoughtful comment. We agree that several topics discussed in this review remain areas of active investigation and debate. Accordingly, we have revised the manuscript to provide a more balanced discussion by explicitly acknowledging the limitations and heterogeneity of the available evidence. Specifically, we expanded the discussion on the light–melatonin–cancer hypothesis, emphasizing that although a substantial body of experimental evidence supports an association between circadian disruption, melatonin suppression, and tumor development, the clinical evidence remains heterogeneous and does not yet allow definitive conclusions regarding causality or the therapeutic role of melatonin (page 12, lines 418-425). We also incorporated a discussion of the variability in responses to light (page 16, lines 585-590) and melatonin interventions (page 17, lines 638-644) among different shift-worker populations, highlighting methodological differences between studies and the need for personalized approaches. Finally, we agree that the previous conclusion did not sufficiently discuss the current limitations of the field. Accordingly, we have created a final section of the manuscript entitled “Current limitations and future perspectives” (page 19) where we point out the methodological heterogeneity of the available literature, current knowledge gaps regarding chronotherapeutic interventions and circadian phase resetting, the need for personalized approaches to shift-work adaptation, and future research priorities.

c) Following the reviewer's valuable suggestion, we incorporated additional findings from our studies on Argentine shift workers, including oil industry workers and medical residents, and by placing these findings within the context of the broader international literature to highlight both their consistency with previous reports and their contribution to understanding shift work in Latin American populations (page 14, lines 474-491).

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript by Mul Fedele and Cardinali makes a compelling review of the parameters affecting the phase of the circadian system and the numerous conditions in which chronodisruption can be induced. The study mainly focuses on data from research in human and provides relevant societal perspectives in the 24/7 present world.

The review is well written and includes abundant references. I am listing below a few points that need to be corrected or clarified in the first chapters dealing with general aspects of circadian rhythms.

-line 32: it is the rotation of the Earth around its own axis that generates the day/night cycle.

-line 33: the sentence is misleading: the changing duration of day and night results from the Earth rotation around the Sun with a tilted axis with respect to the rotation plane.

-Chapter 3 describing the molecular mechanisms: the gene names should be in italic.

-lines 127-131: the first sentence cites Rev-Erb alpha and then Ror alpha. The following sentence states "The proteins encoded by these genes compete to bind to RORE (retinoic acid-related orphan receptor response elements) located in the Bmal1 promoter, thereby activating or inhibiting its transcription, respectively. If respectively refers to the first sentence, the 2 genes should be inverted since Rors are activators and Rev-Erbs repressors for Bmal1. Also, I suggest to mention these elements of the clock feedback loops as plural, like in figure 3, since several members of the 2 families can be involved.

-Figure 3: the arrow indicating activation of RORE element on Bmal1 promoter should be in green. And the CCGs should be written as such in the figure.

-lines 165-166: "This molecular circadian clock operates in virtually...": this sentence would better fit into the previous paragraph that deals with the molecular clock rather than in that one about the circadian system.

-lines 182-184 from the same chapter 4: I would call that the "circadian system" rather than the "circadian clock".

-Figure 5: please clarify the meaning of the vertical dashed lines and of the width of the blue or green arrows.

-Chapter 7: the conditions in which phase response curves are determined in human could be better described and should include more references, in particular regarding the effect of melatonin. 

-Figure 6: please provide more information about the timing and the x-axis. 

Author Response

Comments 1: -line 32: it is the rotation of the Earth around its own axis that generates the day/night cycle.

Response 1: Thank you for pointing this out. The sentence has been revised as follows: The Earth's rotation around its own axis generates a 24-hour light-dark cycle.

Comments 2: -line 33: the sentence is misleading: the changing duration of day and night results from the Earth rotation around the Sun with a tilted axis with respect to the rotation plane.

Response 2: The sentence has been revised as follows: Moreover, the duration of daylight and darkness changes throughout the year as the Earth revolves around the Sun while maintaining a tilted rotational axis relative to the orbital plane.

Comments 3: -Chapter 3 describing the molecular mechanisms: the gene names should be in italic.

Response 3: Thank you for this observation. All gene names have been formatted in italics.

Comments 4: -lines 127-131: the first sentence cites Rev-Erb alpha and then Ror alpha. The following sentence states "The proteins encoded by these genes compete to bind to RORE (retinoic acid-related orphan receptor response elements) located in the Bmal1 promoter, thereby activating or inhibiting its transcription, respectively. If respectively refers to the first sentence, the 2 genes should be inverted since Rors are activators and Rev-Erbs repressors for Bmal1. Also, I suggest to mention these elements of the clock feedback loops as plural, like in figure 3, since several members of the 2 families can be involved.

Response 4: We thank the reviewer for this helpful suggestion. We have revised the text to refer to the ROR and REV-ERB receptor families rather than individual members, corrected the order to reflect that ROR proteins activate, whereas REV-ERB proteins repress, Bmal1 transcription, and changed RORE to ROREs to indicate the response elements in plural, consistent with Figure 3.

Comments 5: -Figure 3: the arrow indicating activation of RORE element on Bmal1 promoter should be in green. And the CCGs should be written as such in the figure.

Response 5: We thank the reviewer for pointing this out, the Figure has been revised accordingly.

Comments 6: -lines 165-166: "This molecular circadian clock operates in virtually...": this sentence would better fit into the previous paragraph that deals with the molecular clock rather than in that one about the circadian system.

Response 6: Thank you for this observation. The sentence has been moved to the previous paragraph, where the molecular clock is described (page 4, lines 133-134).

Comments 7: -lines 182-184 from the same chapter 4: I would call that the "circadian system" rather than the "circadian clock".

Response 7: We agree with the reviewer and have replaced "circadian clock" with "circadian system" in the corresponding sentence.

Comments 8: -Figure 5: please clarify the meaning of the vertical dashed lines and of the width of the blue or green arrows.

Response 8: We thank the reviewer for this helpful suggestion. We have revised the figure legend to clarify that the vertical dashed lines indicate the daytime interval (07:00–19:00), and that the horizontal extent of the blue and green arrows represents the time window during which light or melatonin administration produces a phase advance or phase delay, respectively.

Comments 9: -Chapter 7: the conditions in which phase response curves are determined in human could be better described and should include more references, in particular regarding the effect of melatonin. 

Response 9: We thank the reviewer for this valuable suggestion. We have expanded Chapter 7 to better describe the experimental conditions under which human phase response curves (PRCs) have been determined (page 8-9, lines 280-287). We also included a brief description of type 1 and type 0 PRCs (page 8, lines 274-279), and we incorporated several classical references describing the experimental characterization of the human melatonin PRC (page 9, lines 297-305).

Comments 10: -Figure 6: please provide more information about the timing and the x-axis. 

Response 10: We thank the reviewer for this helpful suggestion. We have revised both the figure legend and the main text (page 9, lines 288-291) to clarify the meaning of the x-axis. Specifically, we now indicate that the x-axis represents the clock time of stimulus administration for an individual with a nocturnal sleep schedule, whereas the reference circadian phase is given by the minimum of the core body temperature rhythm.

Reviewer 3 Report

Comments and Suggestions for Authors

Overall this manuscript provides a nice review on circadian rhythms, the PRC, and the effect of misalignment on health. However, the claim on the role of melatonin to phase shift the clock in adult mammals appears controversial in the circadian rhythm filed. The authors relied on ref [28] to support this claim. Please also discuss other opposing views and experimental results and elaborate the mechanisms (non-photic, directly acting on the SCN?...).

Author Response

Comments: Overall this manuscript provides a nice review on circadian rhythms, the PRC, and the effect of misalignment on health. However, the claim on the role of melatonin to phase shift the clock in adult mammals appears controversial in the circadian rhythm filed. The authors relied on ref [28] to support this claim. Please also discuss other opposing views and experimental results and elaborate the mechanisms (non-photic, directly acting on the SCN?...).

Response: We thank the reviewer for this valuable comment. We have revised the manuscript to provide a more comprehensive and balanced discussion of the chronobiotic actions of melatonin. Specifically, in Section 6 (page 7), we incorporated additional references supporting its phase-shifting effects and expanded the discussion of the underlying mechanisms. We now describe the evidence indicating that melatonin primarily acts through MT1 and MT2 receptors within the suprachiasmatic nucleus, while acknowledging that the relative contribution of additional mechanisms and indirect sleep-promoting effects remains an area of active investigation. In Section 11 (page 17, lines 638-644), we also emphasize that the clinical effectiveness of exogenous melatonin depends on several factors, including the timing of administration relative to the endogenous circadian phase, dose, formulation, and interindividual variability.