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peer_reviews/supplementary_0_Peer Review File NEW__695ec1aa958e104a44be7df10d5fe635817187f04c327e11f9d1c3bd9f672f00/images_list.json

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+[
+  {
+    "type": "image",
+    "img_path": "images/Supplementary_Figure_15.jpg",
+    "caption": "Supplementary Fig. 15 | Heartbeat artifact removal. a, Average time course of blank trials over the center \\(2 \\text{mm} \\times 2 \\text{mm}\\) area of the FOV. The signal fluctuation is caused by the heartbeat. Shaded area ± SEM. b, Average time course of GCaMP response to the flashed gratings (60 degree stimulus) over the",
+    "footnote": [],
+    "bbox": [],
+    "page_idx": 0
+  }
+]

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+
+# nature portfolio  
+
+Peer Review File  
+
+Mesoscopic calcium imaging in a head- unrestrained male non- human primate using a lensless microscope  
+
+![](images/Supplementary_Figure_15.jpg)
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to  
+
+the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+Editorial Note: This manuscript has been previously reviewed at another journal that is not operating a transparent peer review scheme. This document only contains reviewer comments and rebuttal letters for versions considered at Nature Communications.
+
+<--- Page Split --->
+
+Reviewer #1 (Remarks to the Author):  
+
+This is a follow up of from a reviewing thread started in Nature Methods. I will therefore not repeat earlier general comments about the manuscript.  
+
+Thanks for adding qualifiers to the text, particularly in the Discussion, to leave readers with a realistic assessment of the capabilities of this microscope in its current state of development. The overall concept of the microscope is promising, with considerable potential and the text now makes that point well. I have no further comments.  
+
+I look forward to further development of the microscope, e.g with an additional, interdigitated illumination / absorption pathway to help with hemodynamic correction, for extended imaging trials and more realistic experimental setups. Particularly in marmosets.  
+
+(signed) Aniruddha Das  
+
+Reviewer #2 (Remarks to the Author):  
+
+My previous comments reflected, in part, my lack of being convinced that the work as it stands would be sufficiently impactful to a broad audience, and this concern remains unabated for consideration at Nature Communications.  
+
+As it stands, the authors have not made any changes in the manuscript (other than additions to the discussion) in response to my concerns, and I don't see how I can reasonably change my view of the work's impact. Notably, the issues regarding hemodynamic correction are still unaddressed (and were also brought up by reviewer 1).  
+
+1. Whether there is any substantial hemodynamic contamination to the visual stimulus responses in the present paper is somewhat beside the point. I note that their claim this is not an issue for the early response periods studies here, after removal of heartbeat-locked artifacts is not terribly convincing. The authors are trying to demonstrate a highly impactful new method. They are thus obligated to show that the system can and will work under broadly applicable circumstances. For example, can this system collect accurate spontaneous activity data sets that are not time-locked to a stimulus? Can this system collect usable data outside the "1 second" window for sensory-evoked responses? Beyond these questions, the authors don't even provide any raw data showing that there is a heartbeat artifact and that it is successfully removed with their method.  
+
+2. It feels that most of the push-back originates from these data coming from a single animal and the authors are unable or unwilling to do additional experiments to address reviewer concerns. I appreciate the challenges of primate work, but this is simply not acceptable for a study claiming broad impact.  
+
+3. If the biological data (visual responses) are ancillary and simply to show that the camera can collect data, the manuscript ultimately rests on the technical development of a new mesoscopic camera system for primates. I am unconvinced this point alone will be of widespread interest in the absence of demonstrated biological impact. I feel strongly that the authors must present at least some biological findings that (1) are made uniquely possible by this new camera and (2) are rigorously obtained using the current standards in the widefield imaging field.
+
+<--- Page Split --->
+
+We thank the editor and the reviewers for their valuable feedback, which we believe has helped to improve the manuscript. We provide a point- by- point response to reviewers' comments below.  
+
+## Point-by-point responses  
+
+## Reviewer #1  
+
+This is a follow up of from a reviewing thread started in Nature Methods. I will therefore not repeat earlier general comments about the manuscript.  
+
+Thanks for adding qualifiers to the text, particularly in the Discussion, to leave readers with a realistic assessment of the capabilities of this microscope in its current state of development. The overall concept of the microscope is promising, with considerable potential and the text now makes that point well. I have no further comments.  
+
+I look forward to further development of the microscope, e.g with an additional, interdigitated illumination / absorption pathway to help with hemodynamic correction, for extended imaging trials and more realistic experimental setups. Particularly in marmosets.  
+
+We thank the reviewer for the positive assessment and for recognizing the potential of our work. We are especially thankful for the invaluable suggestions and comments from the reviewer throughout the revision process, which significantly help us improve our manuscript.  
+
+## Reviewer #2  
+
+My previous comments reflected, in part, my lack of being convinced that the work as it stands would be sufficiently impactful to a broad audience, and this concern remains unabated for consideration at Nature Communications.  
+
+As it stands, the authors have not made any changes in the manuscript (other than additions to the discussion) in response to my concerns, and I don't see how I can reasonably change my view of the work's impact. Notably, the issues regarding hemodynamic correction are still unaddressed (and were also brought up by reviewer 1).  
+
+1. Whether there is any substantial hemodynamic contamination to the visual stimulus responses in the present paper is somewhat beside the point. I note that their claim this is not an issue for the early response periods studies here, after removal of heartbeat-locked artifacts is not terribly convincing. The authors are trying to demonstrate a highly impactful new method. They are thus obligated to show that the system can and will work under broadly applicable circumstances. For example, can this system collect accurate spontaneous activity data sets that are not time-locked to a stimulus? Can this system collect usable data outside the "1 second" window for sensory-evoked responses? Beyond these questions, the authors don't
+
+<--- Page Split --->
+
+even provide any raw data showing that there is a heartbeat artifact and that it is successfully removed with their method.  
+
+This paper serves as a proof- of- principle demonstrating that the lensless microscope can achieve imaging quality comparable to that of a standard table- top widefield microscope. Moreover, we show the capability of this microscope to extract small neurological signals, such as orientation columns maps from a head- unrestrained animal. To demonstrate the imaging quality of the system, we use the established experimental settings employed in previous work (e.g., Chen, Y. et al. Nature Neuroscience 2006, Seidemann, E. et al. eLife 2016), incorporating a heartbeat triggered visual stimulus.  
+
+Future work can certainly build on this proof- of- concept to develop a more broadly applicable method that could facilitate continuous recording by incorporating an additional imaging path designed to record the hemodynamic signals. This is outside the scope of the current work and involves a change in filters and illumination, while the fundamental concept of lensless microscopy would remain the same. We anticipate that the system can accurately collect spontaneous activity data outside the 1 second time window and without heartbeat triggered stimulus when we simultaneously capture the hemodynamic signals, but this will be the topic of future work. Thanks to the reviewer's previous review we made the changes in the discussion to emphasize this point.  
+
+Removing heartbeat artifact using synced stimulus is widely used and it was first documented in (Grinvald et al., Journal of Neuroscience, 1994). To show the heartbeat artifact removal we added a supplementary figure (Supplementary Figure 15). This shows the raw time traces captured by the table- top widefield microscope, and a comparison before and after the heartbeat artifact correction. Heartbeat artifact is clearly shown in Supplementary Figure 15b, and got removed after the subtraction of averaged blank trials. It's important to highlight that despite the presence of a heartbeat artifact, we still captured a robust 4 Hz stimulus signal, just with a reduced signal- to- noise ratio (SNR).  
+
+![PLACEHOLDER_4_0]
+
+<center>Supplementary Fig. 15 | Heartbeat artifact removal. a, Average time course of blank trials over the center \(2 \text{mm} \times 2 \text{mm}\) area of the FOV. The signal fluctuation is caused by the heartbeat. Shaded area ± SEM. b, Average time course of GCaMP response to the flashed gratings (60 degree stimulus) over the </center>
+
+<--- Page Split --->
+
+center \(2 \text{mm} \times 2 \text{mm}\) area of the FOV before heartbeat artifact correction. Shaded area \(\pm\) SEM. c, Average time course of GCaMP response to the flashed gratings (60 degree stimulus) over the center 2 \(\text{mm} \times 2 \text{mm}\) area of the FOV after heartbeat artifact correction. Shaded area \(\pm\) SEM. Data presented here were captured using the table- top widefield microscope. All traces represent average across 10 repeats. Source data are provided as a Source Data file.  
+
+2. It feels that most of the push-back originates from these data coming from a single animal and the authors are unable or unwilling to do additional experiments to address reviewer concerns. I appreciate the challenges of primate work, but this is simply not acceptable for a study claiming broad impact.  
+
+This paper primarily focused on the technical development of the new lensless microscope. Using this new device, we performed multiple experiments on a macaque over various days spanning a 6-month duration. Specifically, we performed position tuning experiments and orientation tuning experiments more than twice each, consistently obtaining stable results (additional data in Supplementary Information). These consistent outcomes clearly demonstrate the stability of our system. Additional animals and experiments are unlikely to provide any new insights or information.  
+
+3. If the biological data (visual responses) are ancillary and simply to show that the camera can collect data, the manuscript ultimately rests on the technical development of a new mesoscopic camera system for primates. I am unconvinced this point alone will be of widespread interest in the absence of demonstrated biological impact. I feel strongly that the authors must present at least some biological findings that (1) are made uniquely possible by this new camera and (2) are rigorously obtained using the current standards in the widefield imaging field.  
+
+This paper focuses on the development of a miniaturized lensless microscope for non- human primates, capable of achieving similar imaging quality to standard table- top widefield microscopes. Using this microscope, we successfully imaged the first orientation columns map from a head- unrestrained macaque - uniquely made possible by the Bio- FlatScopeNHP. There was no guarantee that these maps would match the head- fixed maps, which is a finding in itself. With the future improvement incorporating hemodynamic correction, this system can assist researchers in observing neurological signals under more naturalistic conditions. This would allow researchers to study the impact of natural head and eye movements on sensory areas like V1 and the auditory cortex, as well as sensory- motor regions such as the premotor cortex and frontal eye fields.
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File NEW__695ec1aa958e104a44be7df10d5fe635817187f04c327e11f9d1c3bd9f672f00/supplementary_0_Peer Review File NEW__695ec1aa958e104a44be7df10d5fe635817187f04c327e11f9d1c3bd9f672f00_det.mmd

@@ -0,0 +1,123 @@
+<|ref|>title<|/ref|><|det|>[[61, 40, 505, 90]]<|/det|>
+# nature portfolio  
+
+<|ref|>text<|/ref|><|det|>[[66, 110, 361, 139]]<|/det|>
+Peer Review File  
+
+<|ref|>text<|/ref|><|det|>[[111, 152, 891, 245]]<|/det|>
+Mesoscopic calcium imaging in a head- unrestrained male non- human primate using a lensless microscope  
+
+<|ref|>image<|/ref|><|det|>[[56, 732, 239, 782]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[250, 732, 911, 784]]<|/det|>
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to  
+
+<|ref|>text<|/ref|><|det|>[[56, 784, 934, 924]]<|/det|>
+the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 84, 864, 136]]<|/det|>
+Editorial Note: This manuscript has been previously reviewed at another journal that is not operating a transparent peer review scheme. This document only contains reviewer comments and rebuttal letters for versions considered at Nature Communications.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[120, 84, 415, 98]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 111, 876, 140]]<|/det|>
+This is a follow up of from a reviewing thread started in Nature Methods. I will therefore not repeat earlier general comments about the manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[118, 153, 878, 210]]<|/det|>
+Thanks for adding qualifiers to the text, particularly in the Discussion, to leave readers with a realistic assessment of the capabilities of this microscope in its current state of development. The overall concept of the microscope is promising, with considerable potential and the text now makes that point well. I have no further comments.  
+
+<|ref|>text<|/ref|><|det|>[[119, 224, 840, 266]]<|/det|>
+I look forward to further development of the microscope, e.g with an additional, interdigitated illumination / absorption pathway to help with hemodynamic correction, for extended imaging trials and more realistic experimental setups. Particularly in marmosets.  
+
+<|ref|>text<|/ref|><|det|>[[119, 280, 300, 294]]<|/det|>
+(signed) Aniruddha Das  
+
+<|ref|>text<|/ref|><|det|>[[119, 335, 414, 350]]<|/det|>
+Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[119, 363, 848, 406]]<|/det|>
+My previous comments reflected, in part, my lack of being convinced that the work as it stands would be sufficiently impactful to a broad audience, and this concern remains unabated for consideration at Nature Communications.  
+
+<|ref|>text<|/ref|><|det|>[[118, 419, 870, 477]]<|/det|>
+As it stands, the authors have not made any changes in the manuscript (other than additions to the discussion) in response to my concerns, and I don't see how I can reasonably change my view of the work's impact. Notably, the issues regarding hemodynamic correction are still unaddressed (and were also brought up by reviewer 1).  
+
+<|ref|>text<|/ref|><|det|>[[118, 490, 868, 616]]<|/det|>
+1. Whether there is any substantial hemodynamic contamination to the visual stimulus responses in the present paper is somewhat beside the point. I note that their claim this is not an issue for the early response periods studies here, after removal of heartbeat-locked artifacts is not terribly convincing. The authors are trying to demonstrate a highly impactful new method. They are thus obligated to show that the system can and will work under broadly applicable circumstances. For example, can this system collect accurate spontaneous activity data sets that are not time-locked to a stimulus? Can this system collect usable data outside the "1 second" window for sensory-evoked responses? Beyond these questions, the authors don't even provide any raw data showing that there is a heartbeat artifact and that it is successfully removed with their method.  
+
+<|ref|>text<|/ref|><|det|>[[118, 629, 866, 685]]<|/det|>
+2. It feels that most of the push-back originates from these data coming from a single animal and the authors are unable or unwilling to do additional experiments to address reviewer concerns. I appreciate the challenges of primate work, but this is simply not acceptable for a study claiming broad impact.  
+
+<|ref|>text<|/ref|><|det|>[[118, 700, 867, 784]]<|/det|>
+3. If the biological data (visual responses) are ancillary and simply to show that the camera can collect data, the manuscript ultimately rests on the technical development of a new mesoscopic camera system for primates. I am unconvinced this point alone will be of widespread interest in the absence of demonstrated biological impact. I feel strongly that the authors must present at least some biological findings that (1) are made uniquely possible by this new camera and (2) are rigorously obtained using the current standards in the widefield imaging field.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 123, 875, 173]]<|/det|>
+We thank the editor and the reviewers for their valuable feedback, which we believe has helped to improve the manuscript. We provide a point- by- point response to reviewers' comments below.  
+
+<|ref|>sub_title<|/ref|><|det|>[[116, 186, 353, 203]]<|/det|>
+## Point-by-point responses  
+
+<|ref|>sub_title<|/ref|><|det|>[[115, 220, 220, 236]]<|/det|>
+## Reviewer #1  
+
+<|ref|>text<|/ref|><|det|>[[115, 253, 839, 287]]<|/det|>
+This is a follow up of from a reviewing thread started in Nature Methods. I will therefore not repeat earlier general comments about the manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[115, 304, 881, 371]]<|/det|>
+Thanks for adding qualifiers to the text, particularly in the Discussion, to leave readers with a realistic assessment of the capabilities of this microscope in its current state of development. The overall concept of the microscope is promising, with considerable potential and the text now makes that point well. I have no further comments.  
+
+<|ref|>text<|/ref|><|det|>[[115, 388, 855, 439]]<|/det|>
+I look forward to further development of the microscope, e.g with an additional, interdigitated illumination / absorption pathway to help with hemodynamic correction, for extended imaging trials and more realistic experimental setups. Particularly in marmosets.  
+
+<|ref|>text<|/ref|><|det|>[[115, 455, 876, 506]]<|/det|>
+We thank the reviewer for the positive assessment and for recognizing the potential of our work. We are especially thankful for the invaluable suggestions and comments from the reviewer throughout the revision process, which significantly help us improve our manuscript.  
+
+<|ref|>sub_title<|/ref|><|det|>[[115, 523, 230, 540]]<|/det|>
+## Reviewer #2  
+
+<|ref|>text<|/ref|><|det|>[[115, 559, 867, 609]]<|/det|>
+My previous comments reflected, in part, my lack of being convinced that the work as it stands would be sufficiently impactful to a broad audience, and this concern remains unabated for consideration at Nature Communications.  
+
+<|ref|>text<|/ref|><|det|>[[115, 626, 872, 694]]<|/det|>
+As it stands, the authors have not made any changes in the manuscript (other than additions to the discussion) in response to my concerns, and I don't see how I can reasonably change my view of the work's impact. Notably, the issues regarding hemodynamic correction are still unaddressed (and were also brought up by reviewer 1).  
+
+<|ref|>text<|/ref|><|det|>[[115, 711, 873, 846]]<|/det|>
+1. Whether there is any substantial hemodynamic contamination to the visual stimulus responses in the present paper is somewhat beside the point. I note that their claim this is not an issue for the early response periods studies here, after removal of heartbeat-locked artifacts is not terribly convincing. The authors are trying to demonstrate a highly impactful new method. They are thus obligated to show that the system can and will work under broadly applicable circumstances. For example, can this system collect accurate spontaneous activity data sets that are not time-locked to a stimulus? Can this system collect usable data outside the "1 second" window for sensory-evoked responses? Beyond these questions, the authors don't
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[113, 123, 858, 156]]<|/det|>
+even provide any raw data showing that there is a heartbeat artifact and that it is successfully removed with their method.  
+
+<|ref|>text<|/ref|><|det|>[[113, 173, 877, 291]]<|/det|>
+This paper serves as a proof- of- principle demonstrating that the lensless microscope can achieve imaging quality comparable to that of a standard table- top widefield microscope. Moreover, we show the capability of this microscope to extract small neurological signals, such as orientation columns maps from a head- unrestrained animal. To demonstrate the imaging quality of the system, we use the established experimental settings employed in previous work (e.g., Chen, Y. et al. Nature Neuroscience 2006, Seidemann, E. et al. eLife 2016), incorporating a heartbeat triggered visual stimulus.  
+
+<|ref|>text<|/ref|><|det|>[[113, 307, 880, 458]]<|/det|>
+Future work can certainly build on this proof- of- concept to develop a more broadly applicable method that could facilitate continuous recording by incorporating an additional imaging path designed to record the hemodynamic signals. This is outside the scope of the current work and involves a change in filters and illumination, while the fundamental concept of lensless microscopy would remain the same. We anticipate that the system can accurately collect spontaneous activity data outside the 1 second time window and without heartbeat triggered stimulus when we simultaneously capture the hemodynamic signals, but this will be the topic of future work. Thanks to the reviewer's previous review we made the changes in the discussion to emphasize this point.  
+
+<|ref|>text<|/ref|><|det|>[[113, 475, 880, 610]]<|/det|>
+Removing heartbeat artifact using synced stimulus is widely used and it was first documented in (Grinvald et al., Journal of Neuroscience, 1994). To show the heartbeat artifact removal we added a supplementary figure (Supplementary Figure 15). This shows the raw time traces captured by the table- top widefield microscope, and a comparison before and after the heartbeat artifact correction. Heartbeat artifact is clearly shown in Supplementary Figure 15b, and got removed after the subtraction of averaged blank trials. It's important to highlight that despite the presence of a heartbeat artifact, we still captured a robust 4 Hz stimulus signal, just with a reduced signal- to- noise ratio (SNR).  
+
+<|ref|>image<|/ref|><|det|>[[111, 640, 884, 803]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[113, 819, 866, 865]]<|/det|>
+<center>Supplementary Fig. 15 | Heartbeat artifact removal. a, Average time course of blank trials over the center \(2 \text{mm} \times 2 \text{mm}\) area of the FOV. The signal fluctuation is caused by the heartbeat. Shaded area ± SEM. b, Average time course of GCaMP response to the flashed gratings (60 degree stimulus) over the </center>
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 123, 880, 201]]<|/det|>
+center \(2 \text{mm} \times 2 \text{mm}\) area of the FOV before heartbeat artifact correction. Shaded area \(\pm\) SEM. c, Average time course of GCaMP response to the flashed gratings (60 degree stimulus) over the center 2 \(\text{mm} \times 2 \text{mm}\) area of the FOV after heartbeat artifact correction. Shaded area \(\pm\) SEM. Data presented here were captured using the table- top widefield microscope. All traces represent average across 10 repeats. Source data are provided as a Source Data file.  
+
+<|ref|>text<|/ref|><|det|>[[115, 216, 852, 285]]<|/det|>
+2. It feels that most of the push-back originates from these data coming from a single animal and the authors are unable or unwilling to do additional experiments to address reviewer concerns. I appreciate the challenges of primate work, but this is simply not acceptable for a study claiming broad impact.  
+
+<|ref|>text<|/ref|><|det|>[[115, 300, 877, 418]]<|/det|>
+This paper primarily focused on the technical development of the new lensless microscope. Using this new device, we performed multiple experiments on a macaque over various days spanning a 6-month duration. Specifically, we performed position tuning experiments and orientation tuning experiments more than twice each, consistently obtaining stable results (additional data in Supplementary Information). These consistent outcomes clearly demonstrate the stability of our system. Additional animals and experiments are unlikely to provide any new insights or information.  
+
+<|ref|>text<|/ref|><|det|>[[115, 434, 875, 536]]<|/det|>
+3. If the biological data (visual responses) are ancillary and simply to show that the camera can collect data, the manuscript ultimately rests on the technical development of a new mesoscopic camera system for primates. I am unconvinced this point alone will be of widespread interest in the absence of demonstrated biological impact. I feel strongly that the authors must present at least some biological findings that (1) are made uniquely possible by this new camera and (2) are rigorously obtained using the current standards in the widefield imaging field.  
+
+<|ref|>text<|/ref|><|det|>[[114, 551, 880, 720]]<|/det|>
+This paper focuses on the development of a miniaturized lensless microscope for non- human primates, capable of achieving similar imaging quality to standard table- top widefield microscopes. Using this microscope, we successfully imaged the first orientation columns map from a head- unrestrained macaque - uniquely made possible by the Bio- FlatScopeNHP. There was no guarantee that these maps would match the head- fixed maps, which is a finding in itself. With the future improvement incorporating hemodynamic correction, this system can assist researchers in observing neurological signals under more naturalistic conditions. This would allow researchers to study the impact of natural head and eye movements on sensory areas like V1 and the auditory cortex, as well as sensory- motor regions such as the premotor cortex and frontal eye fields.
+
+<--- Page Split --->

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+[]

peer_reviews/supplementary_0_Peer Review File.__be834470d518c850b908959fca0cb3295c70157ca34ea00c672f21b5b20c41c8/supplementary_0_Peer Review File.__be834470d518c850b908959fca0cb3295c70157ca34ea00c672f21b5b20c41c8.mmd

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+
+# nature portfolio  
+
+Peer Review File  
+
+Increased lethality in influenza and SARS- CoV- 2 coinfection is prevented by influenza immunity but not SARS- CoV- 2 immunity  
+
+![PLACEHOLDER_0_0]
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+Editorial Note: This manuscript has been previously reviewed at another journal that is not operating a transparent peer review scheme. This document only contains reviewer comments and rebuttal letters for versions considered at Nature Communications.  
+
+Reviewers' Comments:  
+
+Reviewer #1: Remarks to the Author: The authors have addressed my concerns. The additional experiments have strengthened the manuscript.  
+
+Reviewer #2:  
+
+Remarks to the Author:  
+
+The paper by Achdout et al. studies the effect of SARS- CoV- 2/influenza superinfection in a transgenic mouse model expressing human ACE2 under the K18 promoter. The data obtained in this model is compared with that obtained in C57BL/6 mice in which ACE2 is delivery via Adenovirus infection. The study indicates that superinfection results in severe respiratory disease. This is precluded by previous immunity to flu but not SARS- CoV- 2, and this immunity is antibody dependent.  
+
+This is a great study, well conducted and with conclusions of obvious public health relevance. I only have some minor suggestions as indicated below. Well done.  
+
+1- In the abstract (Line 27), I think antibody- dependent is more adequate than humoral- dependent. Alternatively 'dependent on humoral immunity'.  
+
+2- Why do the authors conclude that 'in the human population, coinfection is most likely to occur during the asymptomatic period'? This for sure does not apply for mild cases of COVID- 19 which are the vast majority.  
+
+3- I think that throughout the paper, superinfection is more appropriate than coinfection  
+
+4- Did the authors sequenced their SARS- CoV- 2 stocks? This is important in view of the furing cleavage deletions appearing as a consequence of passage in Vero's. This should be indicated  
+
+5- it would be easier for the reader if the color codes of the graphs were maintained in all the figures
+
+<--- Page Split --->
+
+
+<table><tr><td>REVIEWERS' COMMENTS</td><td>Answer</td></tr><tr><td>REVIEWERS' COMMENTS<br>Reviewer #1 (Remarks to the Author):<br>The authors have addressed my concerns. The<br>additional experiments have strengthened the<br>manuscript.</td><td>We wish to thank the Reviewer for his valuable<br>remarks. We also believe that the additions made<br>following his remarks strengthened the<br>manuscript.</td></tr><tr><td>REVIEWERS' COMMENTS<br>Reviewer #2 (Remarks to the Author):<br>The paper by Achdout et al. studies the effect of<br>SARS-CoV-2/influenza superinfection in a<br>transgenic mouse model expressing human ACE2<br>under the K18 promoter. The data obtained in<br>this model is compared with that obtained in<br>C57BL/6 mice in which ACE2 is delivery via<br>Adenovirus infection. The study indicates that<br>superinfection results in severe respiratory<br>disease. This is precluded by previous immunity to flu but not SARS-CoV-2, and this immunity is<br>antibody dependent.<br>This is a great study, well conducted and with<br>conclusions of obvious public health relevance. I only have some minor suggestions as indicated<br>below. Well done.</td><td>We wish to thank the Reviewer for his comments and remarks. Our replay for his suggestions<br>appears below.<br>The line numbers specified are for the track<br>changes version.</td></tr><tr><td>1- In the abstract (Line 27), I think antibody-dependent is more adequate than humoral-dependent. Alternatively 'dependent on humoral immunity'.</td><td>As suggested, changed to antibody-dependent.</td></tr><tr><td>2- Why do the authors conclude that 'in the<br>human population, coinfection is most likely to<br>occur during the asymptomatic period'? This for sure does not apply for mild cases of COVID-19<br>which are the vast majority.</td><td>We are sorry for the misunderstanding. We<br>added clarification for the meaning of<br>asymptomatic period of influenza (lines 112-113).</td></tr><tr><td>3- I think that throughout the paper,<br>superinfection is more appropriate than<br>coinfection</td><td>We address the terms coinfection and<br>superinfection in lines 81-84.<br>("While the terms coinfection and superinfection are often used interchangeably, the use of<br>'coinfection' here refers to a sequential infection with 2 viruses within a very short time, with the<br>second infection occurring prior to the<br>elimination of the first virus.").</td></tr></table>
+
+<--- Page Split --->
+
+
+<table><tr><td>4- Did the authors sequenced their SARS-CoV-2 stocks? This is important in view of the furing cleavage deletions appearing as a consequence of passage in Vero's. This should be indicated</td><td>Yes. GISAID submission: EPI_ISL_3838266. No deletion in the furin cleavage site is noted. Data was added to the text, lines 262-264.</td></tr><tr><td>5- it would be easier for the reader if the color codes of the graphs were maintained in all the figures</td><td>The color codes of the groups are maintain in all figures when possible.</td></tr></table>
+
+<--- Page Split --->

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@@ -0,0 +1,62 @@
+<|ref|>title<|/ref|><|det|>[[100, 40, 507, 90]]<|/det|>
+# nature portfolio  
+
+<|ref|>text<|/ref|><|det|>[[107, 110, 373, 139]]<|/det|>
+Peer Review File  
+
+<|ref|>text<|/ref|><|det|>[[107, 154, 875, 241]]<|/det|>
+Increased lethality in influenza and SARS- CoV- 2 coinfection is prevented by influenza immunity but not SARS- CoV- 2 immunity  
+
+<|ref|>image<|/ref|><|det|>[[93, 732, 262, 780]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[270, 732, 880, 784]]<|/det|>
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 872, 142]]<|/det|>
+Editorial Note: This manuscript has been previously reviewed at another journal that is not operating a transparent peer review scheme. This document only contains reviewer comments and rebuttal letters for versions considered at Nature Communications.  
+
+<|ref|>text<|/ref|><|det|>[[115, 175, 286, 190]]<|/det|>
+Reviewers' Comments:  
+
+<|ref|>text<|/ref|><|det|>[[115, 207, 812, 270]]<|/det|>
+Reviewer #1: Remarks to the Author: The authors have addressed my concerns. The additional experiments have strengthened the manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[115, 319, 216, 333]]<|/det|>
+Reviewer #2:  
+
+<|ref|>text<|/ref|><|det|>[[115, 336, 291, 350]]<|/det|>
+Remarks to the Author:  
+
+<|ref|>text<|/ref|><|det|>[[115, 350, 872, 429]]<|/det|>
+The paper by Achdout et al. studies the effect of SARS- CoV- 2/influenza superinfection in a transgenic mouse model expressing human ACE2 under the K18 promoter. The data obtained in this model is compared with that obtained in C57BL/6 mice in which ACE2 is delivery via Adenovirus infection. The study indicates that superinfection results in severe respiratory disease. This is precluded by previous immunity to flu but not SARS- CoV- 2, and this immunity is antibody dependent.  
+
+<|ref|>text<|/ref|><|det|>[[115, 444, 860, 476]]<|/det|>
+This is a great study, well conducted and with conclusions of obvious public health relevance. I only have some minor suggestions as indicated below. Well done.  
+
+<|ref|>text<|/ref|><|det|>[[115, 492, 861, 523]]<|/det|>
+1- In the abstract (Line 27), I think antibody- dependent is more adequate than humoral- dependent. Alternatively 'dependent on humoral immunity'.  
+
+<|ref|>text<|/ref|><|det|>[[115, 540, 864, 588]]<|/det|>
+2- Why do the authors conclude that 'in the human population, coinfection is most likely to occur during the asymptomatic period'? This for sure does not apply for mild cases of COVID- 19 which are the vast majority.  
+
+<|ref|>text<|/ref|><|det|>[[115, 604, 775, 620]]<|/det|>
+3- I think that throughout the paper, superinfection is more appropriate than coinfection  
+
+<|ref|>text<|/ref|><|det|>[[115, 636, 820, 668]]<|/det|>
+4- Did the authors sequenced their SARS- CoV- 2 stocks? This is important in view of the furing cleavage deletions appearing as a consequence of passage in Vero's. This should be indicated  
+
+<|ref|>text<|/ref|><|det|>[[112, 683, 866, 700]]<|/det|>
+5- it would be easier for the reader if the color codes of the graphs were maintained in all the figures
+
+<--- Page Split --->
+<|ref|>table<|/ref|><|det|>[[113, 88, 883, 860]]<|/det|>
+
+<table><tr><td>REVIEWERS' COMMENTS</td><td>Answer</td></tr><tr><td>REVIEWERS' COMMENTS<br>Reviewer #1 (Remarks to the Author):<br>The authors have addressed my concerns. The<br>additional experiments have strengthened the<br>manuscript.</td><td>We wish to thank the Reviewer for his valuable<br>remarks. We also believe that the additions made<br>following his remarks strengthened the<br>manuscript.</td></tr><tr><td>REVIEWERS' COMMENTS<br>Reviewer #2 (Remarks to the Author):<br>The paper by Achdout et al. studies the effect of<br>SARS-CoV-2/influenza superinfection in a<br>transgenic mouse model expressing human ACE2<br>under the K18 promoter. The data obtained in<br>this model is compared with that obtained in<br>C57BL/6 mice in which ACE2 is delivery via<br>Adenovirus infection. The study indicates that<br>superinfection results in severe respiratory<br>disease. This is precluded by previous immunity to flu but not SARS-CoV-2, and this immunity is<br>antibody dependent.<br>This is a great study, well conducted and with<br>conclusions of obvious public health relevance. I only have some minor suggestions as indicated<br>below. Well done.</td><td>We wish to thank the Reviewer for his comments and remarks. Our replay for his suggestions<br>appears below.<br>The line numbers specified are for the track<br>changes version.</td></tr><tr><td>1- In the abstract (Line 27), I think antibody-dependent is more adequate than humoral-dependent. Alternatively 'dependent on humoral immunity'.</td><td>As suggested, changed to antibody-dependent.</td></tr><tr><td>2- Why do the authors conclude that 'in the<br>human population, coinfection is most likely to<br>occur during the asymptomatic period'? This for sure does not apply for mild cases of COVID-19<br>which are the vast majority.</td><td>We are sorry for the misunderstanding. We<br>added clarification for the meaning of<br>asymptomatic period of influenza (lines 112-113).</td></tr><tr><td>3- I think that throughout the paper,<br>superinfection is more appropriate than<br>coinfection</td><td>We address the terms coinfection and<br>superinfection in lines 81-84.<br>("While the terms coinfection and superinfection are often used interchangeably, the use of<br>'coinfection' here refers to a sequential infection with 2 viruses within a very short time, with the<br>second infection occurring prior to the<br>elimination of the first virus.").</td></tr></table>
+
+<--- Page Split --->
+<|ref|>table<|/ref|><|det|>[[114, 88, 881, 210]]<|/det|>
+
+<table><tr><td>4- Did the authors sequenced their SARS-CoV-2 stocks? This is important in view of the furing cleavage deletions appearing as a consequence of passage in Vero's. This should be indicated</td><td>Yes. GISAID submission: EPI_ISL_3838266. No deletion in the furin cleavage site is noted. Data was added to the text, lines 262-264.</td></tr><tr><td>5- it would be easier for the reader if the color codes of the graphs were maintained in all the figures</td><td>The color codes of the groups are maintain in all figures when possible.</td></tr></table>
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File.__dbe273a18746a45e22cbb58b83d5a481799589906aa526af76d8caf5b49ae4a6/images_list.json

@@ -0,0 +1,92 @@
+[
+  {
+    "type": "image",
+    "img_path": "images/Figure_1D.jpg",
+    "caption": "Figure S3. The true positive rate increases with the number of convergent substitutions. Simulated data analyzed were from the Convergent scenario of Fig. 1D.",
+    "footnote": [],
+    "bbox": [
+      [
+        191,
+        175,
+        666,
+        291
+      ]
+    ],
+    "page_idx": 8
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_3.jpg",
+    "caption": "Figure 3. Joint convergence of gene expression patterns and protein sequences. (A) Comparison of convergent branch pairs obtained by different methods in the vertebrate dataset. Branch pairs with \\(O_{c}^{B} \\geq 3.0\\) and \\(O_{c}^{B} \\geq 3.0\\) were analyzed. The Venn diagram on the left shows the extent of overlap between the top \\(1\\%\\) convergent branch pairs. The violin plot in the middle shows the lower bootstrap support of the parental branches of the convergent branch pairs. The boxplot on the right compares the rate of synonymous convergence \\((dS_{C})\\) . The stochastic equality of data was tested by a two-sided Brunner–Munzel test (Brunner and Munzel, 2000). (B) A schematic illustration of convergent expression evolution modeled with multi-optima Ornstein-Uhlenbeck process. (C) Venn diagrams showing the extent of overlap between protein and",
+    "footnote": [],
+    "bbox": [
+      [
+        191,
+        174,
+        680,
+        660
+      ]
+    ],
+    "page_idx": 18
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_0.jpg",
+    "caption": "Change in Supplementary Figure:",
+    "footnote": [],
+    "bbox": [
+      [
+        190,
+        200,
+        655,
+        563
+      ]
+    ],
+    "page_idx": 23
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_1.jpg",
+    "caption": "Fig. S10 (continued)",
+    "footnote": [],
+    "bbox": [
+      [
+        187,
+        170,
+        675,
+        666
+      ]
+    ],
+    "page_idx": 24
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_2.jpg",
+    "caption": "Figure S13. Analysis of highly repetitive convergence in 21 animal genomes. (A) Numbers of orthogroups and branch combinations in the higher-order analysis. (B) Falsely detected protein convergence in OG0000136 at \\(K = 10\\) . Combinatorial substitutions are clustered to a limited range of the protein sequence. To the right, all alternative transcripts in human genes annotated in Ensembl are shown. Alternative transcripts from multiple genes harbor the same set of variations, likely generating false convergence. (C) Protein convergence in OG00000062 at \\(K = 10\\) . No evidence was found for shared variations among alternative transcripts. (D) Convergent branch combinations in the OG00000062 tree at \\(K = 6\\) . (E) Positions of higher-order convergent substitutions in the structure of a protocadherin ectodomain (PDB ID: 6VG4) (Harrison et al., 2020).",
+    "footnote": [],
+    "bbox": [
+      [
+        193,
+        175,
+        672,
+        580
+      ]
+    ],
+    "page_idx": 27
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_3.jpg",
+    "caption": "Fig. S13 (continued)",
+    "footnote": [],
+    "bbox": [
+      [
+        191,
+        175,
+        625,
+        640
+      ]
+    ],
+    "page_idx": 28
+  }
+]

peer_reviews/supplementary_0_Peer Review File.__e2e00dd9a6e4e53214fb860ff913a5dbb1f13fad9c449e99635c505512bb7ee8/images_list.json

@@ -0,0 +1,32 @@
+[
+  {
+    "type": "image",
+    "img_path": "images/Figure_1.jpg",
+    "caption": "Figure 1. STIX query resolution depends on the insert size distribution of the cohort under consideration. By shifting the query coordinates of 28,593 deletions called by 1KG up and downstream in 50bp increments, and recalculating the number of samples found to still have evidence for the SV, we find the 1KG STIX queries have a resolution about about 400bp.",
+    "footnote": [],
+    "bbox": [
+      [
+        355,
+        185,
+        636,
+        387
+      ]
+    ],
+    "page_idx": 12
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_0.jpg",
+    "caption": "Figure. The density of VEP annotation types in tumor tissue calls, somatic calls that incorporated normal tissue, and tumor calls filtered using the 1KG STIX index. SVs that are predicted to affect gene function were annotated as HIGH, and those that don't were annotated as MODIFIER. The average per-sample number of SVs annotated as MODIFIER and HIGH in the tumor, tumor/normal, and STIX-filtered calls were 735.0 and 47.5, 28.6 and 22.8, and 10.0 and 10.5, respectively.",
+    "footnote": [],
+    "bbox": [
+      [
+        366,
+        181,
+        630,
+        425
+      ]
+    ],
+    "page_idx": 31
+  }
+]

peer_reviews/supplementary_0_Peer Review File__00c2b94550129fb8f084bb495841a196a9f5afe6d5c14e29f461a9abeaa8a98e/images_list.json

@@ -0,0 +1 @@
+[]

peer_reviews/supplementary_0_Peer Review File__00c2b94550129fb8f084bb495841a196a9f5afe6d5c14e29f461a9abeaa8a98e/supplementary_0_Peer Review File__00c2b94550129fb8f084bb495841a196a9f5afe6d5c14e29f461a9abeaa8a98e.mmd

@@ -0,0 +1,353 @@
+
+# nature portfolio  
+
+Peer Review File  
+
+# Enhancing Combinatorial Optimization with Classical and Quantum Generative Models  
+
+![PLACEHOLDER_0_0]
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+## REVIEWER COMMENTS  
+
+## Reviewer #1 (Remarks to the Author):  
+
+The authors propose a framework that incorporates quantum or classical generative models to tackle optimization problems.  
+
+The main focus of the work is problems related to portfolio optimization and a quantum- inspired instantiation of the framework.  
+
+The paper is generally well- written and presents an interesting approach and results. However in its present form I have difficulty assessing the significance, scope, and impact of the results, which are paramount to my recommendation.  
+
+Hence at present I recommend additional revision of the manuscript. I provide some further comments below.  
+
+## Comments:  
+
+- What is scope and impact of the results? A general framework is described, but what specific instantiations and cases should we expect to provide greatest advantage? (I clearly see, at minimum, a classical-ML approach to portfolio optimization, and would appreciate more clarity regarding more general settings)  
+
+- Results are shown for portfolio optimization, but wasn't clear to what other important problems the approach is best suited for or should be expected to be advantageous.  
+
+- In particular, how common are problems where "cost function evaluation can be very expensive"?  
+
+Can this be quantified? For instance, what is the resource tradeoff between query cost and runtime? Is your approach still competitive if this is relaxed?  
+
+- The results rely on a quantum inspired method, what evidence or support do we have that
+
+<--- Page Split --->
+
+using actual quantum devices will improve further? How should we expect GEO to perform for other quantum or classical models?  
+
+- For the quantum case, is the proposed method not still severely limited by the underlying quantum hardware and quantum model resource requirements? (in contrast, for instance, to problem decomposition approaches where the goal may be to accommodate fewer quantum resources)  
+
+- Regarding "GTS" optimizer in Section III.C and the subsequent reported results for it, I was unclear what was meant. Each of G,T,S refers to an independent optimization strategy, are you running each and reporting the best?  
+
+- Similarly, I was left with follow up questions such as to what degree were these (G,T,S) as well as the other nine "leading SOTA optimizers" tailored/optimized for the problem at hand? (In regards to truly 'fair' comparison in the reported numbers)  
+
+- Table I is not so easy to read with the many entries. Is it worth including first 4 optimizers here? They don't seem competitive (no to mention their columns are mostly "-"s)  
+
+- RCABC appeared to preform comparably to TN-GEO, it would be nice to see more discussion of this or even a more detailed comparison  
+
+## Reviewer #2 (Remarks to the Author):  
+
+\*\* Key results \*\*  
+
+This paper proposes to use a quantum inspired generative model to help more efficiently explore the space of feasible solutions for combinatorial optimization problems. The generative model is used to sample new candidate solutions: from a set of already explored solutions with their associated cost, the generative model is trained to learn a distribution over the solution space for which the probability of each seen solution is proportional to their associated cost, thus potentially making it possible to sample new promising candidate
+
+<--- Page Split --->
+
+solutions. The authors focus on using a quantum inspired generative model based on tensor networks (matrix product states), which have been previously introduced in [15].  
+
+The proposed approach is evaluated on the task of portfolio optimization and compared with state of the art optimization algorithms for this task. The experiments reveal that the approach is competitive with these state of the art solvers (which have been fine tuned for decades), even outperforming some of them on this particular task.  
+
+\*\* Validity \*\*  
+
+The results presented in the paper are valid. Both the methodology and the experiment setup is sound.  
+
+\*\* Data & methodology \*\*  
+
+The paper is overall clearly written and the experiments demonstrate well the potential benefits and usefulness of the method. One concern that I have is that the advantage of using a quantum approach is not clearly demonstrated. In particular, it is not clear how the fact that quantum inspired models where chosen for the generative part is key to obtaining the experimental results. At the very least I believe the proposed approach should be compared with replacing the MPS model with a simple HMM learned using the Baum- Welch (i.e. EM) algorithm. Other generative models should be considered as baselines as well (e.g. a simple RNN trained using backpropagation through time or a more complex model such as NADE [Uria et al]). It may be the case that replacing the MPS model by such an alternative non- quantum inspired model would lead to similar result. To sum up, I believe the authors should experimentally investigate and discuss more in depth to which extent the quantum part of their approach is necessary and beneficial.  
+
+Uria, Benigno, et al. "Neural autoregressive distribution estimation." The Journal of Machine Learning Research 17.1 (2016): 7184- 7220.  
+
+\*\* Appropriate use of statistics and treatment of uncertainties \*\*
+
+<--- Page Split --->
+
+Yes, the results are reported appropriately using classical statistical tools and treatment of uncertainties.  
+
+\*\* Conclusions: Do you find that the conclusions and data interpretation are robust, valid and reliable? \*\*  
+
+Overall the conclusions and interpretation of the experimental results are robust and reliable. Though, as I mentioned above, I believe there is a set of experiments missing which would demonstrate more the relevance of a quantum approach to the generative model part of the proposed method.  
+
+\*\* References \*\*  
+
+The manuscript references previous literature appropriately.  
+
+\*\* Clarity and context \*\*  
+
+The paper is very well written and structured, and easy to follow for someone familiar with tensor network models.  
+
+\*\* Suggested improvements \*\*  
+
+- Include an experiment to compare the proposed approach with non-quantum generative models  
+
+- It could be a nice addition to have experiments on another combinatorial optimization problem than portfolio optimization.  
+
+- Related to the previous point, the authors may consider presenting their approach in a more general context rather than specifically for the portfolio optimization problem. As I understood it, the proposed approach can be applied to many different kind of optimization
+
+<--- Page Split --->
+
+problems but the current presentation can suggest that the method is tailored specifically for portfolio optimization. A more general presentation of the method, as well as a clear explanation of the different kind of problems the approach can be applied to, could make for a more impactful paper (by reaching a wider audience). For example, can the approach be applied to any MIP?
+
+<--- Page Split --->
+
+## Reply to Reviewer #1:  
+
+The authors propose a framework that incorporates quantum or classical generative models to tackle optimization problems. The main focus of the work is problems related to portfolio optimization and a quantum- inspired instantiation of the framework.  
+
+The paper is generally well- written and presents an interesting approach and results. However in its present form I have difficulty assessing the significance, scope, and impact of the results, which are paramount to my recommendation.  
+
+Hence at present I recommend additional revision of the manuscript. I provide some further comments below.  
+
+Reply: We thank the reviewer the careful revision our manuscript, and for the positive feedback about the current form of the manuscript. We address each of the questions below with the hope this helps to clarify the scope and significance of our work.  
+
+- What is scope and impact of the results? A general framework is described, but what specific instantiations and cases should we expect to provide greatest advantage? (I clearly see, at minimum, a classical-ML approach to portfolio optimization, and would appreciate more clarity regarding more general settings)  
+
+- Results are shown for portfolio optimization, but wasn't clear to what other important problems the approach is best suited for or should be expected to be advantageous.  
+
+Reply: We would like to mention that beyond our proposal being a "a classical- ML approach to portfolio optimization", the baseline of our contribution is a ML framework that leverages classical, quantum- inspired, or quantum generative models for solving any combinatorial problems. Although not a requirement for using the framework, having the possibility to easily generate bitstrings in the valid solution space (as the case studied here, i.e., bitstrings with a specified cardinality) can be useful since these synthetic data points can be passed as a warm start for the generative model. This in turn can help guide the trained generative model towards the right support of the probability distribution to be explored.  
+
+Using ML techniques for combinatorial problems is not a straightforward and rather a new research domain. More specifically, although other reinforcement learning and deep learning approaches had been proposed (see e.g., Ref. [1] and [2]), to the best of our knowledge our work is the first proposal using generative models for combinatorial optimization tasks. This is also the first proposal flexible enough to easily incorporate and explore quantum- inspired or quantum generative models. At first, we thought of adapting or enhancing existing reinforcement learning and deep learning proposals with quantum models, but this was not practical or at least not obvious how to do it. Instead, we noticed using generative models could be a very promising approach, given its prominence as candidates for practical quantum advantage. But this required to go beyond the incremental adaptation and to design the algorithm in a completely different framework, which resulted in the GEO proposal here.
+
+<--- Page Split --->
+
+Here are more general settings that make this approach advantageous over other available solvers:  
+
+1) The entire approach is data-driven: what this implies is that the more data is available, either from previous attempts to solve the problem with other state-of-the-art solvers, the better the performance is expected. In the example of GEO as a booster we used data explored by Simulated Annealing (SA) but if we had previous observations from any or many other solvers, we could combine it and give it as a starting point to GEO.  
+
+2) It leverages the power of generative models: The essence of the solver is that it is aiming to unveil non-obvious structure in the data, and once it has captured those correlations, it suggests new outstanding candidates with features similar to the top ones seen until that iteration phase.  
+
+3) the model is cost function agnostic, i.e., it is a black-box solver. This is paramount since any cost function can be solved with our approach. Most of the proposals for quantum or quantum inspired optimization require the cost function of the problem to be mapped to a quadratic or polynomial expression. This black-box feature open the possibility to tackle any discrete optimization problem, regardless of how complicated or expensive it is to compute the cost function. This is possible within GEO since the only information passed to the generative model are the bitstrings who have been explored and their respective cost value.  
+
+4) Nothing is special about the portfolio optimization problem. This follows from the item above. The main motivation for selecting this specific instantiation of portfolio optimization was the availability of concrete benchmarks and an extensive literature of solvers which have been fine-tuned for over the past decades. Every time a new metaheuristic is proposed, chances are this cardinality-constrained portfolio optimization problem is used to benchmark. Other recent independent works have considered other real-world applications of GEO. For example, in Ref. [3], the authors considered an industrial case related to a floor planning NP-hard problem. This black-box feature is one of the most prominent ones which render our approach advantageous compared to other quantum heuristics, such as the quantum approximate optimization algorithm (QAOA), which relies on the cost function to be a polynomial in terms of the binary variables.  
+
+Although the items above only point to features which make the approach valuable and different when compared to optimizers proposed to date, it is still to be explored if similar advantages can be observed in other family of important problems. Although this question is an active research question, there has been some progress in understanding which datasets or problems might benefit the most. In a recent publication, (see Ref. [4]), we showed how the search space of problems with equality constrains, can be represented efficiently in quantum- inspired generative models, but this is not the case for deep- neural- network- based generative models. Besides the demonstration presented here pointing that quantum and quantum- inspired models are a promising route to be explored, this more recent paper poses a more concrete differentiator between the capabilities of quantum- inspired models with respect to traditional deep learning models. These equality constraints appear in a broad family of problems, including the cardinality constrained portfolio optimization studied here, but also it extends to problems with any linear equality constrains.  
+
+Revisions to the manuscript: The new Introduction has been significantly rewritten and restructured for clarity and to indicate more explicitly these salient highlights indicated above. We also revised the Outlook section adding some material related to the recent developments towards the identification of data sets which might benefit from quantum- inspired and quantum models over state- of- the- art generative models
+
+<--- Page Split --->
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+- In particular, how common are problems where "cost function evaluation can be very expensive"? Can this be quantified? For instance, what is the resource tradeoff between query cost and runtime? Is your approach still competitive if this is relaxed?  
+
+Reply: We thank the referee for bringing this point since some clarifications can be added to the manuscript. Note that this desirable condition of having an expensive cost function applies mainly to GEO as a standalone, although it is important to note that even in that setting it is not a necessary condition. The reason is because the overhead of training the generative model might have a larger impact for that setting than in the booster mode, where the initial available data can be seen as a warm start. It can be noted that even in problems with cost function which might be quick to evaluate, as long as the problem is hard and a solution is not reached with a competitor solver, it might be still worth to use GEO as a standalone which might yield a completely different set of solutions, or even better ones, since its search strategy is completely different.  
+
+This condition of an expensive cost function was relaxed altogether in the setting of GEO as a booster (described in Sec. II A) where the comparison criteria was moved from number of cost function evaluations to runtime. In that study, we used a similar criteria as that suggested by the referee, where we used the trade off between query cost and runtime by using the more generic and practical criteria of fixed total wall clock runtime used for each of the algorithmic strategies. There, the time it takes to evaluate the cost function and the training of the generative models are considered, since these are part of the total time it takes to run each algorithm. In that comparison, between Simulated Annealing (SA) and GEO, although the training of the generative model within GEO takes longer, compared to the quick updates in SA, we still see an advantage in adopting GEO as a booster, which consisted of GEO being initialized with partial solutions obtained from SA. We show in that section that it is more efficient to change the strategy to GEO, than to insist and continue performing runs just with SA, if both algorithmic strategies are given the same total wallclock time.  
+
+Revisions to the manuscript: We have added further clarification in the new paragraph right before subsection IIA.  
+
+- The results rely on a quantum inspired method, what evidence or support do we have that using actual quantum devices will improve further? How should we expect GEO to perform for other quantum or classical models?  
+
+Reply: The question of whether a significant advantage can be obtained by using quantum devices is an active research topic and certainly needs to be explored further. One proposal to reach a more systematic and incremental enhancement from the best quantum- inspired solution to an enhanced quantum- hardware realization was recently proposed in Ref. [5]. There, one starts from the best available quantum- inspired tensor- network solution and maps it to a quantum circuit. This can be subsequently modified by adding gates beyond those from the decomposition to increase the plausible correlations beyond those accessible with the quantum- inspired tensor- network- based solution. The access to longer- range correlations enhances, in turn, the expressibility of the quantum generative model while taking it beyond the capabilities of classical simulation. In that work, the specific case of generative models was illustrated, and therefore, these novel decomposition techniques can be directly applied to extend the capabilities of TN- GEO explored here, and, as the technologies mature and the level of noise is reduced, explore these enhanced models directly on quantum devices. Additionally, in Ref. [6] a comparison of quantum
+
+<--- Page Split --->
+
+generative models with state- of- the- art classical generative models was presented, and the results were very encouraging in the data sets studied.  
+
+To the comment of the performance of TN- GEO against other classical models, and as part of the request from the second referee, we added a classical version of GEO, based on the Neural Autoregressive Density Estimation (NADE) model [7]. In this revised version of the manuscript, we showed that although NADE- GEO is competitive with the early solvers (from about a decade ago), it is underperforming compared to TN- GEO and the other state- of- the- art solvers within the past five years. Although we don't expect the results to be universal, in this specific TN- GEO versus NADE- GEO comparison we still see an advantage for the quantum- inspired over the classical generative neural network model, with the advantage as well that the TN- base model has less hyperparameters to fine tune. Each of these comparisons is an extensive amount of work, and we feel that despite being beyond the scope of this particular work, our work opens the possibility to explore quantum and quantum- inspired generative models toward solving arbitrary combinatorial optimization, and as a concrete framework to study quantitatively practical quantum advantage with future quantum technologies (see e.g., Ref. [6] whose framework is inspired in this work)  
+
+Revisions to the manuscript: We complemented the Outlook where we had already mentioned about the opportunities and challenges that arise from thinking of an implementation in quantum devices. In particular, we added some of the discussion above around the new references that appeared after we submitted our work. We have also expanded the Results & Discussion subsection, Sec. IIC, with the respective discussion related to NADE- GEO and TN- GEO.  
+
+- For the quantum case, is the proposed method not still severely limited by the underlying quantum hardware and quantum model resource requirements? (in contrast, for instance, to problem decomposition approaches where the goal may be to accommodate fewer quantum resources)  
+
+Reply: If we correctly understand the proposal from the Reviewer, the reviewer is thinking of problem decomposition techniques which divide the problem into many smaller subproblems to accommodate the number of qubits in near- term technologies. From a practical point of view, for these techniques to be promising for quantum technologies, one still needs the subproblems still be intractable or hard to solve by conventional classical solvers, otherwise, it would not be worth to submit them to a quantum hardware. If the instances happen to be indeed intractable, then GEO can still be used to solve these subproblems, since in the cases of decomposition techniques we are familiar with, it is still required that solutions to the subproblem needs to be gathered before they are combined or used to solve the larger problem (e. g., see Refs. [8], [9], and [10] below and that have been added to the revised manuscript). In other words, we don't see it necessarily as one strategy versus the other, since we think of GEO as a standalone strategy to solving hard optimization problems or subproblems. It is important to note that, with now gate- based devices reaching the level of hundreds of qubits, such techniques and demonstrations can become more relevant. But we can see how GEO can be incorporated as well in the solution of the smaller but intractable partitions which are sent to these quantum devices.  
+
+To the question of the limitation of quantum hardware resources, not only in terms of number of qubits, but also in terms of connectivity, this is one is an important one and we will be addressing that in a theoretical and experimental ongoing work in our team. In that study, noise is also factored as part of the experimental demonstration.
+
+<--- Page Split --->
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+Revisions to the manuscript: We have complemented the Outlook addressing both, the potential interplay with other decomposition techniques, and the open questions and challenges related to hardware limitations.  
+
+- Regarding "GTS" optimizer in Section III.C and the subsequent reported results for it, I was unclear what was meant. Each of G,T,S refers to an independent optimization strategy, are you running each and reporting the best?  
+
+- Similarly, I was left with follow up questions such as to what degree were these (G,T,S) as well as the other nine "leading SOTA optimizers" tailored/optimized for the problem at hand? (In regards to truly 'fair' comparison in the reported numbers)  
+
+Reply: It is important to note that we have not fine- tuned all the solvers ourselves. The Results section of the papers is broken into three subsections, each highlighting different features from GEO. Sec. IIA focuses on GEO as a booster and how it can build from results obtained with other solvers. Sec. IIB focuses on GEO as a standalone and compares its performance to SA and the Bayesian optimization library GPyOpt. Finally, Sec. IIC focuses on a comparison of GEO with state- of- the- art solvers. While in Secs. IIA and IIB we implemented and fine- tuned each solver, in Sec. IIC, we leverage the state- of- the- art results from nine other solvers reported in the literature in the last two decades. In the latter case, each non- GEO solver was thoroughly fine- tuned by the researchers of each reference. This portfolio optimization problem is so canonical that when a new solver is proposed, researchers can compare their results by taking the results from the new proposed solver, as long as the benchmark problems are run in identical conditions. This was one of the main motivations for us to choose this well- established benchmark problem. The "rules of the game" for reporting each market index and performance indicator are reported in Appendix A 2. In contrast, for the other two subsections, the criteria of evaluation are different, and it emphasizes the performance of GEO when one imposes a limit on the total wall- clock time (Sec. IIA) and when there is a limited number of calls to the cost function (Sec. IIB). The latter is a potential scenario when the bottleneck or expensive step is the cost function evaluation itself (e.g., as it is the case of drug discovery where each evaluation (each candidate molecule) might require synthesis in the lab and an expensive and long process towards its Food and Drug Administration (FDA) approval).  
+
+To the specific question of (G,T,S) the Reviewer is correct that the authors from the original reference back in 2000 ran each strategy (Genetic algorithms, Tabu search and Simulated annealing) and reported their best results for each. Since this paper was one of the first ones adopting the current metrics and benchmarking procedure, when the community started creating more sophisticated metaheuristic strategies, they continue referring to the results in this paper as GTS, implying the values correspond to the best result from either G, T, or S  
+
+Revisions to the manuscript: To clarify this to the reader, we have emphasized further in Sec. IIC that the results from the state- of- the- art competitors have been independently fine- tuned and are available in the literature. We have also added more context at the beginning of the Results section to contextualize all the different results subsections and strategies presented in the paper.
+
+<--- Page Split --->
+
+- Table I is not so easy to read with the many entries. Is it worth including first 4 optimizers here? They don't seem competitive (no to mention their columns are mostly ".")
+
+**Reply:** We have followed the Reviewer's suggestion and we have simplified the table accordingly (please see new Table I). The main reason for including all the solvers was to follow the format from previous papers which compared to these early metaheuristics from the early 2000's. Since this cardinality constrained problem has been used for benchmarking solved since the late 1990s, these were the solvers which have been competitive since the last two decades or so ago. As time has passed, and in particular in the last decade, other researchers have proposed other figure of merits to assess the performance of the solvers. Since these were not known in the first papers, they have been conventionally included in the most recent papers with the '- ' since the data is not available. Others have adopted these other metrics as valuable and that is the reason the more recent papers all evaluate them, and we decided to include them here as well.
+
+The original intention from having the complete table was to have the whole spectrum of solvers and help position our quantum-inspired GEO in the historical progress of optimizers for this specific task, clearly showing GEO outperforms these solvers from the early 2000's up to a decade ago, and being on par with the current state-of-the-art optimizers. Note that with the suggestion from the other Reviewer to include a classical version of GEO, called NADE-GEO in the revised version, we can see that NADE-GEO is competitive with solvers from about a decade ago, but not as competitive as TN-GEO or the new solvers.
+
+**Revisions** **to** **the** **manuscript.** We have collapsed the table appearing in the main text as suggested by the reviewer. The original table, expanded now with the NADE-GEO results, has been moved to Appendix C.
+
+-RCABC appeared to preform comparably to TN-GEO, it would be nice to see more discussion of this or even a more detailed comparison
+
+**Reply:** We have expanded the discussion on RCABC at the end of the Results section (Sec. IIC). Note in the current version we have replaced the name RCABC to ABC-HP to match the name of the best performing variant in that 2021 paper cited, and which corresponds to the results reported here.
+
+# References:
+
+[1] Bello, I., Pham, H., Le, Q.V., Norouzi, M. and Bengio, S., Neural combinatorial optimization with reinforcement learning. arXiv preprint arXiv:1611.09940 (2016).
+
+[2] Bengio, Y., Lodi, A. and Prouvost, A., Machine Learning for Combinatorial Optimization: a Methodological Tour d'Horizon. CoRR abs/1811.06128 (2018). arXiv preprint arXiv:1811.06128 (2018).
+
+[3] Banner, W.P., Hadiashar, S.B., Mazur, G., Menke, T., Ziolkowski, M., Kennedy, K., Romero, J., Cao,Y., Grover, J.A. and Oliver, W.D., Quantum Inspired Optimization for Industrial Scale Problems. arXiv preprint arXiv:2305.02179 (2023).
+
+[4] Lopez-Piqueres, J., Chen, J. and Perdomo-Ortiz, A., Symmetric tensor networks for generative modeling and constrained combinatorial optimization. Machine Learning: Science and Technology.4,035009 (2023).
+
+<--- Page Split --->
+
+[5] Rudolph, M.S., Miller, J., Motlagh, D., Chen, J., Acharya, A. and Perdomo-Ortiz, A., Synergy between quantum circuits and tensor networks: Short- cutting the race to practical quantum advantage. arXiv preprint arXiv:2208.13673 (2022).  
+
+[6] Hibat- Allah, M., Mauri, M., Carrasquilla, J. and Perdomo- Ortiz, A., A Framework for Demonstrating Practical Quantum Advantage: Racing Quantum against Classical Generative Models. arXiv preprint arXiv:2303.15626 (2023).  
+
+[7] Uria, B., Côté, M.A., Gregor, K., Murray, I. and Larochelle, H., Neural autoregressive distribution estimation. The Journal of Machine Learning Research, 17(1), pp.7184- 7220 (2016).  
+
+[8] Ponce, M., Herrman, R., Lotshaw, P.C., Powers, S., Siopsis, G., Humble, T. and Ostrowski, J., Graph decomposition techniques for solving combinatorial optimization problems with variational quantum algorithms. arXiv preprint arXiv:2306.00494 (2023).  
+
+[9] Ushijima- Mwesigwa, H., Shaydulin, R., Negre, C.F., Mniszewski, S.M., Alexeev, Y. and Safro, I., Multilevel combinatorial optimization across quantum architectures. ACM Transactions on Quantum Computing, 2(1), pp.1- 29 (2021).  
+
+[10] Zhou, Z., Du, Y., Tian, X. and Tao, D., QAOA- in- QAOA: solving large- scale MaxCut problems on small quantum machines. Physical Review Applied, 19(2), p.024027 (2023).
+
+<--- Page Split --->
+
+## Reply to Reviewer #2  
+
+\*\* Key results \*\*  
+
+This paper proposes to use a quantum inspired generative model to help more efficiently explore the space of feasible solutions for combinatorial optimization problems. The generative model is used to sample new candidate solutions: from a set of already explored solutions with their associated cost, the generative model is trained to learn a distribution over the solution space for which the probability of each seen solution is proportional to their associated cost, thus potentially making it possible to sample new promising candidate solutions. The authors focus on using a quantum inspired generative model based on tensor networks (matrix product states), which have been previously introduced in [15].  
+
+The proposed approach is evaluated on the task of portfolio optimization and compared with state of the art optimization algorithms for this task. The experiments reveal that the approach is competitive with these state of the art solvers (which have been fine tuned for decades), even outperforming some of them on this particular task.  
+
+\*\* Validity \*\*  
+
+The results presented in the paper are valid. Both the methodology and the experiment setup is sound.  
+
+\*\* Data & methodology \*\*  
+
+The paper is overall clearly written and the experiments demonstrate well the potential benefits and usefulness of the method.  
+
+Reply: We thank the referee for the very positive feedback on our proposed framework. Below we address the concerns raised in the comments.  
+
+One concern that I have is that the advantage of using a quantum approach is not clearly demonstrated. In particular, it is not clear how the fact that quantum inspired models where chosen for the generative part is key to obtaining the experimental results. At the very least I believe the proposed approach should be compared with replacing the MPS model with a simple HMM learned using the Baum- Welch (i.e. EM) algorithm. Other generative models should be considered as baselines as well (e.g. a simple RNN trained using backpropagation through time or a more complex model such as NADE [Uria et al]). It may be the case that replacing the MPS model by such an alternative non- quantum inspired model would lead to similar result. To sum up, I believe the authors should experimentally investigate and discuss more in depth to which extent the quantum part of their approach is necessary and beneficial.  
+
+Uria, Benigno, et al. "Neural autoregressive distribution estimation." The Journal of Machine Learning Research 17.1 (2016): 7184- 7220.  
+
+Reply: We agree with the Reviewer this is a critical point that deserves further study since it is in general an open question. One of the highlights for GEO is the flexibility of swapping classical, quantum inspired, and quantum models in the future as quantum technologies mature. We have followed the suggestion from the Reviewer and implemented NADE, which is the most complex model of the ones suggested. In the new results summarized in Table V (computed from the explicit values in Table III), it can be seen that although NADE- GEO is a competitive solver, it is only statistically on par with the solvers which were the best performers up to about a decade ago (GTS, IPSO, IPSO- SA, and PBILD), but statistically different from its quantum- inspired version (TN- GEO), and the solvers proposed from 2015
+
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+and onwards (GRASP, ABCFEIT, AAG, VNSQ, and ABC- HP). Although this does not constitute a proof in any way of the superiority of quantum or quantum- inspired models, this corresponds to the experimental investigation which hints to the value and promising directions we highlight in this work from this flexible framework.  
+
+To the milestone of understanding when quantum or quantum- inspired models could be key to obtaining an advantage over classical models, several subsequent publications have attempted to address this point. For example, in Ref. [1], we have recently published a version of TN- GEO which uses symmetries which are relatively easy in these quantum- inspired TN models. We showed how the search space of problems with equality constraints can be represented efficiently in quantum- inspired generative models, but this is not necessarily the case for classical deep- neural- network- based generative models due to their non- linear activation units. That recent work poses a more concrete differentiator between the capabilities of quantum- inspired models with respect to traditional deep learning generative models. In general, the question of practical quantum advantage in quantum machine learning is still wide open, but in this recent work, Ref. [2] below, the proposed framework there leverages our GEO proposal to establish a clear- cut criteria from optimization problems to concretely test the performance of both classical and quantum generative modeling proposals. From a practical point of view, and in case of real and relevant real- world applications, this question would need to be approached on a case- by- case basis, but that subsequent paper proposes a framework where the performance of quantum generative models can be tested, following similar metrics inspired from this work.  
+
+\*\*Appropriate use of statistics and treatment of uncertainties \*\*Yes, the results are reported appropriately using classical statistical tools and treatment of uncertainties.  
+
+Reply: We thank the referee for the positive feedback on our statistical analysis.  
+
+\*\*Conclusions: Do you find that the conclusions and data interpretation are robust, valid and reliable? \*\*  
+
+Overall the conclusions and interpretation of the experimental results are robust and reliable. Though, as I mentioned above, I believe there is a set of experiments missing which would demonstrate more the relevance of a quantum approach to the generative model part of the proposed method.  
+
+Reply: We thank the referee for the positive feedback on our overall conclusions. We hope the new numerical experiments, including the simulations of NADE- GEO strengthens our claims around the potential benefit of quantum inspired generative models, and in general, of quantum models as an alternative to be explored in the near term.  
+
+\*\*References \*\*The manuscript references previous literature appropriately.  
+
+Reply: We thank the referee for the positive feedback on our literature coverage.  
+
+\*\*Clarity and context \*\*The paper is very well written and structured, and easy to follow for someone familiar with tensor network models.
+
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+
+Reply: We thank the referee for the positive feedback on the content and the structure of the paper.  
+
+\*\* Suggested improvements \*\*  
+
+- Include an experiment to compare the proposed approach with non-quantum generative models  
+
+Reply: Following the advice from the referee, we have implemented NADE- GEO and the comparison with TN- GEO and the other SOTA solvers are included throughout Tables I- V. As previously mentioned, the classical ML model is competitive in the sense that is comparable with models from about a decade ago. But still underperforming TN- GEO, which is on par with the state- of- the- art solvers for this application. This is also included in the new summary Table II in the main text, where it can be seen the superior performance of TN- GEO over NADE- GEO in this specific application.  
+
+- It could be a nice addition to have experiments on another combinatorial optimization problem than portfolio optimization.  
+
+- Related to the previous point, the authors may consider presenting their approach in a more general context rather than specifically for the portfolio optimization problem. As I understood it, the proposed approach can be applied to many different kind of optimization problems but the current presentation can suggest that the method is tailored specifically for portfolio optimization. A more general presentation of the method, as well as a clear explanation of the different kind of problems the approach can be applied to, could make for a more impactful paper (by reaching a wider audience). For example, can the approach be applied to any MIP?  
+
+Reply: We agree with the reviewer that the current presentation hints to GEO as a solution tailored only for the portfolio optimization problem, where in reality, as the referee mentioned, GEO is more in the category of a generic solver. Although we had mentioned in a couple of places its black- box nature, many readers might not immediately associate this concept with a solver that can deal with any cost function, and therefore any combinatorial problem. This black- box feature is one of the most prominent ones which render our approach advantageous compared to other quantum heuristics, such as the quantum approximate optimization algorithm (QAOA), which relies on the cost function to be a polynomial in terms of the binary variables. To mitigate missing these essential points raised by the referee, the introduction has been significantly revised and restructured adding clarity to the main features of our framework.  
+
+Although we did not explore explicitly in this work the case of problems containing both discrete and continuous variables (e.g., MIP) or only continuous variables, we discussed in the Outlook how an approach as the one in Ref. [3] could allow to generalize to these mixed variable cases by using such hybrid quantum- classical generative models within GEO. In a recent publication, Ref. [4], our team addressed this question of how to treat generative models with continuous variables (or a mixture of discrete and continuous) directly with TN models, which would be directly applicable to MIP and other related problems.  
+
+To the question of including other applications, the main motivation for selecting this specific instantiation of portfolio optimization was the availability of concrete benchmarks and an extensive literature of solvers which have been fine tuned for over the past decades. In particular, we wanted to make sure we could make stronger claims in a well- established benchmark problem, and assess in this
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+way where this new quantum- inspired solution TN- GEO fits within the suite of SOTA algorithms. Although this could be done for other well- known benchmarking problems, we felt the paper was extensive enough since it presents the introduction of this new framework plus demonstrating some of its highlights reflected in the three Results subsections: 1) the possibility to leverage data observed with other solvers (Sec. IIA), 2) its high performance in the regime of very limited calls to the cost function evaluation (Sec. IIB) and 3) its performance compared to SOTA algorithms under the rules established by those specific benchmarks (Sec. IIC). Although we considered extending to other application domains is outside of the scope of this work, other recent independent works have considered other real- world applications of GEO. For example, in Ref. [5], the authors considered an industrial case related to a floor planning NP- hard problem. In Ref. [6] the authors considered quantum- inspired generative models similar to the ones we propose here, to explore the solution space of candidates in molecular discovery. We hope the referee considers as well that the work is complete as is, given the new developments supporting the range of applications of our work.  
+
+Revisions to the manuscript: We hope the new Introduction brings more clarity to the essential points raised by the referee. To emphasize the point from the referee that even MIP or problems with continuous variables could be addressed within GEO, we have added some comments in the Outlook about recent work extending and addressing this challenge, and how it fits within our framework.  
+
+## References:  
+
+[1] Lopez- Piqueres, J., Chen, J. and Perdomo- Ortiz, A., Symmetric tensor networks for generative modeling and constrained combinatorial optimization. Machine Learning: Science and Technology. 4 035009 (2023)  
+
+[2] Hibat- Allah, M., Mauri, M., Carrasquilla, J. and Perdomo- Ortiz, A., A Framework for Demonstrating Practical Quantum Advantage: Racing Quantum against Classical Generative Models. arXiv preprint arXiv:2303.15626 (2023).  
+
+[3] Rudolph, M.S., Toussaint, N.B., Katabarwa, A., Johri, S., Peropadre, B. and Perdomo- Ortiz, A., Generation of high- resolution handwritten digits with an ion- trap quantum computer. Physical Review X, 12(3), p.031010 (2022).  
+
+[4] Meiburg, A., Chen, J., Miller, J., Tihon, R., Rabusseau, R., Perdomo- Ortiz, A. Generative Learning of Continuous Data by Tensor Networks. arXiv preprint arXiv: 2310.20498 (2023).  
+
+[5] Banner, W.P., Hadiashar, S.B., Mazur, G., Menke, T., Ziolkowski, M., Kennedy, K., Romero, J., Cao, Y., Grover, J.A. and Oliver, W.D., Quantum- Inspired Optimization for Industrial Scale Problems. arXiv preprint arXiv:2305.02179 (2023).  
+
+[6] Moussa, C., Wang, H., Araya- Polo, M., Bäck, T. and Dunjko, V., 2023. Application of quantum- inspired generative models to small molecular datasets. arXiv preprint arXiv:2304.10867 (2023).
+
+<--- Page Split --->
+
+## REVIEWERS' COMMENTS  
+
+## Reviewer #1 (Remarks to the Author):  
+
+The authors have considered and reasonably addressed the comments of the referees, resulting a significant number of changes and improvements to the manuscript. Hence I am happy to recommend for publication.  
+
+## Reviewer #2 (Remarks to the Author):  
+
+I am overall satisfied by the answers from the authors to my initial review and to the consequent revisions that have been made to the manuscript.  
+
+I find that stylistically speaking, the quality of the introduction has decreased in some of the new paragraphs added in the revision (see detailed comments below). I also give below some relatively minor stylistic comments about the new content that has been added to the revision:  
+
+p. 1, second item: "\\*either\\* from previous..." there is a missing "or" and second part of the sentence.  
+
+p. 2 first item: This open -> This opens  
+
+p. 2 The last item is stylistically very odd and reads more than a rebuttal to the first round of reviews than an actual paragraph from an introduction. More precisely, this list is presented as a list of "salient highlights" of the proposed approach; is "Nothing is special about the portfolio optimization problem" a "salient highlight" of the contribution. I understand the aim but this is done in a very clumsy way, in my opinion. I would suggest rewriting this item and rephrasing in the direction of emphasizing the "versatility" of the method, while justifying the focus on portfolio optimization.
+
+<--- Page Split --->
+
+p.2 last item: "... *as\* the NP hard problem \*as\* the workhorse..." is a bit stylistically clumsy  
+
+p.2 last item: line starting with a comma after "Ref [8]"  
+
+p.6 to be consistent, the item 10)'s sentence in the before last paragraph should end with ";" instead of ".  
+
+p.10 before last paragraph of first column: "it is to use it as" -> "is to use it as"  
+
+p.10 "DATA AVAILABILITY": "have been deposited" -> e.g. "is available"  
+
+p.14 sec 4: "introduced by Uria [37]" -> "introduced by Uria et al. [37]" "is that it models" -> "is to model"
+
+<--- Page Split --->
+
+## Reply to Reviewer #1  
+
+We thank Reviewer #1 for their feedback throughout this review process and for their recommendation to publish this significantly revised version.  
+
+## Reply to Reviewer #2  
+
+We thank Reviewer #2 for their recommendation to accept this latest version for publication. In this resubmission, we have incorporated all the stylistic changes suggested by the reviewer. We thank the reviewer for this valuable feedback throughout this review process.
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__00c2b94550129fb8f084bb495841a196a9f5afe6d5c14e29f461a9abeaa8a98e/supplementary_0_Peer Review File__00c2b94550129fb8f084bb495841a196a9f5afe6d5c14e29f461a9abeaa8a98e_det.mmd

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+<|ref|>title<|/ref|><|det|>[[61, 40, 506, 90]]<|/det|>
+# nature portfolio  
+
+<|ref|>text<|/ref|><|det|>[[67, 110, 362, 139]]<|/det|>
+Peer Review File  
+
+<|ref|>title<|/ref|><|det|>[[108, 154, 888, 210]]<|/det|>
+# Enhancing Combinatorial Optimization with Classical and Quantum Generative Models  
+
+<|ref|>image<|/ref|><|det|>[[57, 732, 240, 783]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[250, 732, 912, 784]]<|/det|>
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[118, 85, 315, 101]]<|/det|>
+## REVIEWER COMMENTS  
+
+<|ref|>sub_title<|/ref|><|det|>[[118, 136, 437, 154]]<|/det|>
+## Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 163, 876, 207]]<|/det|>
+The authors propose a framework that incorporates quantum or classical generative models to tackle optimization problems.  
+
+<|ref|>text<|/ref|><|det|>[[118, 216, 850, 260]]<|/det|>
+The main focus of the work is problems related to portfolio optimization and a quantum- inspired instantiation of the framework.  
+
+<|ref|>text<|/ref|><|det|>[[118, 292, 876, 363]]<|/det|>
+The paper is generally well- written and presents an interesting approach and results. However in its present form I have difficulty assessing the significance, scope, and impact of the results, which are paramount to my recommendation.  
+
+<|ref|>text<|/ref|><|det|>[[118, 396, 686, 440]]<|/det|>
+Hence at present I recommend additional revision of the manuscript. I provide some further comments below.  
+
+<|ref|>sub_title<|/ref|><|det|>[[118, 502, 214, 518]]<|/det|>
+## Comments:  
+
+<|ref|>text<|/ref|><|det|>[[117, 553, 841, 650]]<|/det|>
+- What is scope and impact of the results? A general framework is described, but what specific instantiations and cases should we expect to provide greatest advantage? (I clearly see, at minimum, a classical-ML approach to portfolio optimization, and would appreciate more clarity regarding more general settings)  
+
+<|ref|>text<|/ref|><|det|>[[118, 683, 835, 728]]<|/det|>
+- Results are shown for portfolio optimization, but wasn't clear to what other important problems the approach is best suited for or should be expected to be advantageous.  
+
+<|ref|>text<|/ref|><|det|>[[118, 761, 830, 805]]<|/det|>
+- In particular, how common are problems where "cost function evaluation can be very expensive"?  
+
+<|ref|>text<|/ref|><|det|>[[118, 814, 866, 858]]<|/det|>
+Can this be quantified? For instance, what is the resource tradeoff between query cost and runtime? Is your approach still competitive if this is relaxed?  
+
+<|ref|>text<|/ref|><|det|>[[115, 892, 870, 910]]<|/det|>
+- The results rely on a quantum inspired method, what evidence or support do we have that
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 84, 866, 129]]<|/det|>
+using actual quantum devices will improve further? How should we expect GEO to perform for other quantum or classical models?  
+
+<|ref|>text<|/ref|><|det|>[[117, 162, 866, 260]]<|/det|>
+- For the quantum case, is the proposed method not still severely limited by the underlying quantum hardware and quantum model resource requirements? (in contrast, for instance, to problem decomposition approaches where the goal may be to accommodate fewer quantum resources)  
+
+<|ref|>text<|/ref|><|det|>[[117, 293, 866, 364]]<|/det|>
+- Regarding "GTS" optimizer in Section III.C and the subsequent reported results for it, I was unclear what was meant. Each of G,T,S refers to an independent optimization strategy, are you running each and reporting the best?  
+
+<|ref|>text<|/ref|><|det|>[[117, 397, 860, 468]]<|/det|>
+- Similarly, I was left with follow up questions such as to what degree were these (G,T,S) as well as the other nine "leading SOTA optimizers" tailored/optimized for the problem at hand? (In regards to truly 'fair' comparison in the reported numbers)  
+
+<|ref|>text<|/ref|><|det|>[[117, 501, 860, 546]]<|/det|>
+- Table I is not so easy to read with the many entries. Is it worth including first 4 optimizers here? They don't seem competitive (no to mention their columns are mostly "-"s)  
+
+<|ref|>text<|/ref|><|det|>[[118, 580, 795, 624]]<|/det|>
+- RCABC appeared to preform comparably to TN-GEO, it would be nice to see more discussion of this or even a more detailed comparison  
+
+<|ref|>sub_title<|/ref|><|det|>[[118, 685, 438, 702]]<|/det|>
+## Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 711, 258, 726]]<|/det|>
+\*\* Key results \*\*  
+
+<|ref|>text<|/ref|><|det|>[[117, 761, 877, 911]]<|/det|>
+This paper proposes to use a quantum inspired generative model to help more efficiently explore the space of feasible solutions for combinatorial optimization problems. The generative model is used to sample new candidate solutions: from a set of already explored solutions with their associated cost, the generative model is trained to learn a distribution over the solution space for which the probability of each seen solution is proportional to their associated cost, thus potentially making it possible to sample new promising candidate
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 84, 875, 129]]<|/det|>
+solutions. The authors focus on using a quantum inspired generative model based on tensor networks (matrix product states), which have been previously introduced in [15].  
+
+<|ref|>text<|/ref|><|det|>[[118, 161, 866, 259]]<|/det|>
+The proposed approach is evaluated on the task of portfolio optimization and compared with state of the art optimization algorithms for this task. The experiments reveal that the approach is competitive with these state of the art solvers (which have been fine tuned for decades), even outperforming some of them on this particular task.  
+
+<|ref|>text<|/ref|><|det|>[[118, 292, 230, 308]]<|/det|>
+\*\* Validity \*\*  
+
+<|ref|>text<|/ref|><|det|>[[118, 344, 839, 389]]<|/det|>
+The results presented in the paper are valid. Both the methodology and the experiment setup is sound.  
+
+<|ref|>text<|/ref|><|det|>[[118, 422, 340, 439]]<|/det|>
+\*\* Data & methodology \*\*  
+
+<|ref|>text<|/ref|><|det|>[[115, 474, 880, 781]]<|/det|>
+The paper is overall clearly written and the experiments demonstrate well the potential benefits and usefulness of the method. One concern that I have is that the advantage of using a quantum approach is not clearly demonstrated. In particular, it is not clear how the fact that quantum inspired models where chosen for the generative part is key to obtaining the experimental results. At the very least I believe the proposed approach should be compared with replacing the MPS model with a simple HMM learned using the Baum- Welch (i.e. EM) algorithm. Other generative models should be considered as baselines as well (e.g. a simple RNN trained using backpropagation through time or a more complex model such as NADE [Uria et al]). It may be the case that replacing the MPS model by such an alternative non- quantum inspired model would lead to similar result. To sum up, I believe the authors should experimentally investigate and discuss more in depth to which extent the quantum part of their approach is necessary and beneficial.  
+
+<|ref|>text<|/ref|><|det|>[[118, 814, 877, 858]]<|/det|>
+Uria, Benigno, et al. "Neural autoregressive distribution estimation." The Journal of Machine Learning Research 17.1 (2016): 7184- 7220.  
+
+<|ref|>text<|/ref|><|det|>[[118, 892, 655, 909]]<|/det|>
+\*\* Appropriate use of statistics and treatment of uncertainties \*\*
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 84, 863, 128]]<|/det|>
+Yes, the results are reported appropriately using classical statistical tools and treatment of uncertainties.  
+
+<|ref|>text<|/ref|><|det|>[[118, 162, 855, 205]]<|/det|>
+\*\* Conclusions: Do you find that the conclusions and data interpretation are robust, valid and reliable? \*\*  
+
+<|ref|>text<|/ref|><|det|>[[118, 240, 877, 338]]<|/det|>
+Overall the conclusions and interpretation of the experimental results are robust and reliable. Though, as I mentioned above, I believe there is a set of experiments missing which would demonstrate more the relevance of a quantum approach to the generative model part of the proposed method.  
+
+<|ref|>text<|/ref|><|det|>[[118, 371, 261, 387]]<|/det|>
+\*\* References \*\*  
+
+<|ref|>text<|/ref|><|det|>[[118, 423, 617, 440]]<|/det|>
+The manuscript references previous literature appropriately.  
+
+<|ref|>text<|/ref|><|det|>[[118, 475, 325, 491]]<|/det|>
+\*\* Clarity and context \*\*  
+
+<|ref|>text<|/ref|><|det|>[[118, 526, 866, 571]]<|/det|>
+The paper is very well written and structured, and easy to follow for someone familiar with tensor network models.  
+
+<|ref|>text<|/ref|><|det|>[[118, 631, 378, 648]]<|/det|>
+\*\* Suggested improvements \*\*  
+
+<|ref|>text<|/ref|><|det|>[[118, 683, 863, 727]]<|/det|>
+- Include an experiment to compare the proposed approach with non-quantum generative models  
+
+<|ref|>text<|/ref|><|det|>[[118, 761, 840, 806]]<|/det|>
+- It could be a nice addition to have experiments on another combinatorial optimization problem than portfolio optimization.  
+
+<|ref|>text<|/ref|><|det|>[[118, 839, 875, 911]]<|/det|>
+- Related to the previous point, the authors may consider presenting their approach in a more general context rather than specifically for the portfolio optimization problem. As I understood it, the proposed approach can be applied to many different kind of optimization
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[117, 83, 864, 207]]<|/det|>
+problems but the current presentation can suggest that the method is tailored specifically for portfolio optimization. A more general presentation of the method, as well as a clear explanation of the different kind of problems the approach can be applied to, could make for a more impactful paper (by reaching a wider audience). For example, can the approach be applied to any MIP?
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[115, 91, 285, 107]]<|/det|>
+## Reply to Reviewer #1:  
+
+<|ref|>text<|/ref|><|det|>[[115, 144, 857, 196]]<|/det|>
+The authors propose a framework that incorporates quantum or classical generative models to tackle optimization problems. The main focus of the work is problems related to portfolio optimization and a quantum- inspired instantiation of the framework.  
+
+<|ref|>text<|/ref|><|det|>[[115, 212, 845, 264]]<|/det|>
+The paper is generally well- written and presents an interesting approach and results. However in its present form I have difficulty assessing the significance, scope, and impact of the results, which are paramount to my recommendation.  
+
+<|ref|>text<|/ref|><|det|>[[115, 281, 857, 315]]<|/det|>
+Hence at present I recommend additional revision of the manuscript. I provide some further comments below.  
+
+<|ref|>text<|/ref|><|det|>[[115, 343, 880, 395]]<|/det|>
+Reply: We thank the reviewer the careful revision our manuscript, and for the positive feedback about the current form of the manuscript. We address each of the questions below with the hope this helps to clarify the scope and significance of our work.  
+
+<|ref|>text<|/ref|><|det|>[[115, 439, 854, 509]]<|/det|>
+- What is scope and impact of the results? A general framework is described, but what specific instantiations and cases should we expect to provide greatest advantage? (I clearly see, at minimum, a classical-ML approach to portfolio optimization, and would appreciate more clarity regarding more general settings)  
+
+<|ref|>text<|/ref|><|det|>[[115, 525, 840, 560]]<|/det|>
+- Results are shown for portfolio optimization, but wasn't clear to what other important problems the approach is best suited for or should be expected to be advantageous.  
+
+<|ref|>text<|/ref|><|det|>[[115, 586, 880, 708]]<|/det|>
+Reply: We would like to mention that beyond our proposal being a "a classical- ML approach to portfolio optimization", the baseline of our contribution is a ML framework that leverages classical, quantum- inspired, or quantum generative models for solving any combinatorial problems. Although not a requirement for using the framework, having the possibility to easily generate bitstrings in the valid solution space (as the case studied here, i.e., bitstrings with a specified cardinality) can be useful since these synthetic data points can be passed as a warm start for the generative model. This in turn can help guide the trained generative model towards the right support of the probability distribution to be explored.  
+
+<|ref|>text<|/ref|><|det|>[[115, 718, 880, 890]]<|/det|>
+Using ML techniques for combinatorial problems is not a straightforward and rather a new research domain. More specifically, although other reinforcement learning and deep learning approaches had been proposed (see e.g., Ref. [1] and [2]), to the best of our knowledge our work is the first proposal using generative models for combinatorial optimization tasks. This is also the first proposal flexible enough to easily incorporate and explore quantum- inspired or quantum generative models. At first, we thought of adapting or enhancing existing reinforcement learning and deep learning proposals with quantum models, but this was not practical or at least not obvious how to do it. Instead, we noticed using generative models could be a very promising approach, given its prominence as candidates for practical quantum advantage. But this required to go beyond the incremental adaptation and to design the algorithm in a completely different framework, which resulted in the GEO proposal here.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 822, 107]]<|/det|>
+Here are more general settings that make this approach advantageous over other available solvers:  
+
+<|ref|>text<|/ref|><|det|>[[144, 117, 866, 202]]<|/det|>
+1) The entire approach is data-driven: what this implies is that the more data is available, either from previous attempts to solve the problem with other state-of-the-art solvers, the better the performance is expected. In the example of GEO as a booster we used data explored by Simulated Annealing (SA) but if we had previous observations from any or many other solvers, we could combine it and give it as a starting point to GEO.  
+
+<|ref|>text<|/ref|><|det|>[[144, 204, 865, 255]]<|/det|>
+2) It leverages the power of generative models: The essence of the solver is that it is aiming to unveil non-obvious structure in the data, and once it has captured those correlations, it suggests new outstanding candidates with features similar to the top ones seen until that iteration phase.  
+
+<|ref|>text<|/ref|><|det|>[[144, 255, 870, 375]]<|/det|>
+3) the model is cost function agnostic, i.e., it is a black-box solver. This is paramount since any cost function can be solved with our approach. Most of the proposals for quantum or quantum inspired optimization require the cost function of the problem to be mapped to a quadratic or polynomial expression. This black-box feature open the possibility to tackle any discrete optimization problem, regardless of how complicated or expensive it is to compute the cost function. This is possible within GEO since the only information passed to the generative model are the bitstrings who have been explored and their respective cost value.  
+
+<|ref|>text<|/ref|><|det|>[[144, 375, 880, 548]]<|/det|>
+4) Nothing is special about the portfolio optimization problem. This follows from the item above. The main motivation for selecting this specific instantiation of portfolio optimization was the availability of concrete benchmarks and an extensive literature of solvers which have been fine-tuned for over the past decades. Every time a new metaheuristic is proposed, chances are this cardinality-constrained portfolio optimization problem is used to benchmark. Other recent independent works have considered other real-world applications of GEO. For example, in Ref. [3], the authors considered an industrial case related to a floor planning NP-hard problem. This black-box feature is one of the most prominent ones which render our approach advantageous compared to other quantum heuristics, such as the quantum approximate optimization algorithm (QAOA), which relies on the cost function to be a polynomial in terms of the binary variables.  
+
+<|ref|>text<|/ref|><|det|>[[144, 574, 880, 781]]<|/det|>
+Although the items above only point to features which make the approach valuable and different when compared to optimizers proposed to date, it is still to be explored if similar advantages can be observed in other family of important problems. Although this question is an active research question, there has been some progress in understanding which datasets or problems might benefit the most. In a recent publication, (see Ref. [4]), we showed how the search space of problems with equality constrains, can be represented efficiently in quantum- inspired generative models, but this is not the case for deep- neural- network- based generative models. Besides the demonstration presented here pointing that quantum and quantum- inspired models are a promising route to be explored, this more recent paper poses a more concrete differentiator between the capabilities of quantum- inspired models with respect to traditional deep learning models. These equality constraints appear in a broad family of problems, including the cardinality constrained portfolio optimization studied here, but also it extends to problems with any linear equality constrains.  
+
+<|ref|>text<|/ref|><|det|>[[114, 791, 880, 877]]<|/det|>
+Revisions to the manuscript: The new Introduction has been significantly rewritten and restructured for clarity and to indicate more explicitly these salient highlights indicated above. We also revised the Outlook section adding some material related to the recent developments towards the identification of data sets which might benefit from quantum- inspired and quantum models over state- of- the- art generative models
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 860, 141]]<|/det|>
+- In particular, how common are problems where "cost function evaluation can be very expensive"? Can this be quantified? For instance, what is the resource tradeoff between query cost and runtime? Is your approach still competitive if this is relaxed?  
+
+<|ref|>text<|/ref|><|det|>[[114, 168, 881, 323]]<|/det|>
+Reply: We thank the referee for bringing this point since some clarifications can be added to the manuscript. Note that this desirable condition of having an expensive cost function applies mainly to GEO as a standalone, although it is important to note that even in that setting it is not a necessary condition. The reason is because the overhead of training the generative model might have a larger impact for that setting than in the booster mode, where the initial available data can be seen as a warm start. It can be noted that even in problems with cost function which might be quick to evaluate, as long as the problem is hard and a solution is not reached with a competitor solver, it might be still worth to use GEO as a standalone which might yield a completely different set of solutions, or even better ones, since its search strategy is completely different.  
+
+<|ref|>text<|/ref|><|det|>[[114, 334, 878, 541]]<|/det|>
+This condition of an expensive cost function was relaxed altogether in the setting of GEO as a booster (described in Sec. II A) where the comparison criteria was moved from number of cost function evaluations to runtime. In that study, we used a similar criteria as that suggested by the referee, where we used the trade off between query cost and runtime by using the more generic and practical criteria of fixed total wall clock runtime used for each of the algorithmic strategies. There, the time it takes to evaluate the cost function and the training of the generative models are considered, since these are part of the total time it takes to run each algorithm. In that comparison, between Simulated Annealing (SA) and GEO, although the training of the generative model within GEO takes longer, compared to the quick updates in SA, we still see an advantage in adopting GEO as a booster, which consisted of GEO being initialized with partial solutions obtained from SA. We show in that section that it is more efficient to change the strategy to GEO, than to insist and continue performing runs just with SA, if both algorithmic strategies are given the same total wallclock time.  
+
+<|ref|>text<|/ref|><|det|>[[115, 551, 841, 585]]<|/det|>
+Revisions to the manuscript: We have added further clarification in the new paragraph right before subsection IIA.  
+
+<|ref|>text<|/ref|><|det|>[[115, 622, 864, 673]]<|/det|>
+- The results rely on a quantum inspired method, what evidence or support do we have that using actual quantum devices will improve further? How should we expect GEO to perform for other quantum or classical models?  
+
+<|ref|>text<|/ref|><|det|>[[114, 701, 878, 908]]<|/det|>
+Reply: The question of whether a significant advantage can be obtained by using quantum devices is an active research topic and certainly needs to be explored further. One proposal to reach a more systematic and incremental enhancement from the best quantum- inspired solution to an enhanced quantum- hardware realization was recently proposed in Ref. [5]. There, one starts from the best available quantum- inspired tensor- network solution and maps it to a quantum circuit. This can be subsequently modified by adding gates beyond those from the decomposition to increase the plausible correlations beyond those accessible with the quantum- inspired tensor- network- based solution. The access to longer- range correlations enhances, in turn, the expressibility of the quantum generative model while taking it beyond the capabilities of classical simulation. In that work, the specific case of generative models was illustrated, and therefore, these novel decomposition techniques can be directly applied to extend the capabilities of TN- GEO explored here, and, as the technologies mature and the level of noise is reduced, explore these enhanced models directly on quantum devices. Additionally, in Ref. [6] a comparison of quantum
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 857, 125]]<|/det|>
+generative models with state- of- the- art classical generative models was presented, and the results were very encouraging in the data sets studied.  
+
+<|ref|>text<|/ref|><|det|>[[114, 133, 875, 358]]<|/det|>
+To the comment of the performance of TN- GEO against other classical models, and as part of the request from the second referee, we added a classical version of GEO, based on the Neural Autoregressive Density Estimation (NADE) model [7]. In this revised version of the manuscript, we showed that although NADE- GEO is competitive with the early solvers (from about a decade ago), it is underperforming compared to TN- GEO and the other state- of- the- art solvers within the past five years. Although we don't expect the results to be universal, in this specific TN- GEO versus NADE- GEO comparison we still see an advantage for the quantum- inspired over the classical generative neural network model, with the advantage as well that the TN- base model has less hyperparameters to fine tune. Each of these comparisons is an extensive amount of work, and we feel that despite being beyond the scope of this particular work, our work opens the possibility to explore quantum and quantum- inspired generative models toward solving arbitrary combinatorial optimization, and as a concrete framework to study quantitatively practical quantum advantage with future quantum technologies (see e.g., Ref. [6] whose framework is inspired in this work)  
+
+<|ref|>text<|/ref|><|det|>[[115, 367, 860, 454]]<|/det|>
+Revisions to the manuscript: We complemented the Outlook where we had already mentioned about the opportunities and challenges that arise from thinking of an implementation in quantum devices. In particular, we added some of the discussion above around the new references that appeared after we submitted our work. We have also expanded the Results & Discussion subsection, Sec. IIC, with the respective discussion related to NADE- GEO and TN- GEO.  
+
+<|ref|>text<|/ref|><|det|>[[115, 491, 833, 543]]<|/det|>
+- For the quantum case, is the proposed method not still severely limited by the underlying quantum hardware and quantum model resource requirements? (in contrast, for instance, to problem decomposition approaches where the goal may be to accommodate fewer quantum resources)  
+
+<|ref|>text<|/ref|><|det|>[[114, 569, 874, 812]]<|/det|>
+Reply: If we correctly understand the proposal from the Reviewer, the reviewer is thinking of problem decomposition techniques which divide the problem into many smaller subproblems to accommodate the number of qubits in near- term technologies. From a practical point of view, for these techniques to be promising for quantum technologies, one still needs the subproblems still be intractable or hard to solve by conventional classical solvers, otherwise, it would not be worth to submit them to a quantum hardware. If the instances happen to be indeed intractable, then GEO can still be used to solve these subproblems, since in the cases of decomposition techniques we are familiar with, it is still required that solutions to the subproblem needs to be gathered before they are combined or used to solve the larger problem (e. g., see Refs. [8], [9], and [10] below and that have been added to the revised manuscript). In other words, we don't see it necessarily as one strategy versus the other, since we think of GEO as a standalone strategy to solving hard optimization problems or subproblems. It is important to note that, with now gate- based devices reaching the level of hundreds of qubits, such techniques and demonstrations can become more relevant. But we can see how GEO can be incorporated as well in the solution of the smaller but intractable partitions which are sent to these quantum devices.  
+
+<|ref|>text<|/ref|><|det|>[[115, 821, 870, 890]]<|/det|>
+To the question of the limitation of quantum hardware resources, not only in terms of number of qubits, but also in terms of connectivity, this is one is an important one and we will be addressing that in a theoretical and experimental ongoing work in our team. In that study, noise is also factored as part of the experimental demonstration.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 878, 141]]<|/det|>
+Revisions to the manuscript: We have complemented the Outlook addressing both, the potential interplay with other decomposition techniques, and the open questions and challenges related to hardware limitations.  
+
+<|ref|>text<|/ref|><|det|>[[115, 179, 877, 230]]<|/det|>
+- Regarding "GTS" optimizer in Section III.C and the subsequent reported results for it, I was unclear what was meant. Each of G,T,S refers to an independent optimization strategy, are you running each and reporting the best?  
+
+<|ref|>text<|/ref|><|det|>[[115, 257, 857, 310]]<|/det|>
+- Similarly, I was left with follow up questions such as to what degree were these (G,T,S) as well as the other nine "leading SOTA optimizers" tailored/optimized for the problem at hand? (In regards to truly 'fair' comparison in the reported numbers)  
+
+<|ref|>text<|/ref|><|det|>[[113, 327, 881, 636]]<|/det|>
+Reply: It is important to note that we have not fine- tuned all the solvers ourselves. The Results section of the papers is broken into three subsections, each highlighting different features from GEO. Sec. IIA focuses on GEO as a booster and how it can build from results obtained with other solvers. Sec. IIB focuses on GEO as a standalone and compares its performance to SA and the Bayesian optimization library GPyOpt. Finally, Sec. IIC focuses on a comparison of GEO with state- of- the- art solvers. While in Secs. IIA and IIB we implemented and fine- tuned each solver, in Sec. IIC, we leverage the state- of- the- art results from nine other solvers reported in the literature in the last two decades. In the latter case, each non- GEO solver was thoroughly fine- tuned by the researchers of each reference. This portfolio optimization problem is so canonical that when a new solver is proposed, researchers can compare their results by taking the results from the new proposed solver, as long as the benchmark problems are run in identical conditions. This was one of the main motivations for us to choose this well- established benchmark problem. The "rules of the game" for reporting each market index and performance indicator are reported in Appendix A 2. In contrast, for the other two subsections, the criteria of evaluation are different, and it emphasizes the performance of GEO when one imposes a limit on the total wall- clock time (Sec. IIA) and when there is a limited number of calls to the cost function (Sec. IIB). The latter is a potential scenario when the bottleneck or expensive step is the cost function evaluation itself (e.g., as it is the case of drug discovery where each evaluation (each candidate molecule) might require synthesis in the lab and an expensive and long process towards its Food and Drug Administration (FDA) approval).  
+
+<|ref|>text<|/ref|><|det|>[[115, 647, 864, 750]]<|/det|>
+To the specific question of (G,T,S) the Reviewer is correct that the authors from the original reference back in 2000 ran each strategy (Genetic algorithms, Tabu search and Simulated annealing) and reported their best results for each. Since this paper was one of the first ones adopting the current metrics and benchmarking procedure, when the community started creating more sophisticated metaheuristic strategies, they continue referring to the results in this paper as GTS, implying the values correspond to the best result from either G, T, or S  
+
+<|ref|>text<|/ref|><|det|>[[115, 760, 880, 828]]<|/det|>
+Revisions to the manuscript: To clarify this to the reader, we have emphasized further in Sec. IIC that the results from the state- of- the- art competitors have been independently fine- tuned and are available in the literature. We have also added more context at the beginning of the Results section to contextualize all the different results subsections and strategies presented in the paper.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 860, 125]]<|/det|>
+- Table I is not so easy to read with the many entries. Is it worth including first 4 optimizers here? They don't seem competitive (no to mention their columns are mostly ".")
+
+<|ref|>text<|/ref|><|det|>[[115, 153, 881, 322]]<|/det|>
+**Reply:** We have followed the Reviewer's suggestion and we have simplified the table accordingly (please see new Table I). The main reason for including all the solvers was to follow the format from previous papers which compared to these early metaheuristics from the early 2000's. Since this cardinality constrained problem has been used for benchmarking solved since the late 1990s, these were the solvers which have been competitive since the last two decades or so ago. As time has passed, and in particular in the last decade, other researchers have proposed other figure of merits to assess the performance of the solvers. Since these were not known in the first papers, they have been conventionally included in the most recent papers with the '- ' since the data is not available. Others have adopted these other metrics as valuable and that is the reason the more recent papers all evaluate them, and we decided to include them here as well.
+
+<|ref|>text<|/ref|><|det|>[[115, 336, 880, 437]]<|/det|>
+The original intention from having the complete table was to have the whole spectrum of solvers and help position our quantum-inspired GEO in the historical progress of optimizers for this specific task, clearly showing GEO outperforms these solvers from the early 2000's up to a decade ago, and being on par with the current state-of-the-art optimizers. Note that with the suggestion from the other Reviewer to include a classical version of GEO, called NADE-GEO in the revised version, we can see that NADE-GEO is competitive with solvers from about a decade ago, but not as competitive as TN-GEO or the new solvers.
+
+<|ref|>text<|/ref|><|det|>[[115, 450, 881, 482]]<|/det|>
+**Revisions** **to** **the** **manuscript.** We have collapsed the table appearing in the main text as suggested by the reviewer. The original table, expanded now with the NADE-GEO results, has been moved to Appendix C.
+
+<|ref|>text<|/ref|><|det|>[[115, 520, 874, 553]]<|/det|>
+-RCABC appeared to preform comparably to TN-GEO, it would be nice to see more discussion of this or even a more detailed comparison
+
+<|ref|>text<|/ref|><|det|>[[115, 582, 875, 632]]<|/det|>
+**Reply:** We have expanded the discussion on RCABC at the end of the Results section (Sec. IIC). Note in the current version we have replaced the name RCABC to ABC-HP to match the name of the best performing variant in that 2021 paper cited, and which corresponds to the results reported here.
+
+<|ref|>title<|/ref|><|det|>[[115, 680, 203, 692]]<|/det|>
+# References:
+
+<|ref|>text<|/ref|><|det|>[[115, 706, 852, 737]]<|/det|>
+[1] Bello, I., Pham, H., Le, Q.V., Norouzi, M. and Bengio, S., Neural combinatorial optimization with reinforcement learning. arXiv preprint arXiv:1611.09940 (2016).
+
+<|ref|>text<|/ref|><|det|>[[115, 750, 878, 782]]<|/det|>
+[2] Bengio, Y., Lodi, A. and Prouvost, A., Machine Learning for Combinatorial Optimization: a Methodological Tour d'Horizon. CoRR abs/1811.06128 (2018). arXiv preprint arXiv:1811.06128 (2018).
+
+<|ref|>text<|/ref|><|det|>[[115, 795, 878, 844]]<|/det|>
+[3] Banner, W.P., Hadiashar, S.B., Mazur, G., Menke, T., Ziolkowski, M., Kennedy, K., Romero, J., Cao,Y., Grover, J.A. and Oliver, W.D., Quantum Inspired Optimization for Industrial Scale Problems. arXiv preprint arXiv:2305.02179 (2023).
+
+<|ref|>text<|/ref|><|det|>[[115, 857, 854, 906]]<|/det|>
+[4] Lopez-Piqueres, J., Chen, J. and Perdomo-Ortiz, A., Symmetric tensor networks for generative modeling and constrained combinatorial optimization. Machine Learning: Science and Technology.4,035009 (2023).
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[112, 90, 855, 141]]<|/det|>
+[5] Rudolph, M.S., Miller, J., Motlagh, D., Chen, J., Acharya, A. and Perdomo-Ortiz, A., Synergy between quantum circuits and tensor networks: Short- cutting the race to practical quantum advantage. arXiv preprint arXiv:2208.13673 (2022).  
+
+<|ref|>text<|/ref|><|det|>[[113, 151, 874, 203]]<|/det|>
+[6] Hibat- Allah, M., Mauri, M., Carrasquilla, J. and Perdomo- Ortiz, A., A Framework for Demonstrating Practical Quantum Advantage: Racing Quantum against Classical Generative Models. arXiv preprint arXiv:2303.15626 (2023).  
+
+<|ref|>text<|/ref|><|det|>[[113, 212, 855, 248]]<|/det|>
+[7] Uria, B., Côté, M.A., Gregor, K., Murray, I. and Larochelle, H., Neural autoregressive distribution estimation. The Journal of Machine Learning Research, 17(1), pp.7184- 7220 (2016).  
+
+<|ref|>text<|/ref|><|det|>[[113, 257, 872, 309]]<|/det|>
+[8] Ponce, M., Herrman, R., Lotshaw, P.C., Powers, S., Siopsis, G., Humble, T. and Ostrowski, J., Graph decomposition techniques for solving combinatorial optimization problems with variational quantum algorithms. arXiv preprint arXiv:2306.00494 (2023).  
+
+<|ref|>text<|/ref|><|det|>[[113, 318, 856, 370]]<|/det|>
+[9] Ushijima- Mwesigwa, H., Shaydulin, R., Negre, C.F., Mniszewski, S.M., Alexeev, Y. and Safro, I., Multilevel combinatorial optimization across quantum architectures. ACM Transactions on Quantum Computing, 2(1), pp.1- 29 (2021).  
+
+<|ref|>text<|/ref|><|det|>[[113, 380, 870, 415]]<|/det|>
+[10] Zhou, Z., Du, Y., Tian, X. and Tao, D., QAOA- in- QAOA: solving large- scale MaxCut problems on small quantum machines. Physical Review Applied, 19(2), p.024027 (2023).
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[115, 90, 280, 107]]<|/det|>
+## Reply to Reviewer #2  
+
+<|ref|>text<|/ref|><|det|>[[115, 124, 243, 140]]<|/det|>
+\*\* Key results \*\*  
+
+<|ref|>text<|/ref|><|det|>[[115, 142, 866, 262]]<|/det|>
+This paper proposes to use a quantum inspired generative model to help more efficiently explore the space of feasible solutions for combinatorial optimization problems. The generative model is used to sample new candidate solutions: from a set of already explored solutions with their associated cost, the generative model is trained to learn a distribution over the solution space for which the probability of each seen solution is proportional to their associated cost, thus potentially making it possible to sample new promising candidate solutions. The authors focus on using a quantum inspired generative model based on tensor networks (matrix product states), which have been previously introduced in [15].  
+
+<|ref|>text<|/ref|><|det|>[[115, 279, 872, 348]]<|/det|>
+The proposed approach is evaluated on the task of portfolio optimization and compared with state of the art optimization algorithms for this task. The experiments reveal that the approach is competitive with these state of the art solvers (which have been fine tuned for decades), even outperforming some of them on this particular task.  
+
+<|ref|>text<|/ref|><|det|>[[115, 365, 220, 380]]<|/det|>
+\*\* Validity \*\*  
+
+<|ref|>text<|/ref|><|det|>[[115, 383, 850, 400]]<|/det|>
+The results presented in the paper are valid. Both the methodology and the experiment setup is sound.  
+
+<|ref|>text<|/ref|><|det|>[[115, 416, 315, 432]]<|/det|>
+\*\* Data & methodology \*\*  
+
+<|ref|>text<|/ref|><|det|>[[115, 434, 850, 468]]<|/det|>
+The paper is overall clearly written and the experiments demonstrate well the potential benefits and usefulness of the method.  
+
+<|ref|>text<|/ref|><|det|>[[115, 478, 829, 512]]<|/det|>
+Reply: We thank the referee for the very positive feedback on our proposed framework. Below we address the concerns raised in the comments.  
+
+<|ref|>text<|/ref|><|det|>[[114, 549, 879, 705]]<|/det|>
+One concern that I have is that the advantage of using a quantum approach is not clearly demonstrated. In particular, it is not clear how the fact that quantum inspired models where chosen for the generative part is key to obtaining the experimental results. At the very least I believe the proposed approach should be compared with replacing the MPS model with a simple HMM learned using the Baum- Welch (i.e. EM) algorithm. Other generative models should be considered as baselines as well (e.g. a simple RNN trained using backpropagation through time or a more complex model such as NADE [Uria et al]). It may be the case that replacing the MPS model by such an alternative non- quantum inspired model would lead to similar result. To sum up, I believe the authors should experimentally investigate and discuss more in depth to which extent the quantum part of their approach is necessary and beneficial.  
+
+<|ref|>text<|/ref|><|det|>[[115, 715, 866, 749]]<|/det|>
+Uria, Benigno, et al. "Neural autoregressive distribution estimation." The Journal of Machine Learning Research 17.1 (2016): 7184- 7220.  
+
+<|ref|>text<|/ref|><|det|>[[114, 758, 880, 897]]<|/det|>
+Reply: We agree with the Reviewer this is a critical point that deserves further study since it is in general an open question. One of the highlights for GEO is the flexibility of swapping classical, quantum inspired, and quantum models in the future as quantum technologies mature. We have followed the suggestion from the Reviewer and implemented NADE, which is the most complex model of the ones suggested. In the new results summarized in Table V (computed from the explicit values in Table III), it can be seen that although NADE- GEO is a competitive solver, it is only statistically on par with the solvers which were the best performers up to about a decade ago (GTS, IPSO, IPSO- SA, and PBILD), but statistically different from its quantum- inspired version (TN- GEO), and the solvers proposed from 2015
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 882, 158]]<|/det|>
+and onwards (GRASP, ABCFEIT, AAG, VNSQ, and ABC- HP). Although this does not constitute a proof in any way of the superiority of quantum or quantum- inspired models, this corresponds to the experimental investigation which hints to the value and promising directions we highlight in this work from this flexible framework.  
+
+<|ref|>text<|/ref|><|det|>[[114, 169, 881, 426]]<|/det|>
+To the milestone of understanding when quantum or quantum- inspired models could be key to obtaining an advantage over classical models, several subsequent publications have attempted to address this point. For example, in Ref. [1], we have recently published a version of TN- GEO which uses symmetries which are relatively easy in these quantum- inspired TN models. We showed how the search space of problems with equality constraints can be represented efficiently in quantum- inspired generative models, but this is not necessarily the case for classical deep- neural- network- based generative models due to their non- linear activation units. That recent work poses a more concrete differentiator between the capabilities of quantum- inspired models with respect to traditional deep learning generative models. In general, the question of practical quantum advantage in quantum machine learning is still wide open, but in this recent work, Ref. [2] below, the proposed framework there leverages our GEO proposal to establish a clear- cut criteria from optimization problems to concretely test the performance of both classical and quantum generative modeling proposals. From a practical point of view, and in case of real and relevant real- world applications, this question would need to be approached on a case- by- case basis, but that subsequent paper proposes a framework where the performance of quantum generative models can be tested, following similar metrics inspired from this work.  
+
+<|ref|>text<|/ref|><|det|>[[115, 454, 875, 489]]<|/det|>
+\*\*Appropriate use of statistics and treatment of uncertainties \*\*Yes, the results are reported appropriately using classical statistical tools and treatment of uncertainties.  
+
+<|ref|>text<|/ref|><|det|>[[115, 498, 694, 515]]<|/det|>
+Reply: We thank the referee for the positive feedback on our statistical analysis.  
+
+<|ref|>text<|/ref|><|det|>[[115, 532, 870, 565]]<|/det|>
+\*\*Conclusions: Do you find that the conclusions and data interpretation are robust, valid and reliable? \*\*  
+
+<|ref|>text<|/ref|><|det|>[[115, 568, 880, 619]]<|/det|>
+Overall the conclusions and interpretation of the experimental results are robust and reliable. Though, as I mentioned above, I believe there is a set of experiments missing which would demonstrate more the relevance of a quantum approach to the generative model part of the proposed method.  
+
+<|ref|>text<|/ref|><|det|>[[115, 648, 837, 715]]<|/det|>
+Reply: We thank the referee for the positive feedback on our overall conclusions. We hope the new numerical experiments, including the simulations of NADE- GEO strengthens our claims around the potential benefit of quantum inspired generative models, and in general, of quantum models as an alternative to be explored in the near term.  
+
+<|ref|>text<|/ref|><|det|>[[115, 733, 555, 768]]<|/det|>
+\*\*References \*\*The manuscript references previous literature appropriately.  
+
+<|ref|>text<|/ref|><|det|>[[115, 795, 696, 812]]<|/det|>
+Reply: We thank the referee for the positive feedback on our literature coverage.  
+
+<|ref|>text<|/ref|><|det|>[[115, 840, 822, 890]]<|/det|>
+\*\*Clarity and context \*\*The paper is very well written and structured, and easy to follow for someone familiar with tensor network models.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 830, 108]]<|/det|>
+Reply: We thank the referee for the positive feedback on the content and the structure of the paper.  
+
+<|ref|>text<|/ref|><|det|>[[118, 134, 341, 150]]<|/det|>
+\*\* Suggested improvements \*\*  
+
+<|ref|>text<|/ref|><|det|>[[115, 179, 819, 197]]<|/det|>
+- Include an experiment to compare the proposed approach with non-quantum generative models  
+
+<|ref|>text<|/ref|><|det|>[[115, 205, 877, 310]]<|/det|>
+Reply: Following the advice from the referee, we have implemented NADE- GEO and the comparison with TN- GEO and the other SOTA solvers are included throughout Tables I- V. As previously mentioned, the classical ML model is competitive in the sense that is comparable with models from about a decade ago. But still underperforming TN- GEO, which is on par with the state- of- the- art solvers for this application. This is also included in the new summary Table II in the main text, where it can be seen the superior performance of TN- GEO over NADE- GEO in this specific application.  
+
+<|ref|>text<|/ref|><|det|>[[115, 336, 850, 372]]<|/det|>
+- It could be a nice addition to have experiments on another combinatorial optimization problem than portfolio optimization.  
+
+<|ref|>text<|/ref|><|det|>[[114, 388, 880, 510]]<|/det|>
+- Related to the previous point, the authors may consider presenting their approach in a more general context rather than specifically for the portfolio optimization problem. As I understood it, the proposed approach can be applied to many different kind of optimization problems but the current presentation can suggest that the method is tailored specifically for portfolio optimization. A more general presentation of the method, as well as a clear explanation of the different kind of problems the approach can be applied to, could make for a more impactful paper (by reaching a wider audience). For example, can the approach be applied to any MIP?  
+
+<|ref|>text<|/ref|><|det|>[[114, 518, 879, 691]]<|/det|>
+Reply: We agree with the reviewer that the current presentation hints to GEO as a solution tailored only for the portfolio optimization problem, where in reality, as the referee mentioned, GEO is more in the category of a generic solver. Although we had mentioned in a couple of places its black- box nature, many readers might not immediately associate this concept with a solver that can deal with any cost function, and therefore any combinatorial problem. This black- box feature is one of the most prominent ones which render our approach advantageous compared to other quantum heuristics, such as the quantum approximate optimization algorithm (QAOA), which relies on the cost function to be a polynomial in terms of the binary variables. To mitigate missing these essential points raised by the referee, the introduction has been significantly revised and restructured adding clarity to the main features of our framework.  
+
+<|ref|>text<|/ref|><|det|>[[114, 700, 861, 821]]<|/det|>
+Although we did not explore explicitly in this work the case of problems containing both discrete and continuous variables (e.g., MIP) or only continuous variables, we discussed in the Outlook how an approach as the one in Ref. [3] could allow to generalize to these mixed variable cases by using such hybrid quantum- classical generative models within GEO. In a recent publication, Ref. [4], our team addressed this question of how to treat generative models with continuous variables (or a mixture of discrete and continuous) directly with TN models, which would be directly applicable to MIP and other related problems.  
+
+<|ref|>text<|/ref|><|det|>[[115, 832, 853, 902]]<|/det|>
+To the question of including other applications, the main motivation for selecting this specific instantiation of portfolio optimization was the availability of concrete benchmarks and an extensive literature of solvers which have been fine tuned for over the past decades. In particular, we wanted to make sure we could make stronger claims in a well- established benchmark problem, and assess in this
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[113, 90, 881, 313]]<|/det|>
+way where this new quantum- inspired solution TN- GEO fits within the suite of SOTA algorithms. Although this could be done for other well- known benchmarking problems, we felt the paper was extensive enough since it presents the introduction of this new framework plus demonstrating some of its highlights reflected in the three Results subsections: 1) the possibility to leverage data observed with other solvers (Sec. IIA), 2) its high performance in the regime of very limited calls to the cost function evaluation (Sec. IIB) and 3) its performance compared to SOTA algorithms under the rules established by those specific benchmarks (Sec. IIC). Although we considered extending to other application domains is outside of the scope of this work, other recent independent works have considered other real- world applications of GEO. For example, in Ref. [5], the authors considered an industrial case related to a floor planning NP- hard problem. In Ref. [6] the authors considered quantum- inspired generative models similar to the ones we propose here, to explore the solution space of candidates in molecular discovery. We hope the referee considers as well that the work is complete as is, given the new developments supporting the range of applications of our work.  
+
+<|ref|>text<|/ref|><|det|>[[115, 323, 880, 392]]<|/det|>
+Revisions to the manuscript: We hope the new Introduction brings more clarity to the essential points raised by the referee. To emphasize the point from the referee that even MIP or problems with continuous variables could be addressed within GEO, we have added some comments in the Outlook about recent work extending and addressing this challenge, and how it fits within our framework.  
+
+<|ref|>sub_title<|/ref|><|det|>[[115, 456, 206, 472]]<|/det|>
+## References:  
+
+<|ref|>text<|/ref|><|det|>[[115, 484, 850, 535]]<|/det|>
+[1] Lopez- Piqueres, J., Chen, J. and Perdomo- Ortiz, A., Symmetric tensor networks for generative modeling and constrained combinatorial optimization. Machine Learning: Science and Technology. 4 035009 (2023)  
+
+<|ref|>text<|/ref|><|det|>[[115, 545, 875, 597]]<|/det|>
+[2] Hibat- Allah, M., Mauri, M., Carrasquilla, J. and Perdomo- Ortiz, A., A Framework for Demonstrating Practical Quantum Advantage: Racing Quantum against Classical Generative Models. arXiv preprint arXiv:2303.15626 (2023).  
+
+<|ref|>text<|/ref|><|det|>[[115, 607, 874, 659]]<|/det|>
+[3] Rudolph, M.S., Toussaint, N.B., Katabarwa, A., Johri, S., Peropadre, B. and Perdomo- Ortiz, A., Generation of high- resolution handwritten digits with an ion- trap quantum computer. Physical Review X, 12(3), p.031010 (2022).  
+
+<|ref|>text<|/ref|><|det|>[[115, 669, 880, 704]]<|/det|>
+[4] Meiburg, A., Chen, J., Miller, J., Tihon, R., Rabusseau, R., Perdomo- Ortiz, A. Generative Learning of Continuous Data by Tensor Networks. arXiv preprint arXiv: 2310.20498 (2023).  
+
+<|ref|>text<|/ref|><|det|>[[115, 714, 878, 765]]<|/det|>
+[5] Banner, W.P., Hadiashar, S.B., Mazur, G., Menke, T., Ziolkowski, M., Kennedy, K., Romero, J., Cao, Y., Grover, J.A. and Oliver, W.D., Quantum- Inspired Optimization for Industrial Scale Problems. arXiv preprint arXiv:2305.02179 (2023).  
+
+<|ref|>text<|/ref|><|det|>[[111, 776, 842, 810]]<|/det|>
+[6] Moussa, C., Wang, H., Araya- Polo, M., Bäck, T. and Dunjko, V., 2023. Application of quantum- inspired generative models to small molecular datasets. arXiv preprint arXiv:2304.10867 (2023).
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[118, 85, 330, 101]]<|/det|>
+## REVIEWERS' COMMENTS  
+
+<|ref|>sub_title<|/ref|><|det|>[[118, 164, 439, 181]]<|/det|>
+## Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 189, 833, 260]]<|/det|>
+The authors have considered and reasonably addressed the comments of the referees, resulting a significant number of changes and improvements to the manuscript. Hence I am happy to recommend for publication.  
+
+<|ref|>sub_title<|/ref|><|det|>[[118, 320, 439, 336]]<|/det|>
+## Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 345, 815, 389]]<|/det|>
+I am overall satisfied by the answers from the authors to my initial review and to the consequent revisions that have been made to the manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[117, 422, 877, 520]]<|/det|>
+I find that stylistically speaking, the quality of the introduction has decreased in some of the new paragraphs added in the revision (see detailed comments below). I also give below some relatively minor stylistic comments about the new content that has been added to the revision:  
+
+<|ref|>text<|/ref|><|det|>[[117, 580, 868, 624]]<|/det|>
+p. 1, second item: "\\*either\\* from previous..." there is a missing "or" and second part of the sentence.  
+
+<|ref|>text<|/ref|><|det|>[[118, 658, 433, 675]]<|/det|>
+p. 2 first item: This open -> This opens  
+
+<|ref|>text<|/ref|><|det|>[[116, 709, 878, 885]]<|/det|>
+p. 2 The last item is stylistically very odd and reads more than a rebuttal to the first round of reviews than an actual paragraph from an introduction. More precisely, this list is presented as a list of "salient highlights" of the proposed approach; is "Nothing is special about the portfolio optimization problem" a "salient highlight" of the contribution. I understand the aim but this is done in a very clumsy way, in my opinion. I would suggest rewriting this item and rephrasing in the direction of emphasizing the "versatility" of the method, while justifying the focus on portfolio optimization.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 83, 877, 103]]<|/det|>
+p.2 last item: "... *as\* the NP hard problem \*as\* the workhorse..." is a bit stylistically clumsy  
+
+<|ref|>text<|/ref|><|det|>[[117, 136, 569, 154]]<|/det|>
+p.2 last item: line starting with a comma after "Ref [8]"  
+
+<|ref|>text<|/ref|><|det|>[[117, 188, 857, 232]]<|/det|>
+p.6 to be consistent, the item 10)'s sentence in the before last paragraph should end with ";" instead of ".  
+
+<|ref|>text<|/ref|><|det|>[[117, 266, 762, 285]]<|/det|>
+p.10 before last paragraph of first column: "it is to use it as" -> "is to use it as"  
+
+<|ref|>text<|/ref|><|det|>[[117, 318, 700, 336]]<|/det|>
+p.10 "DATA AVAILABILITY": "have been deposited" -> e.g. "is available"  
+
+<|ref|>text<|/ref|><|det|>[[117, 369, 698, 414]]<|/det|>
+p.14 sec 4: "introduced by Uria [37]" -> "introduced by Uria et al. [37]" "is that it models" -> "is to model"
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[118, 84, 303, 100]]<|/det|>
+## Reply to Reviewer #1  
+
+<|ref|>text<|/ref|><|det|>[[118, 117, 878, 152]]<|/det|>
+We thank Reviewer #1 for their feedback throughout this review process and for their recommendation to publish this significantly revised version.  
+
+<|ref|>sub_title<|/ref|><|det|>[[118, 170, 304, 187]]<|/det|>
+## Reply to Reviewer #2  
+
+<|ref|>text<|/ref|><|det|>[[118, 204, 878, 256]]<|/det|>
+We thank Reviewer #2 for their recommendation to accept this latest version for publication. In this resubmission, we have incorporated all the stylistic changes suggested by the reviewer. We thank the reviewer for this valuable feedback throughout this review process.
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__00d0f482762f2f37431ca49a939480fc54bdd5eb053d5ac8ce0b474c9dacda22/images_list.json

@@ -0,0 +1,145 @@
+[
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_0.jpg",
+    "caption": "Fig S2. H. Western blot showed the absence of AAVR at the protein level at its expected size in the KO clone. I. VCN and luciferase activities of AAV_ITGs in 293_WT and 293_AAVR-KO cells ( \\(n = 4 - 6\\) , one-way ANOVA).",
+    "footnote": [],
+    "bbox": [],
+    "page_idx": 0
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_1.jpg",
+    "caption": "Fig S1. C. The cell type-enriched transcriptome profiles of ITGAV and ITGB6 in a list of cell types from multiple tissues (graphs were generated from The Human Protein Atlas). MYH7 is a positive control that is known to highly enrich in the cardiac and skeletal muscles.",
+    "footnote": [],
+    "bbox": [
+      [
+        125,
+        84,
+        872,
+        490
+      ]
+    ],
+    "page_idx": 5
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_5.jpg",
+    "caption": "Fig 5. D. Immuno-staining of DAPI (blue) and SGCA (red) in Quadriceps muscles.",
+    "footnote": [],
+    "bbox": [
+      [
+        128,
+        660,
+        880,
+        805
+      ]
+    ],
+    "page_idx": 7
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_6.jpg",
+    "caption": "Fig. 6: LICA1 showed cross-species enhanced transduction in the skeletal muscle.",
+    "footnote": [],
+    "bbox": [
+      [
+        120,
+        85,
+        870,
+        740
+      ]
+    ],
+    "page_idx": 8
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_2.jpg",
+    "caption": "Fig. S6: LICA1 showed improved transduction across species. A. Predicted structures of \\(\\alpha \\beta \\beta\\) heterocomplex from multiple species by AlphaFold2 showed very high similarity with the crystal structure of human form (pdb code: 4um9). The ITGAV and ITGB6 of human (ITGAV: aa31-467, ITGB6: 132-373), primate (ITGAV: aa31-467, ITGB6: 132-373), pig (ITGAV: aa31-",
+    "footnote": [],
+    "bbox": [
+      [
+        120,
+        88,
+        875,
+        850
+      ]
+    ],
+    "page_idx": 10
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_3G.jpg",
+    "caption": "Fig S3. F-G. VCN (F) and gene expression (G) (GFP mRNA level in liver and luciferase activity in other organs) of AAV9rh74_4um9, MYOAAV2, and MYOAAV3 18 in liver, skeletal muscles, heart, lung, kidney, and brain (n=4, one-way ANOVA). VCN and mRNA levels of AAV9 and MYOAAV1A in the previous comparison (Fig 3G-H) were added as reference. H-I. Ratio of VCN found in skeletal muscle; TA (H) and Qua (I); to the level in the liver one-month post-injection (n=4, one-way ANOVA).",
+    "footnote": [],
+    "bbox": [
+      [
+        122,
+        192,
+        875,
+        562
+      ]
+    ],
+    "page_idx": 12
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_3.jpg",
+    "caption": "Figure R1: Tissue Cell Type enrichment score of different ITGB subunits' mRNA in human biopsies (Figures generated by The Human Protein Atlas).",
+    "footnote": [],
+    "bbox": [
+      [
+        120,
+        81,
+        880,
+        600
+      ]
+    ],
+    "page_idx": 16
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_4.jpg",
+    "caption": "Fig 4. Comparison of transduction efficacy between AAV9 and LICA1 in three skeletal muscles and heart, in terms of VCN (A), and \\(\\mu\\) Dys mRNA level (B).",
+    "footnote": [],
+    "bbox": [
+      [
+        120,
+        262,
+        878,
+        368
+      ]
+    ],
+    "page_idx": 18
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_5.jpg",
+    "caption": "Fig 5. Comparison of transduction efficacy between AAV9 and LICA1 in three skeletal muscles and heart, in terms of VCN (A), and hSGCA mRNA level (B).",
+    "footnote": [],
+    "bbox": [
+      [
+        120,
+        490,
+        875,
+        633
+      ]
+    ],
+    "page_idx": 21
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_4.jpg",
+    "caption": "Figure R2: The bulk mRNA expression of ITGB6 in skeletal muscle, heart, and liver from human biopsies (data was reanalyzed from GTEx.V8).",
+    "footnote": [],
+    "bbox": [
+      [
+        117,
+        81,
+        234,
+        280
+      ]
+    ],
+    "page_idx": 21
+  }
+]

peer_reviews/supplementary_0_Peer Review File__00d0f482762f2f37431ca49a939480fc54bdd5eb053d5ac8ce0b474c9dacda22/supplementary_0_Peer Review File__00d0f482762f2f37431ca49a939480fc54bdd5eb053d5ac8ce0b474c9dacda22.mmd

@@ -0,0 +1,435 @@
+
+# nature portfolio  
+
+Peer Review File  
+
+An engineered AAV targeting integrin alpha V beta 6 presents improved myotropism across species  
+
+![](images/Figure_unknown_0.jpg)
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+## REVIEWER COMMENTS  
+
+Reviewer #1 (Remarks to the Author):  
+
+This manuscript reports the design and characterization of a novel myotropic AAV vector, LICA1, by grafting natural \(\alpha \nabla \beta 6\) binding peptides to the VR4 of a liver- detargeted Cap9rh74 hybrid capsid. The authors demonstrate the efficient delivery of therapeutic transgenes to rescue dystrophic phenotypes in two mouse models with a lower dose compared to AAV9. As compared to the recently reported AAVMYO and MYOAAV capsids, the LICA1 showed comparable efficiency in striated muscles but had greatly reduced liver tropism. The experiments are well designed and the novel LICA1 is very promising for clinical translation. The following major and minor points should be addressed.  
+
+1. The authors chose VR4 instead of the more commonly pursued VR8 to insert the integrin binding peptide. VR4 region show some overlapping with AAVR binding site. An interesting question should be addressed is whether the novel LICA1 capsid requires AAVR for cellular entry and transduction. This should be experimentally tested.  
+
+2. According to the data shown in Supplementary Figure 1B, ITGAV exhibits a broad expression across different tissues, while ITGAB6 shows relatively restrictive expression in bladder, breast, esophagus, kidney, lung, muscle, prostate, testis, thyroid and vagina, with low expression in the heart. These expression patterns do not match well the in vivo biodistribution/transduction of LICA1. For example, kidney and lung show quite high level of \(\alpha \nabla\) and \(\beta 6\) integrin expression, but LICA1 shows very little transduction in the kidney and lung. Please provide an explanation in the discussion.  
+
+Minor points:  
+
+1. Line 219, MYOAAV should be "AAVMYO"?  
+2. Why are the vector copy number units shown in Fig. S3E (10^-4) several orders of magnitude different from that in Fig. S3D (eg. 10^6)?  
+3. In Figure 4 and 5, please use dot plot to present the data in the graphs.  
+4. In Figure5D, DAPI staining is hardly visible.  
+
+Reviewer #2 (Remarks to the Author):  
+
+Hong et al expand on previously published reports that the integrin binding RGD motif can confer high heart and skeletal muscle transduction by AAV capsids. Here, instead of a peptide insert based capsid library screening method, the authors utilize rational modification of an AAV capsid surface loop (IV) and molecular docking/modeling to determine optimal flanking residues and presentation of the RGD motif. In addition, the authors utilize a chimeric AAV9/rh74 chimeric capsid which is equivalent to AAV9 but displays liver detargeting potential. The study convincingly demonstrates that the new, rationally engineered AAV capsid variant, LICA- 1 can transduce muscle and avoid liver sequestration effectively. Therapeutic applications in DMD and LGMD mouse models are demonstrated. Overall the study is
+
+<--- Page Split --->
+
+thorough and technically well executed, the methods/experimentation and comparative analysis are robust and the manuscript well written. Some advantages such as improved liver detargeting over other AAVMyo or MyoAAV casids is demonstrated. However, no new mechanistic insight is evident or whether this is selective to mice. A related and significant concern is the lack of large animal translational data (as has been demonstrated with other myotropic AAVs). It is therefore unclear, whether LICA- 1 (although well characterized) presents a significant conceptual advance or alternative to existing engineered AAV vectors.  
+
+Reviewer #3 (Remarks to the Author):  
+
+In this paper, Vu Hong et al. generated and characterized a new muscle- tropic AAV capsid (designated as LICA1) using on an innovative approach based on molecular modelling to integrate alphaV- beta6 (aVb6) binding RGD motifs into the capsid. The study is overall well designed with comprehensive testing of biodistribution and improved therapeutic efficacy of AAV vectors based on this new LICA1 capsid (compared to AAV9) in two different mouse disease models of hereditary muscle disorders, namely Duchenne muscular dystrophy and limb girdle muscular dystrophy.  
+
+The results show improved muscle- targeted transduction and increased liver detargeting compared to AAV9 as a benchmark. Additional muscle tropic AAV variants were used that were recently described, namely AAVMYO, MYOAAV1A and MYOAAV2A. The increased muscle transduction obtained with LICA1 capsid is comparable to that attained by the previously described AAV MYO, MYOAAV1A and MYOAAV2A variants (while the latter is even more efficient for cardiac gene delivery).  
+
+## Major comments  
+
+1) Though this novel capsid appears to offer no clear advantage over AAVMYO, MYOAAV1A and MYOAAV2A in terms of muscle targeting, the authors claim that their capsid exhibits significantly reduced liver targeting compared to the state of the art (at least in comparison to AAV MYO, MYOAAV1A and MYOAAV2A). Unfortunately, the authors did not include the AAVMYO2 and AAVMYO3 capsids described by El-Andari (Ref. 16 for comparison which are at least as potent as AAVMYO but shows pronounced liver detargeting compared to AAVMYO (or AAV8 and AAV9). Hence, in the absence of these AAVMYO2 and/or AAVMYO3 capsids as benchmark, the superiority of LICA1 over existing muscle-targeted and liver-detargeted capsids has not been unequivocally demonstrated.  
+
+To ascertain the potential superior properties of LICA1 over the state of the art it is therefore necessary to compare vector performance head to head with AAVMYO2 and AAVMYO3. The available data in the current study along with the comprehensive analyses performed by El- Andari et al. suggest that this new AAV LICA1 capsid may offer only limited advantage compared to these AAVMYO2 and AAVMYO3. From a mechanistic perspective, the LICA1 variant also exploit the RGD motif for muscle targeting as in the case of AAVMYO, MYOAAV1A and MYOAAV2A, essentially confirming the crucial role of RGD and integrin- mediated muscle targeting as demonstrated by the previously published studies (Ref. 15, 16 & 17).  
+
+2) The therapeutic efficacy of the LICA1 variant in the DMD and LGMD mouse models was only
+
+<--- Page Split --->
+
+compared to AAV9 (Fig. 4 & 5) instead of also including the known myotropic variants AAV MYO, MYOAAV1A and MYOAAV2A that were used in the current study in the wild- type mice. To demonstrate the superiority of LICA1 over the state of the art, it would have been more compelling to also include the AAVMYO2 and AAVMYO3 variants for comparison.  
+
+3) It would appear that the increase in muscle transduction obtained with LICA1 (vs AAV9) based on vector copy number (VCN) in the dystrophic mdx model (at least in the quadriceps) is relatively modest (2-fold) (Fig. 4) compared to the relative increase transduction in wildtype mice (Fig. 3). What is the reason for this apparent discrepancy?  
+
+4) There appears to be no significant difference in vector copy number (VCN) in the diaphragm when comparing LICA1 with AAV9 in the mdx mice (Fig. 4). Why is there a significant difference in microDYS mRNA levels and DYS+ fibers despite the lack of any difference in VCN?  
+
+5) Fig. 3C suggest increased cardiac transduction with the LICA1 variant. Does this translate into increased cardiac transduction and phenotypic correction in the DMDM and LGMD disease models?  
+
+## Minor comments  
+
+1) Why is there only a partial inhibition of transduction (about 30%) in the blocking experiment with recombinant aVb6 protein (Fig. 2E)? Were different concentrations tested? Were equimolar amounts of the rSGCA control used?  
+
+2) The term 'infection' should not be used in the context of replication-defective viral vectors. The correct term is 'transduction'.
+
+<--- Page Split --->
+
+## Authors' responses to the reviewers  
+
+## Color code in this document:  
+
+Reviewers text in black  Author responses in blue  
+
+## General comments:  
+
+We thank the three reviewers for spending time with our manuscript and appreciate all the constructive comments and questions. We have carefully addressed all the comments and made the necessary revisions. This helped to improve considerably the manuscript. Please find below our point- by- point responses. Besides, there are some major changes in our revised manuscript.  
+
+In response to reviewer #2 concern about translatability of LICA1 in other species and the standard of this fast- moving field, we added a new Result section and new Fig 6/S6 where we examined whether LICA1 can improve transduction across species. The data includes characterization of \(\alpha \mathrm{V}\beta 6\) receptor cross- species in vitro and comparative study in multiple species, including in non- human primates. The result confirms the improved myotropic properties of LICA1 vector. We modified the title and abstract to a shorter version following the journal formatting guidelines while including the additional results. Additionally, we have made minor editorial changes throughout the manuscript to improve clarity and readability.
+
+<--- Page Split --->
+
+## REVIEWER COMMENTS  
+
+## Reviewer #1 (Remarks to the Author):  
+
+This manuscript reports the design and characterization of a novel myotropic AAV vector, LICA1, by grafting natural \(\alpha \mathrm{V}\beta 6\) binding peptides to the VR4 of a liver- detargeted Cap9rh74 hybrid capsid. The authors demonstrate the efficient delivery of therapeutic transgenes to rescue dystrophic phenotypes in two mouse models with a lower dose compared to AAV9. As compared to the recently reported AAVMYO and MYOAAV capsids, the LICA1 showed comparable efficiency in striated muscles but had greatly reduced liver tropism. The experiments are well designed and the novel LICA1 is very promising for clinical translation. The following major and minor points should be addressed.  
+
+We thank the reviewer for the supportive and helpful comments.  
+
+1. The authors chose VR4 instead of the more commonly pursued VR8 to insert the integrin binding peptide. VR4 region show some overlapping with AAVR binding site. An interesting question should be addressed is whether the novel LICA1 capsid requires AAVR for cellular entry and transduction. This should be experimentally tested.  
+
+Response: To answer this question, we created an AAVR- KO cell line (293_AAVR- KO) and evaluated the transduction efficiency of AAV_ITGs in this cell compared to 293_WT. Three gRNAs were designed to target exon 2 of AAVR, a common exon of all AAVR mRNA isoforms (genome- euro.ucsc.edu), co- transfected with SpCas9 in RNP form, and the individual clones were screened by western blot to confirm AAVR- KO at protein level (Fig S2H). In 293_AAVR- KO, the AAV entry was significantly reduced for both 9rh74_5f0 and 9rh74_4um9 by \(65.37\%\) and \(74.73\%\) , respectively, and their transgene activity was reduced by \(94.1\%\) and \(96.9\%\) , respectively, compared to 293_WT (Fig S2I). This result indicates that both AAV_ITGs require AAVR for successful transduction, similar to natural and other VR8-peptide- inserted AAV capsids \(^{1 - 4}\) .  
+
+We are reproducing Fig S2H- I below for the reviewer's convenience:  
+
+![](images/Figure_unknown_1.jpg)
+
+<center>Fig S2. H. Western blot showed the absence of AAVR at the protein level at its expected size in the KO clone. I. VCN and luciferase activities of AAV_ITGs in 293_WT and 293_AAVR-KO cells ( \(n = 4 - 6\) , one-way ANOVA). </center>  
+
+We also added the results in the revised manuscript (line 170- 178):
+
+<--- Page Split --->
+
+"We next examined the dependence of AAV_ITGs on the AAV's human cell entry receptor (AAVR) \(^{40}\) . Structural and molecular studies have previously highlighted the importance of AAVR binding in cellular transduction of multiple natural \(^{40,41}\) and engineered capsids \(^{17,42}\) where VR4 plays a partial role in AAVR binding. We created a 293_AAVR-KO cell line (Fig S2H) and test the viral transduction efficiency in comparison to 293_WT. Similar to other engineered capsids with peptide- inserted VR8 \(^{17}\) , AAV9rh74_4um9 and _5ff0 reduced the AAV entry by 65.37% and 74.73%, reduced the transgene activity by 94.10% and 96.90% with the absence of AAVR (Fig S2I, n=4- 6, p<0.0001). It suggests that AAV_ITGs both require AAVR for effective transduction."  
+
+As reviewer mentioned, AAVR binds to multiple AAV capsid serotypes at the 3- fold protrusions, mainly VR4, and 2/5- fold wall, despite diverse structural conformation within these regions across serotypes \(^{2}\) . Our result suggests that VR4- modified capsid can still bind to AAVR, presumably by other known AAVR- binding region including VR1, VR3, and VR8 \(^{2,5}\) . It was shown that altering the VR sequences/structures can negatively impact the binding affinity of AAV capsid with AAVR \(^{3,6}\) . However, binding affinity of AAVR with natural AAV, for example AAV2 (K<sub>d</sub> = \(\sim 150 \text{nm}\) ) \(^{1,3}\) , is much lower than the interaction between AAV2 and heparan sulfate (K<sub>d</sub> = 0.1- 3.7 nm) \(^{7}\) . It suggests that AAVR can function by interacting weakly with AAV capsid, therefore might explain the dependence of AAV_ITGs on AAVR for cellular transduction.  
+
+2. According to the data shown in Supplementary Figure 1B, ITGAV exhibits a broad expression across different tissues, while ITGAB6 shows relatively restrictive expression in bladder, breast, esophagus, kidney, lung, muscle, prostate, testis, thyroid and vagina, with low expression in the heart. These expression patterns do not match well the in vivo biodistribution/transduction of LICA1. For example, kidney and lung show quite high level of \(\alpha \mathrm{V}\) and \(\beta 6\) integrin expression, but LICA1 shows very little transduction in the kidney and lung. Please provide an explanation in the discussion.  
+
+Response: The mean expression level of ITGB6 in Fig S1B is admittedly high in a number of tissues, as reviewer pointed out. However, the graph presents only the mean, therefore does not illustrate sufficiently the distribution of the data and no tissue enrichment analysis was included. To compensate, we added new Fig S1C is the cell- type expression specificity analysis, which was performed in the same bulk GTEx RNA- seq dataset by an integrative co- expression analysis with known reference transcripts in each cell types for all tissues (The Human Protein Atlas) \(^{8,9}\) . This analysis therefore allows the comparison of gene expression in different cell types across tissues. The analysis showed that ITGB6 is highly enriched in both skeletal myocytes and cardiomyocytes, and ITGAV is highly enriched in cardiomyocytes. This might explain the enhanced transduction in the heart of AAV_ITGs and MYOAAVs \(^{4}\) .  
+
+We are reproducing Fig S1C below for the reviewer's convenience:
+
+<--- Page Split --->
+![](images/Figure_5.jpg)
+
+<center>Fig S1. C. The cell type-enriched transcriptome profiles of ITGAV and ITGB6 in a list of cell types from multiple tissues (graphs were generated from The Human Protein Atlas). MYH7 is a positive control that is known to highly enrich in the cardiac and skeletal muscles. </center>  
+
+We modified in the revised manuscript (line 96- 100):  
+
+"Furthermore, the cell type-enriched transcriptome profiles from bulk RNA sequencing data of multiple human tissues \(^{31}\) confirmed high expression and enrichment of both Itgav and Itgb6 in skeletal and cardiac muscles, and low expression of Itgb6 in the liver and spleen, two preferred targets of natural AAV (Figure S1B- C, GTEx V8, dbGaP Accession phs000424.v8.p2)."  
+
+We further discussed on the low transduction level seen in kidney and lung in the revised manuscript (line 485- 499).  
+
+"Despite high enrichment in the human skeletal muscle, ITGB6 – the subunit with more binding interaction with TGFβ-derived motifs, also shows relatively high enrichment in gastric mucous cells (stomach), urothelial cells (prostate), ductal cells (pancreas), and alveolar cells type 2 (AT2, lung), colon enterocytes (colon) and high bulk expression also in kidney, esophagus, and bladder (Fig S1B- C). There was very low AAV transduction observed in both kidney and lung, two organs with highest ITGB6 expression. Poor kidney and lung transduction despite high expression of targeted receptor in these two organs was also observed with other neurotropic engineered AAVs \(^{52, 53}\) . Route of administration in targeting kidney might explain the low transduction of AAV_ITGs in the kidney where local delivery via
+
+<--- Page Split --->
+
+the renal vein or the renal pelvis showed much better transduction than IV injection for multiple AAV serotypes \(^{54}\) . On the other hand, AT2 cells in the lung comprise only \(< 5\%\) of the alveolar surface area \(^{55}\) , therefore they have limited contact with pulmonary capillary blood, which might explain poor transduction efficiency in this organ. Natural AAVs were previously shown to transduce the gastrointestinal tract \(^{56}\) and pancreas \(^{57}\) , suggesting a potential transduction of AAV_ITGs in these organs. Further validation of these tissues is therefore required in future studies to confirm AAV_ITGs' specificity."  
+
+Minor points:  
+
+1. Line 219, MYOAAV should be "AAVMYO"?  
+
+Response: This was corrected.  
+
+2. Why are the vector copy number units shown in Fig. S3E (10^-4) several orders of magnitude different from that in Fig. S3D (eg. 10^6)?  
+
+Response: This is due to the difference in the normalization used and units being presented in the graph: VCN in Fig S3B-D was presented as number of AAV genome copies per \(10\mu \mathrm{l}\) assay while the VCN values in Fig S3E was calculated as total of viral genome detected normalized to the total \(1\mathrm{E}10\mathrm{vg}\) AAV initially infected (AAV copies per \(10\mu \mathrm{l}*20 / 10^{10}\) ). Details about the represented units were added in the graph in the revised manuscript.  
+
+3. In Figure 4 and 5, please use dot plot to present the data in the graphs.  
+
+Response: This was modified.  
+
+4. In Figure5D, DAPI staining is hardly visible.  
+
+Response: The images were now modified for better visibility in DAPI channel.  
+
+We are reproducing Fig 5D below for the reviewer's convenience.  
+
+![](images/Figure_6.jpg)
+
+<center>Fig 5. D. Immuno-staining of DAPI (blue) and SGCA (red) in Quadriceps muscles. </center>  
+
+Reviewer #2 (Remarks to the Author):
+
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+Hong et al expand on previously published reports that the integrin binding RGD motif can confer high heart and skeletal muscle transduction by AAV capsids. Here, instead of a peptide insert based capsid library screening method, the authors utilize rational modification of an AAV capsid surface loop (IV) and molecular docking/modeling to determine optimal flanking residues and presentation of the RGD motif. In addition, the authors utilize a chimeric AAV9/rh74 chimeric capsid which is equivalent to AAV9 but displays liver detargeting potential. The study convincingly demonstrates that the new, rationally engineered AAV capsid variant, LICA- 1 can transduce muscle and avoid liver sequestration effectively. Therapeutic applications in DMD and LGMD mouse models are demonstrated. Overall the study is thorough and technically well executed, the methods/experimentation and comparative analysis are robust and the manuscript well written. Some advantages such as improved liver detargeting over other AAVMyo or MyoAAV casids is demonstrated. However, no new mechanistic insight is evident or whether this is selective to mice. A related and significant concern is the lack of large animal translational data (as has been demonstrated with other myotropic AAVs). It is therefore unclear, whether LICA- 1 (although well characterized) presents a significant conceptual advance or alternative to existing engineered AAV vectors.  
+
+Response: We thank the reviewer for the supportive and helpful comments. We are agree that the translatability of LICA1 myotropic properties in different species is an important and reasonable concern, given that engineered capsids for brain (PHP.B variant \(^{10 - 12}\) ) and muscle (AAVMYO/- 2/- 3 family - ASGCT 2024, Abstract 214) can be selective to mouse. To answer this question, we used two different approaches: 1. Investigate the impact on cellular transduction of AAV_ITGs binding with its receptor, \(\alpha \nabla \beta 6\) , from different species; and 2. Validation of enhanced transduction in skeletal muscle in various models, including non- human primate (NHP). The data was added in the manuscript in Fig 6, Fig S6, and the text in line 382- 446.  
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+We are reproducing Fig 6 and Fig S6 below for the reviewer's convenience.
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+<center>Fig. 6: LICA1 showed cross-species enhanced transduction in the skeletal muscle. </center>  
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+A. Structure of human \(\alpha \mathrm{V}\beta 6\) binding to human TGF-β3-derived motif (pdb code: 4um9). The binding interface, defined as all amino acid in \(\alpha \mathrm{V}\beta 6\) with distance to the binding motif of less than 8Å, is highlighted. B. Alignment of ITGAV (upper panel) and ITGB6 (lower panel) protein sequence around the binding interface from multiple species. The binding interface defined in A is highlighted, and is identical across species being examined. Amino acids with distance to the binding motif of less than 6Å, are in bold and boxed. The sequence mismatches, only found outside the binding interface, are colored in red. C. Transduction efficiency, measured by luciferase activity, of AAV_ITGs but not AAV9 was inhibited by pre-incubating AAVs before infection with recombinant \(\alpha \mathrm{V}\beta 6\) protein from both human, rat, and mouse. 2E8vg AAVs were incubated with different \(\alpha \mathrm{V}\beta 6\) concentrations (0-120 nM) for 1 hour at \(37^{\circ}\mathrm{C}\) before added directly into cell medium (dose: 1E4 vg/cell, 96-well plate, duration: 24 hours, cell
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+line: 293_WT, \(n = 3\) ). The statistics were performed to compare with the condition of no aVβ6 protein during incubation (0 nM aVβ6) by using two- way ANOVA ( \(\sim\) AAV serotypes \* aVβ6 treatments). Same incubation conditions using 120nM of recombinant SGCA protein were used as the control, which showed no significant difference with 0nM aVβ6 condition. D. Scheme of comparative study between multiple AAV capsids in multiple species. Transgene expression cassette flanked between ITR sequences include the ORF of human inactivate CAPN3 protein under the control of skeletal muscle- specific promoter tMCK 49, SV40 polyA sequence, and DNA barcode (BC). BCs were placed between ORF and polyA sequence, which allow quantitative measurements of transgene expression mRNA level by RT- qPCR or NGS 17. Two or three barcodes were used for each capsid to minimize the sequence bias which might impact AAV transduction and PCR bias during RT- qPCR or NGS library preparation. AAV production and purification of each capsid variant was done separately, before pooled together at the equimolar amount before AAV transduction in vitro (F, human myotube, \(n = 2\) , dose: 2E10/2E11 vg per 12wp well, duration: 48 hours), or in vivo injection in C57Bl6 mice (E, \(n = 3\) , dose: 5E12 vg/kg per capsid variant, route: intravenous injection, duration: 21 days) and Macaca fascicularis NHP (G, \(n = 3\) , dose: 3.2E12 vg/kg per capsid variant, route: intravenous injection, duration: 45 days). E. The mRNA enrichment (BC_mRNA/Rplp0/ BC_AAV) of different capsid variants in the liver and three skeletal muscles in C57Bl6 mice measured by RT- qPCR. Each dot corresponds to the individual barcode. The color of each barcode corresponds to the individual mouse in Fig S6C. The statistics were performed on the average on different barcodes (using for the same capsid variant in each mouse) by using two- way ANOVA ( \(\sim\) mice \* capsids). F. The mRNA enrichment, measured by NGS, of different capsid variants in in vitro human myotubes at two AAV concentration. The log2 fold- change compared to AAV9 of all variants is presented. Each dot corresponds to the individual barcode. The colors correspond to different biological replicates. G. The mRNA enrichment (BC_mRNA/Rplp0/BC_AAV) of different capsid variants in the liver and skeletal muscles in NHP measured by RT- qPCR. Each dot corresponds to the individual barcode. The data was presented for individual primates (P1-3). The statistics were to compare the mRNA level of LICA1 with natural capsids, AAV8/AAV9, performed on the fold change of LICA1 to AAV8/AAV9 (averages of barcodes using for the same capsid variant in each primate were used to calculate the fold- change) by using two- tailed one- sample t- test.
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+<center>Fig. S6: LICA1 showed improved transduction across species. A. Predicted structures of \(\alpha \beta \beta\) heterocomplex from multiple species by AlphaFold2 showed very high similarity with the crystal structure of human form (pdb code: 4um9). The ITGAV and ITGB6 of human (ITGAV: aa31-467, ITGB6: 132-373), primate (ITGAV: aa31-467, ITGB6: 132-373), pig (ITGAV: aa31- </center>
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+466, ITGB6: 132- 373), rat (ITGAV: aa31- 466, ITGB6: 132- 373) and mouse (ITGAV: aa31- 467, ITGB6: 132- 373) were used for the prediction. The Alphafold2 hyperparameters includes: model=alphafold2_multimer_v3, num-recycle=8, templates=True. The prediction of the ITGAV-B6 heterodimer from all species were highly confident (pLDDT=96.06/96.00/95.72/95.71/95.66 for human, primate, pig, rat, and mouse, respectively). The superimpose of predicted structures colored by pLDDT values and experimental human structure in light grey are shown. RMSD values were calculated by ChimeraX software. B. Transduction efficiency, measured by luciferase activity, of AAV_ITGs but not AAV9 was inhibited by pre-incubating AAVs before infection with recombinant \(\alpha \mathrm{V}\beta 6\) protein from both human, rat, and mouse. 2E8vg AAVs were incubated with different \(\alpha \mathrm{V}\beta 6\) concentrations (0- 120 nM) for 1 hour at \(37^{\circ}C\) before added directly into cell medium (dose: 1E4 vg/cell, 96- well plate, duration: 24 hours, cell line: 293_ \(\alpha \mathrm{V}\beta 6\) , \(n = 3\) ). The statistics were performed to compare with the condition of no \(\alpha \mathrm{V}\beta 6\) protein during incubation (0 nM \(\alpha \mathrm{V}\beta 6\) ) by using two- way ANOVA ( \(\sim\) AAV serotypes \* \(\alpha \mathrm{V}\beta 6\) treatments). Same incubation conditions using 120nM of recombinant SGCA protein were used as the control, which showed no significant difference with 0nM \(\alpha \mathrm{V}\beta 6\) condition. C. Relative mRNA level of total hC3i to Rplp0 in different tissues in C57Bl6 mice, measured by RT- qPCR. The same colors were used as in Fig 6E, which correspond to the individual mice. D. In vitro assay to quantify the titers of neutralizing antibodies in the primate serum against AAV8, AAV9, and LICA1 before AAV injection. Different AAVs with GFP/Luc reporter transgene were incubated with primate serum at different dilutions or FBS for 1 hour before added in 293_E3 cells (n=1). Luciferase activities were quantified 24- hour post- transduction. E. Relative mRNA level of total hC3i to Rplp0 in different tissues in NHPs, measured by RT- qPCR. The colors of each dot correspond to the individual primates. DRG - dorsal root ganglia, LN - lymph node.  
+
+We believe that one of main strength of the study is that both AAV_ITGs were initially designed to bind to human form of \(\alpha \mathrm{V}\beta 6\) , where they were incorporated with binding motifs derived from human TGFβ1/3. In conventional in vivo directed evolution, AAV variants are identified based on the selective experimental pressure. Therefore, it lacks the interpretability of the transduction improvement and consequently whether the improvement can translate outside screening setup, for example other species or other uninvestigated organs/cell types. AAV- PHP.B is an example of AAV variant whose improvement is restricted in C57Bl6 mouse but not in other strains or species due to the lack of its receptor \(^{10 - 12}\) . Our approach is primarily different in which we aim to target a receptor that highly conserved across species, and more importantly start the design targeting the human form. This approach recently showed a great efficacy, with validation in either humanized mouse model \(^{13}\) (ASGCT 2024 – Abstract 120/991) or NHP (ASGCT 2023 – Abstract 42/468, ASGCT 2024 – Abstract 118/217/638). Besides, there is an increasing need for AAV receptor identification for interpretability \(^{4,10,14}\) (ASGCT 2024 – Abstract 975). It therefore indicates that the bottom- up approach might better translate in cross- species transduction.  
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+However, the characterization of \(\alpha \mathrm{V}\beta 6\) receptor and AAV- \(\alpha \mathrm{V}\beta 6\) interaction might be not sufficient in the previous version of manuscript and was added in the current version. We compared the sequences and predicted structures of aVb6 from human, primate, pig, rat, and mouse, and found identical sequence alignment and near- identical structure at the binding interface with TGFβ- derived peptides (Fig 6A- B, Fig S6A). It suggests similar binding interaction of AAV_ITGs with \(\alpha \mathrm{V}\beta 6\) in different species. We next tested how binding between AAV_ITGs and \(\alpha \mathrm{V}\beta 6\) in different species impact transduction by the inhibition assay where AAVs were incubated with different recombinant \(\alpha \mathrm{V}\beta 6\) proteins before infection.
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+It was shown in two cell lines that transduction level of AAV_ITGs, but not AAV9, were abolished with the \(\alpha \mathrm{V}\beta 6\) - preincubation, suggesting these interactions between AAV_ITGs and \(\alpha \mathrm{V}\beta 6\) is essential for the enhanced transduction. Besides, the improvement of AAV_ITGs, especially LICA1, was seen in human cell lines (293_ \(\alpha \mathrm{V}\beta 6\) and in vitro human differentiated myotubes) and skeletal muscle of mouse models. It indicates LICA1 functions similarly across species.  
+
+Furthermore, we compared LICA1 transduction level across species at mRNA level, similar to what has been described for other myotropic serotypes in NHP 4 (Fig 6D- G). Barcoded LICA1 and other serotypes were mixed at equal viral genome number before being co- infected/injected in in vitro differentiated human myotube, C57Bl6 mouse, and non- human primate. First, the multiplex comparison of LICA1 with AAV8/AAV9/AAV9rh74, 9rh74_5ffo and other myotropic AAVs, AAVMYO and MYOAAV2A showed similar results with individual comparison (Fig 6E and Fig 3). However, a similar pool of capsids (except not including AAVMYO) used in in vitro differentiated human myotube showed contradicting results with data obtained in mouse and previous individual comparison also performed in the same model. Despite again confirming better transduction level than AAV8/AAV9/AAV9rh74 of all myotropic variants, including LICA1, 9rh74_5ffo showed unexpected considerable higher transduction level than both LICA1 and MYOAAV2A in all biological replicates and 2 conditions with different AAV doses infected (Fig 6F). This observation together with a more significant inhibition by human \(\alpha \mathrm{V}\beta 6\) pre- incubation in 9rh74_5ffo than LICA1 (Fig 2E, 6C), it suggested a competition of myotropic AAVs over the \(\alpha \mathrm{V}\beta 6\) receptor – a putative receptor for both LICA1, 9rh74_5ffo, and MYOAAV2A. The competition between capsid variants during pooled transduction has been reported before 15. With the potential competition of \(\alpha \mathrm{V}\beta 6\) - binding myotropic AAVs and the profile of Nab against multiple tested capsid variants (Table S3, Fig S6D, no primate was found to be Nab- negative for all variants), we decided to conduct a comparison of LICA1 with only AAV8 or AAV9, 2 capsid variants currently used in clinical trials for muscular diseases 16, in 3 NHPs with lowest LICA1- Nab levels. In these NHPs, we confirmed the significant improved expression in skeletal muscle and strong liver detargeting of LICA1 compared to AAV8 and AAV9 benchmarks (Fig 6G).  
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+Altogether, we believe that we have provided sufficient evidence to support the translatability of LICA variant across species, including non- human primate.  
+
+## Reviewer #3 (Remarks to the Author):  
+
+In this paper, Vu Hong et al. generated and characterized a new muscle- tropic AAV capsid (designated as LICA1) using on an innovative approach based on molecular modelling to integrate alphaV- beta6 (aVb6) binding RGD motifs into the capsid. The study is overall well designed with comprehensive testing of biodistribution and improved therapeutic efficacy of AAV vectors based on this new LICA1
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+capsid (compared to AAV9) in two different mouse disease models of hereditary muscle disorders, namely Duchenne muscular dystrophy and limb girdle muscular dystrophy.  
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+The results show improved muscle- targeted transduction and increased liver detargeting compared to AAV9 as a benchmark. Additional muscle tropic AAV variants were used that were recently described, namely AAVMYO, MYOAAV1A and MYOAAV2A. The increased muscle transduction obtained with LICA1 capsid is comparable to that attained by the previously described AAV MYO, MYOAAV1A and MYOAAV2A variants (while the latter is even more efficient for cardiac gene delivery).  
+
+We thank the reviewer for the supportive and helpful comments.  
+
+Major comments  
+
+1) Though this novel capsid appears to offer no clear advantage over AAVMYO, MYOAAV1A and MYOAAV2A in terms of muscle targeting, the authors claim that their capsid exhibits significantly reduced liver targeting compared to the state of the art (at least in comparison to AAV MYO, MYOAAV1A and MYOAAV2A). Unfortunately, the authors did not include the AAVMYO2 and AAVMYO3 capsids described by El-Andari (Ref. 16 for comparison which are at least as potent as AAVMYO but shows pronounced liver detargeting compared to AAVMYO (or AAV8 and AAV9). Hence, in the absence of these AAVMYO2 and/or AAVMYO3 capsids as benchmark, the superiority of LICA1 over existing muscle-targeted and liver-detargeted capsids has not been unequivocally demonstrated.  
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+To ascertain the potential superior properties of LICA1 over the state of the art it is therefore necessary to compare vector performance head to head with AAVMYO2 and AAVMYO3. The available data in the current study along with the comprehensive analyses performed by El- Andari et al. suggest that this new AAV LICA1 capsid may offer only limited advantage compared to these AAVMYO2 and AAVMYO3. From a mechanistic perspective, the LICA1 variant also exploit the RGD motif for muscle targeting as in the case of AAVMYO, MYOAAV1A and MYOAAV2A, essentially confirming the crucial role of RGD and integrin- mediated muscle targeting as demonstrated by the previously published studies (Ref. 15, 16 & 17).  
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+Response: To response to the request of reviewer, we performed head- to- head comparison of LICA1 with AAVMYO2 and AAVMYO3 17 as presented in Fig 3G- H. We found no significant difference between the level of virus accumulation in the liver of LICA1 and AAVMYO- 2/3, yet mRNA level of AAVMYO- 2/3 is significantly lower than LICA1. However, in a more important aspect of a myotropic capsid – ability to transduce skeletal muscle, LICA1 is considerably better than both AAVMYO- 2/3 in all skeletal and cardiac muscles tested, in both virus entry and transgene activity. Of note, AAVMYO- 2/3 were also shown in the original publication to have lower muscle transduction than AAVMYO in most muscle tested (Fig 3D, Fig S5 from 17). The data was now added as Fig S3F- I and the text was modified in the revised manuscript. We also modified our previous claim of “AAV9rh74_4um9
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+transduced skeletal muscle similarly, but detargeted the liver more strongly than other myotropic AAVs" to "AAV9rh74_4um9 exhibits highest tropism towards the skeletal muscle among tested myotropic AAVs in mice" (line 207- 208).  
+
+We are reproducing Fig S3F- I below for the reviewer's convenience.  
+
+![](images/Figure_unknown_3.jpg)
+
+<center>Fig S3. F-G. VCN (F) and gene expression (G) (GFP mRNA level in liver and luciferase activity in other organs) of AAV9rh74_4um9, MYOAAV2, and MYOAAV3 18 in liver, skeletal muscles, heart, lung, kidney, and brain (n=4, one-way ANOVA). VCN and mRNA levels of AAV9 and MYOAAV1A in the previous comparison (Fig 3G-H) were added as reference. H-I. Ratio of VCN found in skeletal muscle; TA (H) and Qua (I); to the level in the liver one-month post-injection (n=4, one-way ANOVA). </center>  
+
+We added the results section (line 236- 247):  
+
+"Recently, AAVMYO2 and AAVMYO3 myotropic variants were identified to be more liver- detargeted while transducing skeletal and cardiac muscles less effectively than AAVMYO<sup>16</sup>. We then conducted a similar head- to- head comparison of these two variants with 9rh74_4um9 (6- week- old WT mice, dose: 1E13 vg/kg, n=4). All three capsids showed very low transduction in kidney, lung and brain (Fig S3F). Similar to previous AAVMYO comparison<sup>16</sup>, AAVMYO2 and AAVMYO3 both showed significantly lower VCN (3.24- 7.44/2.61- 6.16 times, respectively, \(p< 0.001\) ) and transgene activity (12.41- 18.42/11.1- 15.91 times, respectively, \(p< 0.05\) ) in all tested skeletal/cardiac muscles compared to LICA1 (Fig S3F- G). On another hand, the liver transduction of AAVMYO2/3 is not significantly different from that of LICA1, yet the transgene expression is significantly lower (Fig S3F- G). Altogether, LICA1 and AAVMYO2/3 showed
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+highest ratio of muscle- to- liver transduction in all tested myotropic capsids (Fig S3H- I), yet LICA1 is considerably better in transgene delivery and expression in skeletal muscles than AAVMYO2/3."  
+
+An important question is whether the RGD- containing capsids can show improved transduction in the skeletal muscle across species, especially in non- human primate (NHP). MYOAAV family has been discovered and validated in NHP \(^{4}\) . In this revised manuscript, we also validated significantly enhanced transduction of LICA1 in NHP (please see the discussion with Reviewer #2 for the attention of reviewer). However, in the latest data presented at ASGCT 2024 (Abstract 214), researchers showed no improvement of either AAVMYO, AAVMYO2, AAVMYO3, all shared P1 peptide – RGDLGLS, compared to natural capsids, AAV8, AAV9. It is worth noting that while LICA1 and MYOAAV family both target \(\alpha \mathrm{V}\beta 6\) while P1 peptide from AAVMYO/AAVMYO- 2/3 potentially targets \(\alpha \mathrm{V}\beta 8\) or \(\beta 3 / \beta 5^{18}\) , which is lowly enriched in human skeletal myocyte (Figure R1 below for the attention of the reviewer). It suggests that the integrin subunits that RGD- containing capsid target is essentially important for cross- species transduction. Besides, an alternative explanation is a potential competition between AAV serotypes being pooled and co- injected, as seen in Fig S6D or by others \(^{15}\) . Since different AAV serotypes were identified by various methodologies (LICA1 – rational design, MYOAAVs – directed evolution which is known to perform very well in competing conditions such as library transduction, and AAVMYO/- 2/- 3 – seminational design), evaluation methods should be carefully taken into account.
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+<center>Figure R1: Tissue Cell Type enrichment score of different ITGB subunits' mRNA in human biopsies (Figures generated by The Human Protein Atlas). </center>  
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+2) The therapeutic efficacy of the LICA1 variant in the DMD and LGMD mouse models was only compared to AAV9 (Fig. 4 & 5) instead of also including the known myotropic variants AAV MYO, MYOAAV1A and MYOAAV2A that were used in the current study in the wild-type mice. To demonstrate the superiority of LICA1 over the state of the art, it would have been more compelling to also include the AAVMYO2 and AAVMYO3 variants for comparison.  
+
+Response: We strongly believe that we have provided sufficient evidence to support the myotropic properties of LICA1 in various cellular and animal models. Besides, we only claimed the equivalent transduction level in murine skeletal muscle between LICA1, AAVMYO and MYOAAV- 1A/2A. The difference between myotropic serotypes was only observed in the level of liver detargeting, which aims to reduce the risk of hepatoxicity mainly reported at very high- dose AAV injection, in human patients 19, 20 or NHP 21. Therefore, we do not expect any difference in muscle transduction level as well as liver
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+toxicity phenotypes (not observed in the present study) between LICA1 and other mAAVs at this low-dose treatment (5E12 vg/kg). Besides, we observed a considerably better transduction level of LICA1 than AAVMYO- 2/3 as added in the revised manuscript, while no improvement of AAVMYO- 2/3 has been observed in NHP as reported in ASGCT 2024 (Abstract 214). We therefore think that the comparison is unnecessary for the present manuscript because there would be no added value for translatability. However, we completely agree with the reviewer that the comparison of LICA1 with other myotropic AAVs at higher dose regarding adverse events, especially in the liver, is important for future studies.  
+
+3) It would appear that the increase in muscle transduction obtained with LICA1 (vs AAV9) based on vector copy number (VCN) in the dystrophic mdx model (at least in the quadriceps) is relatively modest (2-fold) (Fig. 4) compared to the relative increase transduction in wildtype mice (Fig. 3). What is the reason for this apparent discrepancy?  
+
+Response: We have two hypotheses for the discrepancy of VCN transduction level between WT and dystrophic muscle.  
+
+First, the VCN data was presented as vector genome found per nucleus. In dystrophic muscle compared to WT, number of nuclei significantly increases due to the infiltration of immune cells, newly formed myocytes, proliferated satellite cells and fibrotic cells. Similarly, the nuclei number differs also between effective or ineffective treated muscles, where cellular composition can alter according to immune response, fibrosis level, and muscle regeneration. The discrepancy of denominator between conditions therefore makes it difficult to compare between two conditions, in addition to other technical differences between protocols (age of animals: 6-week versus 4-week, duration of the treatment: 21-day versus 28- day, and the dose: 1E13 versus 5E12 vg/kg). Normalization of the viral entry in skeletal muscle should be more appropriate by dividing to number of myocyte nuclei only, but rather practically difficult.  
+
+Second, discrepancy of transduction efficiency between WT and dystrophic muscle or injury- induced muscle regeneration has been reported with AAV1<sup>22</sup>. It suggests that the muscle physiology plays an important role in viral transduction. Since LICA1 targets specifically \(\alpha \mathrm{V}\beta 6\) , which is shown to constantly trafficked actively participate in endocytic- exocytic pathways in cells<sup>23</sup>, we hypothesize that \(\alpha \mathrm{V}\beta 6\) - dependent intracellular trafficking might explain the global transduction improvement of LICA1, in addition to improved cellular internalization at the plasma membrane. Indeed, we and others have recently reported dysregulated endocytosis in multiple muscular dystrophies<sup>24, 25</sup> (Myology 2024 - Presentation S5L3). Concurrently, we have observed upregulation of ITGB6 in dystrophic myocytes (Fig S1A). This might also explain the discrepancy in the \(\mathrm{FC}_{\mathrm{LICA1 / AAV9}}\) between models. Nonetheless, the impact of dysregulated endocytosis on LICA1 transduction in dystrophic muscles remains inadequately understood and will be a focus of our future research. We added this point in the discussion of the revised manuscript.
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+We added elements of discussion in the revised manuscript (line 500- 510):  
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+"Mechanism of enhanced transduction by \(\alpha \mathrm{V}\beta 6\) binding remains unclear. In most cases, the improved transduction of AAV_ITGs and other mAAVs was evident at the VCN level, indicating better cell entry via \(\alpha \mathrm{V}\beta 6\) binding. In some cases, for example observed in Dia and Heart of dystrophic mice, the improvement is only seen at the transgene expression level, suggesting the role of \(\alpha \mathrm{V}\beta 6\) beyond cellular internalization. Indeed, integrins are constantly trafficked in cells, initially internalized by either clathrin- mediated or clathrin- independent endocytosis to enter the endocytic- exocytic pathway before being recycled \(^{58}\) . The foot- and- mouth disease virus, which contains \(\alpha \mathrm{V}\beta 6\) - binding RGD motif to infect cells, also requires EEA1/Rab5- positive vesicles for intracellular trafficking \(^{59,60}\) , suggesting a similar trafficking mechanism with \(\alpha \mathrm{V}\beta 6\) - targeting AAVs. Future studies are necessary to better understand the \(\alpha \mathrm{V}\beta 6\) 's role in intracellular myotropic AAV trafficking within endocytic system."  
+
+4) There appears to be no significant difference in vector copy number (VCN) in the diaphragm when comparing LICA1 with AAV9 in the mdx mice (Fig. 4). Why is there a significant difference in microDYS mRNA levels and DYS+ fibers despite the lack of any difference in VCN?  
+
+Response: The \(\mathrm{FC}_{\mathrm{LICA1 / AAV9}}\) of mRNA is much higher than that of VCN in all muscles tested ( \(\mathrm{FC}_{\mathrm{TA:VCN}} = 1.86\) versus \(\mathrm{FC}_{\mathrm{TA:mRNA}} = 4.56\) ; \(\mathrm{FC}_{\mathrm{Qua:VCN}} = 2.03\) versus \(\mathrm{FC}_{\mathrm{Qua:mRNA}} = 5.56\) ; \(\mathrm{FC}_{\mathrm{Dia:VCN}} = 1.06\) versus \(\mathrm{FC}_{\mathrm{Dia:mRNA}} = 7.57\) ). In the Fig S3B- E, there was also discrepancy between VCN and transgene expression of all tested myotropic AAVs in multiple cell lines. Similarly, in the library screening, no correlation between VCN and mRNA level of AAV variants was observed in multiple tissues \(^{4}\) . We hypothesize that, as discussed in point 3, the transduction improvement of LICA1 is not only from cell entry by better internalization, but also largely due to the \(\alpha \mathrm{V}\beta 6\) - dependent intracellular endocytic trafficking. The foot- and- mouth disease virus, which contains RGD motif to bind to \(\alpha \mathrm{V}\beta 6\) to infect cells \(^{26,27}\) , also requires EEA1/Rab5- positive vesicles for intracellular trafficking, suggesting a similar trafficking mechanism with \(\alpha \mathrm{V}\beta 6\) - targeting AAVs. Besides, it was also suggested that the role of AAV universal receptor, AAVR, might lie more within the endosome trafficking than virus uptake in the cell surface \(^{28}\) . Besides trafficking, another possible explanation is that the capsid can influence the epigenetic profile the vector genome, subsequently can impact the transgene expression \(^{29}\) . Further studies are therefore required to explore the mechanisms underlying this phenomenon.  
+
+5) Fig. 3C suggest increased cardiac transduction with the LICA1 variant. Does this translate into increased cardiac transduction and phenotypic correction in the DMDM and LGMD disease models?  
+
+Response: We want to clarify that we initially did not expect an improvement in heart transduction of LICA1 since its receptor, ITGB6, expresses relatively lowly in heart compared to skeletal muscle (Figure R2 below for the attention of reviewer), despite high enrichment score comparing to other cell types from other tissues (Fig S1C in the revised manuscript in a response to reviewer #1). It might
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+explain a lower improvement of LICA1 in heart \((\mathrm{FC}_{\mathrm{Luc}|\mathrm{Heart:LICA1 / AAV9}} = 4.65)\) compared to what seen in skeletal muscle \((\mathrm{FC}_{\mathrm{Luc}|\mathrm{Qua:LICA1 / AAV9}} = 17.52)\) .  
+
+In response to the question of reviewer, we compared the VCN and mRNA level in the heart, and showed improvement in heart transduction of LICA1 over AAV9 in both models. Again, the improvement and expression level in heart is modest compared to what seen in skeletal muscles. We added the data in Fig 4A, 4B, 5B, 5C, and modified the text in the revised manuscript.  
+
+We are reproducing Fig 4A- B and Fig 5B- C below for the reviewer's convenience.  
+
+![](images/Figure_5.jpg)
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+<center>Fig 4. Comparison of transduction efficacy between AAV9 and LICA1 in three skeletal muscles and heart, in terms of VCN (A), and \(\mu\) Dys mRNA level (B). </center>  
+
+We added in the main text (line 284- 286): "In the heart, there were no difference in VCN but slightly significantly higher in mRNA level in LICA1- treated group compared to AAV9 (Fig 4A- B, \(FC_{VCN} = 0.72\) , \(p = 0.073\) ; \(FC_{mRNA} = 1.39\) , \(p = 0.021\) )."  
+
+![](images/Figure_unknown_4.jpg)
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+<center>Fig 5. Comparison of transduction efficacy between AAV9 and LICA1 in three skeletal muscles and heart, in terms of VCN (A), and hSGCA mRNA level (B). </center>  
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+We added in the main text (line 342- 344): "Besides, slight significant increase was observed in both VCN and mRNA level in LICA1- treated group compared to AAV9 in the heart (Fig 5B- C, \(FC_{VCN} = 2.01\) , \(p = 0.0069\) ; \(FC_{mRNA} = 5.11\) , \(p = 0.018\) )."  
+
+There is no cardiac pathology reported at 4- week of age in both mouse models \(^{30,31}\) . We therefore did not examine the cardiac phenotype after AAV injection in the present study. However, we agree that it is important to access cardiac phenotypes of different AAV treatment in more advanced pathological stages.
+
+<--- Page Split --->
+![PLACEHOLDER_22_0]
+
+<center>Figure R2: The bulk mRNA expression of ITGB6 in skeletal muscle, heart, and liver from human biopsies (data was reanalyzed from GTEx.V8). </center>  
+
+Minor comments  
+
+1) Why is there only a partial inhibition of transduction (about \(30\%\) ) in the blocking experiment with recombinant aVb6 protein (Fig. 2E)? Were different concentrations tested? Were equimolar amounts of the rSGCA control used?  
+
+Response: We added a more comprehensive experiment in Fig 6C and Fig S6B, with different concentrations of recombinant \(\alpha \mathrm{V}\beta 6\) from different species in responding to Reviewer #2. In Fig 2E, \(2\mu \mathrm{g}\) of recombinant protein, both for SGCA control and \(\alpha \mathrm{V}\beta 6\) , were incubated with \(1\times 10^{10}\mathrm{vg}\) AAV. In Fig 6C, SCGA control was used at the same equimolar amount as the highest concentration tested with \(\alpha \mathrm{V}\beta 6\) ( \(120\mathrm{nM}\) ). Details about the concentration was added in the figure legends. The reason for partial inhibition in LICA1 in Fig 2E is due to the low concentration of recombinant protein, which of equivalent of \(\sim 1.5\mathrm{nM}\) of what has been presented in Fig 6C, S6B. For comparison, LICA1 transduction level was reduced by \(81.64\%\) at \(30\mathrm{nM}\) and by \(94.2\%\) at \(60\mathrm{nM}\) .  
+
+2) The term 'infection' should not be used in the context of replication-defective viral vectors. The correct term is 'transduction'.  
+
+Response: This was modified in the revised manuscript.
+
+<--- Page Split --->
+
+## REFERENCES  
+
+1. Pillay, S. et al. An essential receptor for adeno-associated virus infection. Nature 530, 108-112 (2016).  
+2. Zhang, R. et al. Divergent engagements between adeno-associated viruses with their cellular receptor AAVR. Nature communications 10, 3760 (2019).  
+3. Xu, G. et al. Structural basis for the neurotropic AAV9 and the engineered AAVPHP.eB recognition with cellular receptors. Molecular therapy. Methods & clinical development 26, 52-60 (2022).  
+4. Tabebordbar, M. et al. Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell 184, 4919-4938 e4922 (2021).  
+5. Meyer, N.L. et al. Structure of the gene therapy vector, adeno-associated virus with its cell receptor, AAVR. Elife 8 (2019).  
+6. Jang, S., Shen, H.K., Ding, X., Miles, T.F. & Gradinaru, V. Structural basis of receptor usage by the engineered capsid AAV-PHP.eB. Molecular therapy. Methods & clinical development 26, 343-354 (2022).  
+7. Zhang, F. et al. Characterization of interactions between heparin/glycosaminoglycan and adeno-associated virus. Biochemistry 52, 6275-6285 (2013).  
+8. Dusart, P. et al. A tissue centric atlas of cell type transcriptome enrichment signatures. bioRxiv, 2023.2001.2010.520698 (2023).  
+9. Norreen-Thorsen, M. et al. A human adipose tissue cell-type transcriptome atlas. Cell Rep 40, 111046 (2022).  
+10. Hordeaux, J. et al. The GPI-Linked Protein LY6A Drives AAV-PHP.B Transport across the Blood-Brain Barrier. Molecular therapy : the journal of the American Society of Gene Therapy 27, 912-921 (2019).  
+11. Hordeaux, J. et al. The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL/6J Mice. Molecular therapy : the journal of the American Society of Gene Therapy 26, 664-668 (2018).  
+12. Liguore, W.A. et al. AAV-PHP.B Administration Results in a Differential Pattern of CNS Biodistribution in Non-human Primates Compared with Mice. Molecular therapy : the journal of the American Society of Gene Therapy 27, 2018-2037 (2019).  
+13. Huang, Q. et al. An AAV capsid reprogrammed to bind human Transferrin Receptor mediates brain-wide gene delivery. bioRxiv, 2023.2012.2020.572615 (2023).  
+14. Shay, T.F. et al. Primate-conserved carbonic anhydrase IV and murine-restricted LY6C1 enable blood-brain barrier crossing by engineered viral vectors. Science advances 9, eadg6618 (2023).  
+15. de Alencastro, G. et al. Tracking Adeno-Associated Virus Capsid Evolution by High-Throughput Sequencing. Human gene therapy 31, 553-564 (2020).  
+16. Manini, A., Abati, E., Nuredini, A., Corti, S. & Comi, G.P. Adeno-Associated Virus (AAV)-Mediated Gene Therapy for Duchenne Muscular Dystrophy: The Issue of Transgene Persistence. Frontiers in neurology 12, 814174 (2021).  
+17. El Andari, J. et al. Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders. Science advances 8, eabn4704 (2022).  
+18. Bauer, A. et al. Molecular Signature of Astrocytes for Gene Delivery by the Synthetic Adeno-Associated Viral Vector rAAV9P1. Adv Sci (Weinh) 9, e2104979 (2022).  
+19. Chand, D. et al. Hepatotoxicity following administration of onasemnogene abeparvovec (AVXS-101) for the treatment of spinal muscular atrophy. J Hepatol 74, 560-566 (2021).  
+20. Duan, D. Lethal immunotoxicity in high-dose systemic AAV therapy. Molecular therapy : the journal of the American Society of Gene Therapy 31, 3123-3126 (2023).  
+21. Hudry, E. et al. Liver injury in cynomolgus monkeys following intravenous and intrathecal scAAV9 gene therapy delivery. Molecular therapy : the journal of the American Society of Gene Therapy 31, 2999-3014 (2023).  
+22. Mollard, A. et al. Muscle regeneration affects Adeno Associated Virus 1 mediated transgene transcription. Sci Rep 12, 9674 (2022).  
+23. Moreno-Layseca, P., Icha, J., Hamidi, H. & Ivaska, J. Integrin trafficking in cells and tissues. Nat Cell Biol 21, 122-132 (2019).
+
+<--- Page Split --->
+
+24. Amor, F. et al. Cholesterol metabolism is a potential therapeutic target in Duchenne muscular dystrophy. J Cachexia Sarcopenia Muscle 12, 677-693 (2021).  
+
+25. Yu, L. et al. Small-molecule activation of lysosomal TRP channels ameliorates Duchenne muscular dystrophy in mouse models. Science advances 6, eaaz2736 (2020).  
+
+26. Jackson, T., Sheppard, D., Denyer, M., Blakemore, W. & King, A.M. The epithelial integrin alphavbeta6 is a receptor for foot-and-mouth disease virus. Journal of virology 74, 4949-4956 (2000).  
+
+27. Kotecha, A. et al. Rules of engagement between alphavbeta6 integrin and foot-and-mouth disease virus. Nature communications 8, 15408 (2017).  
+
+28. Summerford, C., Johnson, J.S. & Samulski, R.J. AAVR: A Multi-Serotype Receptor for AAV. Molecular therapy: the journal of the American Society of Gene Therapy 24, 663-666 (2016).  
+
+29. Gonzalez-Sandoval, A. et al. The AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner. Nature communications 14, 2448 (2023).  
+
+30. Duclos, F. et al. Progressive muscular dystrophy in alpha-sarcoglycan-deficient mice. The Journal of cell biology 142, 1461-1471 (1998).  
+
+31. Stedman, H.H. et al. The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature 352, 536-539 (1991).
+
+<--- Page Split --->
+
+## REVIEWERS' COMMENTS  
+
+Reviewer #1 (Remarks to the Author):  
+
+The authors have done an excellent job of addressing the issues that were raised.  
+
+Reviewer #2 (Remarks to the Author):  
+
+The authors have carried out additional experiments and satisfactorily addressed concerns.  
+
+Reviewer #3 (Remarks to the Author):  
+
+The authors have significantly improved the manuscript and addressed most of my comments. In particular, the authors have included non- human primate studies and extended upon the potential translatability of their work which is a very important addition to the paper that further strengthens the study. There were still a few issues that were not completely resolved but the authors provided possible explanations that could account for the observations and that were adequately discussed, without requiring further experimentation. This is an important and timely study that contributes to a better understanding of developing muscle- tropic vectors for gene therapy.  
+
+There are just a few remaining minor comments that the authors should address, which is relatively straightforward:  
+
+Minor comments  
+
+1. Some of the figures are too small and may be very difficult to read in the final version of the manuscript once it is printed. I would suggest to improve those and increase the font sizes significantly.  
+
+2. The exact nature of the capsid DNA sequence and the corresponding myotropic target peptide is not \(100\%\) clear. In particular, some amino acids from the AAV9 rh74 loop were deleted, whereas some additional amino acids were included as linker. It would appear that the precise sequencing conferring muscle-specificity was not clearly disclosed, only the "consensus motif" from the TGF-β1 and TGF-β3 domains.  
+
+To exclude any remaining ambiguity it is therefore important to include the actual DNA sequence of the relevant capsid sequence with the muscle-tropic targeting sequence, the deleted AAV9 rh74 sequence and the additional flanking sequences would be included in the paper along with the corresponding translated amino acid sequence.
+
+<--- Page Split --->
+
+# 1. 1. 1. 1. 1. 1. 1. 2. 2. 2. 2. 2. 2. 2.
+
+<--- Page Split --->
+
+## Authors' responses to the reviewers  
+
+## Color code in this document:  
+
+Reviewers text in black  Author responses in blue  
+
+## Reviewer #1 (Remarks to the Author):  
+
+The authors have done an excellent job of addressing the issues that were raised.  
+
+Response: We would like to thank the reviewer for your time and for recommending our manuscript for publication.  
+
+## Reviewer #2 (Remarks to the Author):  
+
+The authors have carried out additional experiments and satisfactorily addressed concerns.  
+
+Response: We would like to thank the reviewer for your time and for recommending our manuscript for publication.  
+
+## Reviewer #3 (Remarks to the Author):  
+
+The authors have significantly improved the manuscript and addressed most of my comments. In particular, the authors have included non- human primate studies and extended upon the potential translatability of their work which is a very important addition to the paper that further strengthens the study. There were still a few issues that were not completely resolved but the authors provided possible explanations that could account for the observations and that were adequately discussed, without requiring further experimentation. This is an important and timely study that contributes to a better understanding of developing muscle- tropic vectors for gene therapy.  
+
+Response: We would like to thank the reviewer for your time and for recommending our manuscript for publication.  
+
+There are just a few remaining minor comments that the authors should address, which is relatively straightforward:  
+
+Minor comments  
+
+1. Some of the figures are too small and may be very difficult to read in the final version of the manuscript once it is printed. I would suggest to improve those and increase the font sizes significantly. Response: We have reformatted the figures to comply with Nature Communications' submission guidelines and enhance readability.  
+
+2. The exact nature of the capsid DNA sequence and the corresponding myotropic target peptide is not \(100\%\) clear. In particular, some amino acids from the AAV9 rh74 loop were deleted, whereas some additional amino acids were included as linker. It would appear that the precise sequencing conferring muscle-specificity was not clearly disclosed, only the "consensus motif" from the TGF-β1 and TGF-β3 domains.  
+
+To exclude any remaining ambiguity it is therefore important to include the actual DNA sequence of the relevant capsid sequence with the muscle-tropic targeting sequence, the deleted AAV9 rh74 sequence
+
+<--- Page Split --->
+
+and the additional flanking sequences would be included in the paper along with the corresponding translated amino acid sequence.  
+
+Response: We have added Data File S3, which includes the DNA and amino acid sequences of all capsid variants generated in this study, with sequence differences between constructs highlighted.  
+
+In the Data Availability section, we have added:  
+
+"The DNA and amino acid sequences of AAV capsid variants used in this study are provided in the data file S3".
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__00d0f482762f2f37431ca49a939480fc54bdd5eb053d5ac8ce0b474c9dacda22/supplementary_0_Peer Review File__00d0f482762f2f37431ca49a939480fc54bdd5eb053d5ac8ce0b474c9dacda22_det.mmd

@@ -0,0 +1,567 @@
+<|ref|>title<|/ref|><|det|>[[61, 40, 508, 90]]<|/det|>
+# nature portfolio  
+
+<|ref|>text<|/ref|><|det|>[[70, 110, 362, 139]]<|/det|>
+Peer Review File  
+
+<|ref|>text<|/ref|><|det|>[[70, 154, 916, 212]]<|/det|>
+An engineered AAV targeting integrin alpha V beta 6 presents improved myotropism across species  
+
+<|ref|>image<|/ref|><|det|>[[57, 732, 240, 782]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[250, 732, 912, 785]]<|/det|>
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[116, 90, 290, 106]]<|/det|>
+## REVIEWER COMMENTS  
+
+<|ref|>text<|/ref|><|det|>[[116, 127, 392, 143]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[114, 162, 877, 290]]<|/det|>
+This manuscript reports the design and characterization of a novel myotropic AAV vector, LICA1, by grafting natural \(\alpha \nabla \beta 6\) binding peptides to the VR4 of a liver- detargeted Cap9rh74 hybrid capsid. The authors demonstrate the efficient delivery of therapeutic transgenes to rescue dystrophic phenotypes in two mouse models with a lower dose compared to AAV9. As compared to the recently reported AAVMYO and MYOAAV capsids, the LICA1 showed comparable efficiency in striated muscles but had greatly reduced liver tropism. The experiments are well designed and the novel LICA1 is very promising for clinical translation. The following major and minor points should be addressed.  
+
+<|ref|>text<|/ref|><|det|>[[115, 309, 864, 381]]<|/det|>
+1. The authors chose VR4 instead of the more commonly pursued VR8 to insert the integrin binding peptide. VR4 region show some overlapping with AAVR binding site. An interesting question should be addressed is whether the novel LICA1 capsid requires AAVR for cellular entry and transduction. This should be experimentally tested.  
+
+<|ref|>text<|/ref|><|det|>[[115, 400, 857, 510]]<|/det|>
+2. According to the data shown in Supplementary Figure 1B, ITGAV exhibits a broad expression across different tissues, while ITGAB6 shows relatively restrictive expression in bladder, breast, esophagus, kidney, lung, muscle, prostate, testis, thyroid and vagina, with low expression in the heart. These expression patterns do not match well the in vivo biodistribution/transduction of LICA1. For example, kidney and lung show quite high level of \(\alpha \nabla\) and \(\beta 6\) integrin expression, but LICA1 shows very little transduction in the kidney and lung. Please provide an explanation in the discussion.  
+
+<|ref|>text<|/ref|><|det|>[[115, 530, 215, 545]]<|/det|>
+Minor points:  
+
+<|ref|>text<|/ref|><|det|>[[115, 565, 877, 656]]<|/det|>
+1. Line 219, MYOAAV should be "AAVMYO"?  
+2. Why are the vector copy number units shown in Fig. S3E (10^-4) several orders of magnitude different from that in Fig. S3D (eg. 10^6)?  
+3. In Figure 4 and 5, please use dot plot to present the data in the graphs.  
+4. In Figure5D, DAPI staining is hardly visible.  
+
+<|ref|>text<|/ref|><|det|>[[116, 711, 392, 727]]<|/det|>
+Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 747, 878, 893]]<|/det|>
+Hong et al expand on previously published reports that the integrin binding RGD motif can confer high heart and skeletal muscle transduction by AAV capsids. Here, instead of a peptide insert based capsid library screening method, the authors utilize rational modification of an AAV capsid surface loop (IV) and molecular docking/modeling to determine optimal flanking residues and presentation of the RGD motif. In addition, the authors utilize a chimeric AAV9/rh74 chimeric capsid which is equivalent to AAV9 but displays liver detargeting potential. The study convincingly demonstrates that the new, rationally engineered AAV capsid variant, LICA- 1 can transduce muscle and avoid liver sequestration effectively. Therapeutic applications in DMD and LGMD mouse models are demonstrated. Overall the study is
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[114, 90, 880, 216]]<|/det|>
+thorough and technically well executed, the methods/experimentation and comparative analysis are robust and the manuscript well written. Some advantages such as improved liver detargeting over other AAVMyo or MyoAAV casids is demonstrated. However, no new mechanistic insight is evident or whether this is selective to mice. A related and significant concern is the lack of large animal translational data (as has been demonstrated with other myotropic AAVs). It is therefore unclear, whether LICA- 1 (although well characterized) presents a significant conceptual advance or alternative to existing engineered AAV vectors.  
+
+<|ref|>text<|/ref|><|det|>[[116, 254, 392, 270]]<|/det|>
+Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[114, 290, 877, 400]]<|/det|>
+In this paper, Vu Hong et al. generated and characterized a new muscle- tropic AAV capsid (designated as LICA1) using on an innovative approach based on molecular modelling to integrate alphaV- beta6 (aVb6) binding RGD motifs into the capsid. The study is overall well designed with comprehensive testing of biodistribution and improved therapeutic efficacy of AAV vectors based on this new LICA1 capsid (compared to AAV9) in two different mouse disease models of hereditary muscle disorders, namely Duchenne muscular dystrophy and limb girdle muscular dystrophy.  
+
+<|ref|>text<|/ref|><|det|>[[114, 418, 880, 510]]<|/det|>
+The results show improved muscle- targeted transduction and increased liver detargeting compared to AAV9 as a benchmark. Additional muscle tropic AAV variants were used that were recently described, namely AAVMYO, MYOAAV1A and MYOAAV2A. The increased muscle transduction obtained with LICA1 capsid is comparable to that attained by the previously described AAV MYO, MYOAAV1A and MYOAAV2A variants (while the latter is even more efficient for cardiac gene delivery).  
+
+<|ref|>sub_title<|/ref|><|det|>[[116, 530, 243, 545]]<|/det|>
+## Major comments  
+
+<|ref|>text<|/ref|><|det|>[[114, 565, 880, 710]]<|/det|>
+1) Though this novel capsid appears to offer no clear advantage over AAVMYO, MYOAAV1A and MYOAAV2A in terms of muscle targeting, the authors claim that their capsid exhibits significantly reduced liver targeting compared to the state of the art (at least in comparison to AAV MYO, MYOAAV1A and MYOAAV2A). Unfortunately, the authors did not include the AAVMYO2 and AAVMYO3 capsids described by El-Andari (Ref. 16 for comparison which are at least as potent as AAVMYO but shows pronounced liver detargeting compared to AAVMYO (or AAV8 and AAV9). Hence, in the absence of these AAVMYO2 and/or AAVMYO3 capsids as benchmark, the superiority of LICA1 over existing muscle-targeted and liver-detargeted capsids has not been unequivocally demonstrated.  
+
+<|ref|>text<|/ref|><|det|>[[114, 728, 880, 857]]<|/det|>
+To ascertain the potential superior properties of LICA1 over the state of the art it is therefore necessary to compare vector performance head to head with AAVMYO2 and AAVMYO3. The available data in the current study along with the comprehensive analyses performed by El- Andari et al. suggest that this new AAV LICA1 capsid may offer only limited advantage compared to these AAVMYO2 and AAVMYO3. From a mechanistic perspective, the LICA1 variant also exploit the RGD motif for muscle targeting as in the case of AAVMYO, MYOAAV1A and MYOAAV2A, essentially confirming the crucial role of RGD and integrin- mediated muscle targeting as demonstrated by the previously published studies (Ref. 15, 16 & 17).  
+
+<|ref|>text<|/ref|><|det|>[[112, 875, 805, 893]]<|/det|>
+2) The therapeutic efficacy of the LICA1 variant in the DMD and LGMD mouse models was only
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 89, 880, 161]]<|/det|>
+compared to AAV9 (Fig. 4 & 5) instead of also including the known myotropic variants AAV MYO, MYOAAV1A and MYOAAV2A that were used in the current study in the wild- type mice. To demonstrate the superiority of LICA1 over the state of the art, it would have been more compelling to also include the AAVMYO2 and AAVMYO3 variants for comparison.  
+
+<|ref|>text<|/ref|><|det|>[[115, 180, 870, 254]]<|/det|>
+3) It would appear that the increase in muscle transduction obtained with LICA1 (vs AAV9) based on vector copy number (VCN) in the dystrophic mdx model (at least in the quadriceps) is relatively modest (2-fold) (Fig. 4) compared to the relative increase transduction in wildtype mice (Fig. 3). What is the reason for this apparent discrepancy?  
+
+<|ref|>text<|/ref|><|det|>[[115, 272, 857, 327]]<|/det|>
+4) There appears to be no significant difference in vector copy number (VCN) in the diaphragm when comparing LICA1 with AAV9 in the mdx mice (Fig. 4). Why is there a significant difference in microDYS mRNA levels and DYS+ fibers despite the lack of any difference in VCN?  
+
+<|ref|>text<|/ref|><|det|>[[115, 345, 845, 381]]<|/det|>
+5) Fig. 3C suggest increased cardiac transduction with the LICA1 variant. Does this translate into increased cardiac transduction and phenotypic correction in the DMDM and LGMD disease models?  
+
+<|ref|>sub_title<|/ref|><|det|>[[115, 419, 244, 435]]<|/det|>
+## Minor comments  
+
+<|ref|>text<|/ref|><|det|>[[115, 455, 872, 509]]<|/det|>
+1) Why is there only a partial inhibition of transduction (about 30%) in the blocking experiment with recombinant aVb6 protein (Fig. 2E)? Were different concentrations tested? Were equimolar amounts of the rSGCA control used?  
+
+<|ref|>text<|/ref|><|det|>[[112, 527, 877, 563]]<|/det|>
+2) The term 'infection' should not be used in the context of replication-defective viral vectors. The correct term is 'transduction'.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[117, 83, 425, 100]]<|/det|>
+## Authors' responses to the reviewers  
+
+<|ref|>sub_title<|/ref|><|det|>[[117, 130, 345, 145]]<|/det|>
+## Color code in this document:  
+
+<|ref|>text<|/ref|><|det|>[[115, 153, 304, 191]]<|/det|>
+Reviewers text in black  Author responses in blue  
+
+<|ref|>sub_title<|/ref|><|det|>[[117, 220, 272, 235]]<|/det|>
+## General comments:  
+
+<|ref|>text<|/ref|><|det|>[[115, 242, 884, 327]]<|/det|>
+We thank the three reviewers for spending time with our manuscript and appreciate all the constructive comments and questions. We have carefully addressed all the comments and made the necessary revisions. This helped to improve considerably the manuscript. Please find below our point- by- point responses. Besides, there are some major changes in our revised manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[114, 333, 884, 530]]<|/det|>
+In response to reviewer #2 concern about translatability of LICA1 in other species and the standard of this fast- moving field, we added a new Result section and new Fig 6/S6 where we examined whether LICA1 can improve transduction across species. The data includes characterization of \(\alpha \mathrm{V}\beta 6\) receptor cross- species in vitro and comparative study in multiple species, including in non- human primates. The result confirms the improved myotropic properties of LICA1 vector. We modified the title and abstract to a shorter version following the journal formatting guidelines while including the additional results. Additionally, we have made minor editorial changes throughout the manuscript to improve clarity and readability.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[117, 82, 337, 99]]<|/det|>
+## REVIEWER COMMENTS  
+
+<|ref|>sub_title<|/ref|><|det|>[[117, 112, 420, 128]]<|/det|>
+## Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 141, 884, 295]]<|/det|>
+This manuscript reports the design and characterization of a novel myotropic AAV vector, LICA1, by grafting natural \(\alpha \mathrm{V}\beta 6\) binding peptides to the VR4 of a liver- detargeted Cap9rh74 hybrid capsid. The authors demonstrate the efficient delivery of therapeutic transgenes to rescue dystrophic phenotypes in two mouse models with a lower dose compared to AAV9. As compared to the recently reported AAVMYO and MYOAAV capsids, the LICA1 showed comparable efficiency in striated muscles but had greatly reduced liver tropism. The experiments are well designed and the novel LICA1 is very promising for clinical translation. The following major and minor points should be addressed.  
+
+<|ref|>text<|/ref|><|det|>[[117, 307, 593, 323]]<|/det|>
+We thank the reviewer for the supportive and helpful comments.  
+
+<|ref|>text<|/ref|><|det|>[[115, 336, 883, 421]]<|/det|>
+1. The authors chose VR4 instead of the more commonly pursued VR8 to insert the integrin binding peptide. VR4 region show some overlapping with AAVR binding site. An interesting question should be addressed is whether the novel LICA1 capsid requires AAVR for cellular entry and transduction. This should be experimentally tested.  
+
+<|ref|>text<|/ref|><|det|>[[115, 433, 884, 630]]<|/det|>
+Response: To answer this question, we created an AAVR- KO cell line (293_AAVR- KO) and evaluated the transduction efficiency of AAV_ITGs in this cell compared to 293_WT. Three gRNAs were designed to target exon 2 of AAVR, a common exon of all AAVR mRNA isoforms (genome- euro.ucsc.edu), co- transfected with SpCas9 in RNP form, and the individual clones were screened by western blot to confirm AAVR- KO at protein level (Fig S2H). In 293_AAVR- KO, the AAV entry was significantly reduced for both 9rh74_5f0 and 9rh74_4um9 by \(65.37\%\) and \(74.73\%\) , respectively, and their transgene activity was reduced by \(94.1\%\) and \(96.9\%\) , respectively, compared to 293_WT (Fig S2I). This result indicates that both AAV_ITGs require AAVR for successful transduction, similar to natural and other VR8-peptide- inserted AAV capsids \(^{1 - 4}\) .  
+
+<|ref|>text<|/ref|><|det|>[[117, 643, 634, 660]]<|/det|>
+We are reproducing Fig S2H- I below for the reviewer's convenience:  
+
+<|ref|>image<|/ref|><|det|>[[123, 675, 880, 802]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 809, 881, 856]]<|/det|>
+<center>Fig S2. H. Western blot showed the absence of AAVR at the protein level at its expected size in the KO clone. I. VCN and luciferase activities of AAV_ITGs in 293_WT and 293_AAVR-KO cells ( \(n = 4 - 6\) , one-way ANOVA). </center>  
+
+<|ref|>text<|/ref|><|det|>[[115, 862, 614, 878]]<|/det|>
+We also added the results in the revised manuscript (line 170- 178):
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 81, 884, 258]]<|/det|>
+"We next examined the dependence of AAV_ITGs on the AAV's human cell entry receptor (AAVR) \(^{40}\) . Structural and molecular studies have previously highlighted the importance of AAVR binding in cellular transduction of multiple natural \(^{40,41}\) and engineered capsids \(^{17,42}\) where VR4 plays a partial role in AAVR binding. We created a 293_AAVR-KO cell line (Fig S2H) and test the viral transduction efficiency in comparison to 293_WT. Similar to other engineered capsids with peptide- inserted VR8 \(^{17}\) , AAV9rh74_4um9 and _5ff0 reduced the AAV entry by 65.37% and 74.73%, reduced the transgene activity by 94.10% and 96.90% with the absence of AAVR (Fig S2I, n=4- 6, p<0.0001). It suggests that AAV_ITGs both require AAVR for effective transduction."  
+
+<|ref|>text<|/ref|><|det|>[[115, 269, 884, 467]]<|/det|>
+As reviewer mentioned, AAVR binds to multiple AAV capsid serotypes at the 3- fold protrusions, mainly VR4, and 2/5- fold wall, despite diverse structural conformation within these regions across serotypes \(^{2}\) . Our result suggests that VR4- modified capsid can still bind to AAVR, presumably by other known AAVR- binding region including VR1, VR3, and VR8 \(^{2,5}\) . It was shown that altering the VR sequences/structures can negatively impact the binding affinity of AAV capsid with AAVR \(^{3,6}\) . However, binding affinity of AAVR with natural AAV, for example AAV2 (K<sub>d</sub> = \(\sim 150 \text{nm}\) ) \(^{1,3}\) , is much lower than the interaction between AAV2 and heparan sulfate (K<sub>d</sub> = 0.1- 3.7 nm) \(^{7}\) . It suggests that AAVR can function by interacting weakly with AAV capsid, therefore might explain the dependence of AAV_ITGs on AAVR for cellular transduction.  
+
+<|ref|>text<|/ref|><|det|>[[115, 479, 884, 609]]<|/det|>
+2. According to the data shown in Supplementary Figure 1B, ITGAV exhibits a broad expression across different tissues, while ITGAB6 shows relatively restrictive expression in bladder, breast, esophagus, kidney, lung, muscle, prostate, testis, thyroid and vagina, with low expression in the heart. These expression patterns do not match well the in vivo biodistribution/transduction of LICA1. For example, kidney and lung show quite high level of \(\alpha \mathrm{V}\) and \(\beta 6\) integrin expression, but LICA1 shows very little transduction in the kidney and lung. Please provide an explanation in the discussion.  
+
+<|ref|>text<|/ref|><|det|>[[115, 620, 884, 840]]<|/det|>
+Response: The mean expression level of ITGB6 in Fig S1B is admittedly high in a number of tissues, as reviewer pointed out. However, the graph presents only the mean, therefore does not illustrate sufficiently the distribution of the data and no tissue enrichment analysis was included. To compensate, we added new Fig S1C is the cell- type expression specificity analysis, which was performed in the same bulk GTEx RNA- seq dataset by an integrative co- expression analysis with known reference transcripts in each cell types for all tissues (The Human Protein Atlas) \(^{8,9}\) . This analysis therefore allows the comparison of gene expression in different cell types across tissues. The analysis showed that ITGB6 is highly enriched in both skeletal myocytes and cardiomyocytes, and ITGAV is highly enriched in cardiomyocytes. This might explain the enhanced transduction in the heart of AAV_ITGs and MYOAAVs \(^{4}\) .  
+
+<|ref|>text<|/ref|><|det|>[[117, 854, 619, 871]]<|/det|>
+We are reproducing Fig S1C below for the reviewer's convenience:
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[125, 84, 872, 490]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 494, 881, 542]]<|/det|>
+<center>Fig S1. C. The cell type-enriched transcriptome profiles of ITGAV and ITGB6 in a list of cell types from multiple tissues (graphs were generated from The Human Protein Atlas). MYH7 is a positive control that is known to highly enrich in the cardiac and skeletal muscles. </center>  
+
+<|ref|>text<|/ref|><|det|>[[115, 555, 512, 571]]<|/det|>
+We modified in the revised manuscript (line 96- 100):  
+
+<|ref|>text<|/ref|><|det|>[[115, 577, 883, 664]]<|/det|>
+"Furthermore, the cell type-enriched transcriptome profiles from bulk RNA sequencing data of multiple human tissues \(^{31}\) confirmed high expression and enrichment of both Itgav and Itgb6 in skeletal and cardiac muscles, and low expression of Itgb6 in the liver and spleen, two preferred targets of natural AAV (Figure S1B- C, GTEx V8, dbGaP Accession phs000424.v8.p2)."  
+
+<|ref|>text<|/ref|><|det|>[[115, 675, 881, 713]]<|/det|>
+We further discussed on the low transduction level seen in kidney and lung in the revised manuscript (line 485- 499).  
+
+<|ref|>text<|/ref|><|det|>[[115, 719, 883, 896]]<|/det|>
+"Despite high enrichment in the human skeletal muscle, ITGB6 – the subunit with more binding interaction with TGFβ-derived motifs, also shows relatively high enrichment in gastric mucous cells (stomach), urothelial cells (prostate), ductal cells (pancreas), and alveolar cells type 2 (AT2, lung), colon enterocytes (colon) and high bulk expression also in kidney, esophagus, and bladder (Fig S1B- C). There was very low AAV transduction observed in both kidney and lung, two organs with highest ITGB6 expression. Poor kidney and lung transduction despite high expression of targeted receptor in these two organs was also observed with other neurotropic engineered AAVs \(^{52, 53}\) . Route of administration in targeting kidney might explain the low transduction of AAV_ITGs in the kidney where local delivery via
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 81, 884, 235]]<|/det|>
+the renal vein or the renal pelvis showed much better transduction than IV injection for multiple AAV serotypes \(^{54}\) . On the other hand, AT2 cells in the lung comprise only \(< 5\%\) of the alveolar surface area \(^{55}\) , therefore they have limited contact with pulmonary capillary blood, which might explain poor transduction efficiency in this organ. Natural AAVs were previously shown to transduce the gastrointestinal tract \(^{56}\) and pancreas \(^{57}\) , suggesting a potential transduction of AAV_ITGs in these organs. Further validation of these tissues is therefore required in future studies to confirm AAV_ITGs' specificity."  
+
+<|ref|>text<|/ref|><|det|>[[115, 248, 220, 264]]<|/det|>
+Minor points:  
+
+<|ref|>text<|/ref|><|det|>[[115, 277, 480, 293]]<|/det|>
+1. Line 219, MYOAAV should be "AAVMYO"?  
+
+<|ref|>text<|/ref|><|det|>[[115, 307, 346, 323]]<|/det|>
+Response: This was corrected.  
+
+<|ref|>text<|/ref|><|det|>[[115, 336, 883, 376]]<|/det|>
+2. Why are the vector copy number units shown in Fig. S3E (10^-4) several orders of magnitude different from that in Fig. S3D (eg. 10^6)?  
+
+<|ref|>text<|/ref|><|det|>[[115, 388, 883, 496]]<|/det|>
+Response: This is due to the difference in the normalization used and units being presented in the graph: VCN in Fig S3B-D was presented as number of AAV genome copies per \(10\mu \mathrm{l}\) assay while the VCN values in Fig S3E was calculated as total of viral genome detected normalized to the total \(1\mathrm{E}10\mathrm{vg}\) AAV initially infected (AAV copies per \(10\mu \mathrm{l}*20 / 10^{10}\) ). Details about the represented units were added in the graph in the revised manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[115, 508, 654, 525]]<|/det|>
+3. In Figure 4 and 5, please use dot plot to present the data in the graphs.  
+
+<|ref|>text<|/ref|><|det|>[[115, 538, 344, 554]]<|/det|>
+Response: This was modified.  
+
+<|ref|>text<|/ref|><|det|>[[115, 568, 471, 584]]<|/det|>
+4. In Figure5D, DAPI staining is hardly visible.  
+
+<|ref|>text<|/ref|><|det|>[[115, 597, 712, 614]]<|/det|>
+Response: The images were now modified for better visibility in DAPI channel.  
+
+<|ref|>text<|/ref|><|det|>[[115, 626, 608, 643]]<|/det|>
+We are reproducing Fig 5D below for the reviewer's convenience.  
+
+<|ref|>image<|/ref|><|det|>[[128, 660, 880, 805]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 808, 732, 824]]<|/det|>
+<center>Fig 5. D. Immuno-staining of DAPI (blue) and SGCA (red) in Quadriceps muscles. </center>  
+
+<|ref|>text<|/ref|><|det|>[[115, 866, 420, 882]]<|/det|>
+Reviewer #2 (Remarks to the Author):
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 81, 884, 415]]<|/det|>
+Hong et al expand on previously published reports that the integrin binding RGD motif can confer high heart and skeletal muscle transduction by AAV capsids. Here, instead of a peptide insert based capsid library screening method, the authors utilize rational modification of an AAV capsid surface loop (IV) and molecular docking/modeling to determine optimal flanking residues and presentation of the RGD motif. In addition, the authors utilize a chimeric AAV9/rh74 chimeric capsid which is equivalent to AAV9 but displays liver detargeting potential. The study convincingly demonstrates that the new, rationally engineered AAV capsid variant, LICA- 1 can transduce muscle and avoid liver sequestration effectively. Therapeutic applications in DMD and LGMD mouse models are demonstrated. Overall the study is thorough and technically well executed, the methods/experimentation and comparative analysis are robust and the manuscript well written. Some advantages such as improved liver detargeting over other AAVMyo or MyoAAV casids is demonstrated. However, no new mechanistic insight is evident or whether this is selective to mice. A related and significant concern is the lack of large animal translational data (as has been demonstrated with other myotropic AAVs). It is therefore unclear, whether LICA- 1 (although well characterized) presents a significant conceptual advance or alternative to existing engineered AAV vectors.  
+
+<|ref|>text<|/ref|><|det|>[[115, 427, 884, 602]]<|/det|>
+Response: We thank the reviewer for the supportive and helpful comments. We are agree that the translatability of LICA1 myotropic properties in different species is an important and reasonable concern, given that engineered capsids for brain (PHP.B variant \(^{10 - 12}\) ) and muscle (AAVMYO/- 2/- 3 family - ASGCT 2024, Abstract 214) can be selective to mouse. To answer this question, we used two different approaches: 1. Investigate the impact on cellular transduction of AAV_ITGs binding with its receptor, \(\alpha \nabla \beta 6\) , from different species; and 2. Validation of enhanced transduction in skeletal muscle in various models, including non- human primate (NHP). The data was added in the manuscript in Fig 6, Fig S6, and the text in line 382- 446.  
+
+<|ref|>text<|/ref|><|det|>[[116, 615, 683, 632]]<|/det|>
+We are reproducing Fig 6 and Fig S6 below for the reviewer's convenience.
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[120, 85, 870, 740]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 748, 744, 764]]<|/det|>
+<center>Fig. 6: LICA1 showed cross-species enhanced transduction in the skeletal muscle. </center>  
+
+<|ref|>text<|/ref|><|det|>[[115, 764, 883, 914]]<|/det|>
+A. Structure of human \(\alpha \mathrm{V}\beta 6\) binding to human TGF-β3-derived motif (pdb code: 4um9). The binding interface, defined as all amino acid in \(\alpha \mathrm{V}\beta 6\) with distance to the binding motif of less than 8Å, is highlighted. B. Alignment of ITGAV (upper panel) and ITGB6 (lower panel) protein sequence around the binding interface from multiple species. The binding interface defined in A is highlighted, and is identical across species being examined. Amino acids with distance to the binding motif of less than 6Å, are in bold and boxed. The sequence mismatches, only found outside the binding interface, are colored in red. C. Transduction efficiency, measured by luciferase activity, of AAV_ITGs but not AAV9 was inhibited by pre-incubating AAVs before infection with recombinant \(\alpha \mathrm{V}\beta 6\) protein from both human, rat, and mouse. 2E8vg AAVs were incubated with different \(\alpha \mathrm{V}\beta 6\) concentrations (0-120 nM) for 1 hour at \(37^{\circ}\mathrm{C}\) before added directly into cell medium (dose: 1E4 vg/cell, 96-well plate, duration: 24 hours, cell
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 81, 883, 504]]<|/det|>
+line: 293_WT, \(n = 3\) ). The statistics were performed to compare with the condition of no aVβ6 protein during incubation (0 nM aVβ6) by using two- way ANOVA ( \(\sim\) AAV serotypes \* aVβ6 treatments). Same incubation conditions using 120nM of recombinant SGCA protein were used as the control, which showed no significant difference with 0nM aVβ6 condition. D. Scheme of comparative study between multiple AAV capsids in multiple species. Transgene expression cassette flanked between ITR sequences include the ORF of human inactivate CAPN3 protein under the control of skeletal muscle- specific promoter tMCK 49, SV40 polyA sequence, and DNA barcode (BC). BCs were placed between ORF and polyA sequence, which allow quantitative measurements of transgene expression mRNA level by RT- qPCR or NGS 17. Two or three barcodes were used for each capsid to minimize the sequence bias which might impact AAV transduction and PCR bias during RT- qPCR or NGS library preparation. AAV production and purification of each capsid variant was done separately, before pooled together at the equimolar amount before AAV transduction in vitro (F, human myotube, \(n = 2\) , dose: 2E10/2E11 vg per 12wp well, duration: 48 hours), or in vivo injection in C57Bl6 mice (E, \(n = 3\) , dose: 5E12 vg/kg per capsid variant, route: intravenous injection, duration: 21 days) and Macaca fascicularis NHP (G, \(n = 3\) , dose: 3.2E12 vg/kg per capsid variant, route: intravenous injection, duration: 45 days). E. The mRNA enrichment (BC_mRNA/Rplp0/ BC_AAV) of different capsid variants in the liver and three skeletal muscles in C57Bl6 mice measured by RT- qPCR. Each dot corresponds to the individual barcode. The color of each barcode corresponds to the individual mouse in Fig S6C. The statistics were performed on the average on different barcodes (using for the same capsid variant in each mouse) by using two- way ANOVA ( \(\sim\) mice \* capsids). F. The mRNA enrichment, measured by NGS, of different capsid variants in in vitro human myotubes at two AAV concentration. The log2 fold- change compared to AAV9 of all variants is presented. Each dot corresponds to the individual barcode. The colors correspond to different biological replicates. G. The mRNA enrichment (BC_mRNA/Rplp0/BC_AAV) of different capsid variants in the liver and skeletal muscles in NHP measured by RT- qPCR. Each dot corresponds to the individual barcode. The data was presented for individual primates (P1-3). The statistics were to compare the mRNA level of LICA1 with natural capsids, AAV8/AAV9, performed on the fold change of LICA1 to AAV8/AAV9 (averages of barcodes using for the same capsid variant in each primate were used to calculate the fold- change) by using two- tailed one- sample t- test.
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[120, 88, 875, 850]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 850, 881, 910]]<|/det|>
+<center>Fig. S6: LICA1 showed improved transduction across species. A. Predicted structures of \(\alpha \beta \beta\) heterocomplex from multiple species by AlphaFold2 showed very high similarity with the crystal structure of human form (pdb code: 4um9). The ITGAV and ITGB6 of human (ITGAV: aa31-467, ITGB6: 132-373), primate (ITGAV: aa31-467, ITGB6: 132-373), pig (ITGAV: aa31- </center>
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 81, 883, 415]]<|/det|>
+466, ITGB6: 132- 373), rat (ITGAV: aa31- 466, ITGB6: 132- 373) and mouse (ITGAV: aa31- 467, ITGB6: 132- 373) were used for the prediction. The Alphafold2 hyperparameters includes: model=alphafold2_multimer_v3, num-recycle=8, templates=True. The prediction of the ITGAV-B6 heterodimer from all species were highly confident (pLDDT=96.06/96.00/95.72/95.71/95.66 for human, primate, pig, rat, and mouse, respectively). The superimpose of predicted structures colored by pLDDT values and experimental human structure in light grey are shown. RMSD values were calculated by ChimeraX software. B. Transduction efficiency, measured by luciferase activity, of AAV_ITGs but not AAV9 was inhibited by pre-incubating AAVs before infection with recombinant \(\alpha \mathrm{V}\beta 6\) protein from both human, rat, and mouse. 2E8vg AAVs were incubated with different \(\alpha \mathrm{V}\beta 6\) concentrations (0- 120 nM) for 1 hour at \(37^{\circ}C\) before added directly into cell medium (dose: 1E4 vg/cell, 96- well plate, duration: 24 hours, cell line: 293_ \(\alpha \mathrm{V}\beta 6\) , \(n = 3\) ). The statistics were performed to compare with the condition of no \(\alpha \mathrm{V}\beta 6\) protein during incubation (0 nM \(\alpha \mathrm{V}\beta 6\) ) by using two- way ANOVA ( \(\sim\) AAV serotypes \* \(\alpha \mathrm{V}\beta 6\) treatments). Same incubation conditions using 120nM of recombinant SGCA protein were used as the control, which showed no significant difference with 0nM \(\alpha \mathrm{V}\beta 6\) condition. C. Relative mRNA level of total hC3i to Rplp0 in different tissues in C57Bl6 mice, measured by RT- qPCR. The same colors were used as in Fig 6E, which correspond to the individual mice. D. In vitro assay to quantify the titers of neutralizing antibodies in the primate serum against AAV8, AAV9, and LICA1 before AAV injection. Different AAVs with GFP/Luc reporter transgene were incubated with primate serum at different dilutions or FBS for 1 hour before added in 293_E3 cells (n=1). Luciferase activities were quantified 24- hour post- transduction. E. Relative mRNA level of total hC3i to Rplp0 in different tissues in NHPs, measured by RT- qPCR. The colors of each dot correspond to the individual primates. DRG - dorsal root ganglia, LN - lymph node.  
+
+<|ref|>text<|/ref|><|det|>[[115, 427, 883, 738]]<|/det|>
+We believe that one of main strength of the study is that both AAV_ITGs were initially designed to bind to human form of \(\alpha \mathrm{V}\beta 6\) , where they were incorporated with binding motifs derived from human TGFβ1/3. In conventional in vivo directed evolution, AAV variants are identified based on the selective experimental pressure. Therefore, it lacks the interpretability of the transduction improvement and consequently whether the improvement can translate outside screening setup, for example other species or other uninvestigated organs/cell types. AAV- PHP.B is an example of AAV variant whose improvement is restricted in C57Bl6 mouse but not in other strains or species due to the lack of its receptor \(^{10 - 12}\) . Our approach is primarily different in which we aim to target a receptor that highly conserved across species, and more importantly start the design targeting the human form. This approach recently showed a great efficacy, with validation in either humanized mouse model \(^{13}\) (ASGCT 2024 – Abstract 120/991) or NHP (ASGCT 2023 – Abstract 42/468, ASGCT 2024 – Abstract 118/217/638). Besides, there is an increasing need for AAV receptor identification for interpretability \(^{4,10,14}\) (ASGCT 2024 – Abstract 975). It therefore indicates that the bottom- up approach might better translate in cross- species transduction.  
+
+<|ref|>text<|/ref|><|det|>[[115, 750, 883, 902]]<|/det|>
+However, the characterization of \(\alpha \mathrm{V}\beta 6\) receptor and AAV- \(\alpha \mathrm{V}\beta 6\) interaction might be not sufficient in the previous version of manuscript and was added in the current version. We compared the sequences and predicted structures of aVb6 from human, primate, pig, rat, and mouse, and found identical sequence alignment and near- identical structure at the binding interface with TGFβ- derived peptides (Fig 6A- B, Fig S6A). It suggests similar binding interaction of AAV_ITGs with \(\alpha \mathrm{V}\beta 6\) in different species. We next tested how binding between AAV_ITGs and \(\alpha \mathrm{V}\beta 6\) in different species impact transduction by the inhibition assay where AAVs were incubated with different recombinant \(\alpha \mathrm{V}\beta 6\) proteins before infection.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 82, 884, 190]]<|/det|>
+It was shown in two cell lines that transduction level of AAV_ITGs, but not AAV9, were abolished with the \(\alpha \mathrm{V}\beta 6\) - preincubation, suggesting these interactions between AAV_ITGs and \(\alpha \mathrm{V}\beta 6\) is essential for the enhanced transduction. Besides, the improvement of AAV_ITGs, especially LICA1, was seen in human cell lines (293_ \(\alpha \mathrm{V}\beta 6\) and in vitro human differentiated myotubes) and skeletal muscle of mouse models. It indicates LICA1 functions similarly across species.  
+
+<|ref|>text<|/ref|><|det|>[[114, 202, 884, 670]]<|/det|>
+Furthermore, we compared LICA1 transduction level across species at mRNA level, similar to what has been described for other myotropic serotypes in NHP 4 (Fig 6D- G). Barcoded LICA1 and other serotypes were mixed at equal viral genome number before being co- infected/injected in in vitro differentiated human myotube, C57Bl6 mouse, and non- human primate. First, the multiplex comparison of LICA1 with AAV8/AAV9/AAV9rh74, 9rh74_5ffo and other myotropic AAVs, AAVMYO and MYOAAV2A showed similar results with individual comparison (Fig 6E and Fig 3). However, a similar pool of capsids (except not including AAVMYO) used in in vitro differentiated human myotube showed contradicting results with data obtained in mouse and previous individual comparison also performed in the same model. Despite again confirming better transduction level than AAV8/AAV9/AAV9rh74 of all myotropic variants, including LICA1, 9rh74_5ffo showed unexpected considerable higher transduction level than both LICA1 and MYOAAV2A in all biological replicates and 2 conditions with different AAV doses infected (Fig 6F). This observation together with a more significant inhibition by human \(\alpha \mathrm{V}\beta 6\) pre- incubation in 9rh74_5ffo than LICA1 (Fig 2E, 6C), it suggested a competition of myotropic AAVs over the \(\alpha \mathrm{V}\beta 6\) receptor – a putative receptor for both LICA1, 9rh74_5ffo, and MYOAAV2A. The competition between capsid variants during pooled transduction has been reported before 15. With the potential competition of \(\alpha \mathrm{V}\beta 6\) - binding myotropic AAVs and the profile of Nab against multiple tested capsid variants (Table S3, Fig S6D, no primate was found to be Nab- negative for all variants), we decided to conduct a comparison of LICA1 with only AAV8 or AAV9, 2 capsid variants currently used in clinical trials for muscular diseases 16, in 3 NHPs with lowest LICA1- Nab levels. In these NHPs, we confirmed the significant improved expression in skeletal muscle and strong liver detargeting of LICA1 compared to AAV8 and AAV9 benchmarks (Fig 6G).  
+
+<|ref|>text<|/ref|><|det|>[[117, 682, 881, 722]]<|/det|>
+Altogether, we believe that we have provided sufficient evidence to support the translatability of LICA variant across species, including non- human primate.  
+
+<|ref|>sub_title<|/ref|><|det|>[[117, 763, 420, 780]]<|/det|>
+## Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 792, 883, 877]]<|/det|>
+In this paper, Vu Hong et al. generated and characterized a new muscle- tropic AAV capsid (designated as LICA1) using on an innovative approach based on molecular modelling to integrate alphaV- beta6 (aVb6) binding RGD motifs into the capsid. The study is overall well designed with comprehensive testing of biodistribution and improved therapeutic efficacy of AAV vectors based on this new LICA1
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[117, 82, 880, 122]]<|/det|>
+capsid (compared to AAV9) in two different mouse disease models of hereditary muscle disorders, namely Duchenne muscular dystrophy and limb girdle muscular dystrophy.  
+
+<|ref|>text<|/ref|><|det|>[[115, 134, 883, 242]]<|/det|>
+The results show improved muscle- targeted transduction and increased liver detargeting compared to AAV9 as a benchmark. Additional muscle tropic AAV variants were used that were recently described, namely AAVMYO, MYOAAV1A and MYOAAV2A. The increased muscle transduction obtained with LICA1 capsid is comparable to that attained by the previously described AAV MYO, MYOAAV1A and MYOAAV2A variants (while the latter is even more efficient for cardiac gene delivery).  
+
+<|ref|>text<|/ref|><|det|>[[117, 254, 593, 270]]<|/det|>
+We thank the reviewer for the supportive and helpful comments.  
+
+<|ref|>text<|/ref|><|det|>[[117, 284, 245, 300]]<|/det|>
+Major comments  
+
+<|ref|>text<|/ref|><|det|>[[115, 313, 884, 510]]<|/det|>
+1) Though this novel capsid appears to offer no clear advantage over AAVMYO, MYOAAV1A and MYOAAV2A in terms of muscle targeting, the authors claim that their capsid exhibits significantly reduced liver targeting compared to the state of the art (at least in comparison to AAV MYO, MYOAAV1A and MYOAAV2A). Unfortunately, the authors did not include the AAVMYO2 and AAVMYO3 capsids described by El-Andari (Ref. 16 for comparison which are at least as potent as AAVMYO but shows pronounced liver detargeting compared to AAVMYO (or AAV8 and AAV9). Hence, in the absence of these AAVMYO2 and/or AAVMYO3 capsids as benchmark, the superiority of LICA1 over existing muscle-targeted and liver-detargeted capsids has not been unequivocally demonstrated.  
+
+<|ref|>text<|/ref|><|det|>[[115, 522, 884, 698]]<|/det|>
+To ascertain the potential superior properties of LICA1 over the state of the art it is therefore necessary to compare vector performance head to head with AAVMYO2 and AAVMYO3. The available data in the current study along with the comprehensive analyses performed by El- Andari et al. suggest that this new AAV LICA1 capsid may offer only limited advantage compared to these AAVMYO2 and AAVMYO3. From a mechanistic perspective, the LICA1 variant also exploit the RGD motif for muscle targeting as in the case of AAVMYO, MYOAAV1A and MYOAAV2A, essentially confirming the crucial role of RGD and integrin- mediated muscle targeting as demonstrated by the previously published studies (Ref. 15, 16 & 17).  
+
+<|ref|>text<|/ref|><|det|>[[115, 710, 884, 908]]<|/det|>
+Response: To response to the request of reviewer, we performed head- to- head comparison of LICA1 with AAVMYO2 and AAVMYO3 17 as presented in Fig 3G- H. We found no significant difference between the level of virus accumulation in the liver of LICA1 and AAVMYO- 2/3, yet mRNA level of AAVMYO- 2/3 is significantly lower than LICA1. However, in a more important aspect of a myotropic capsid – ability to transduce skeletal muscle, LICA1 is considerably better than both AAVMYO- 2/3 in all skeletal and cardiac muscles tested, in both virus entry and transgene activity. Of note, AAVMYO- 2/3 were also shown in the original publication to have lower muscle transduction than AAVMYO in most muscle tested (Fig 3D, Fig S5 from 17). The data was now added as Fig S3F- I and the text was modified in the revised manuscript. We also modified our previous claim of “AAV9rh74_4um9
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 81, 881, 144]]<|/det|>
+transduced skeletal muscle similarly, but detargeted the liver more strongly than other myotropic AAVs" to "AAV9rh74_4um9 exhibits highest tropism towards the skeletal muscle among tested myotropic AAVs in mice" (line 207- 208).  
+
+<|ref|>text<|/ref|><|det|>[[115, 156, 630, 173]]<|/det|>
+We are reproducing Fig S3F- I below for the reviewer's convenience.  
+
+<|ref|>image<|/ref|><|det|>[[122, 192, 875, 562]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 577, 881, 656]]<|/det|>
+<center>Fig S3. F-G. VCN (F) and gene expression (G) (GFP mRNA level in liver and luciferase activity in other organs) of AAV9rh74_4um9, MYOAAV2, and MYOAAV3 18 in liver, skeletal muscles, heart, lung, kidney, and brain (n=4, one-way ANOVA). VCN and mRNA levels of AAV9 and MYOAAV1A in the previous comparison (Fig 3G-H) were added as reference. H-I. Ratio of VCN found in skeletal muscle; TA (H) and Qua (I); to the level in the liver one-month post-injection (n=4, one-way ANOVA). </center>  
+
+<|ref|>text<|/ref|><|det|>[[117, 668, 445, 684]]<|/det|>
+We added the results section (line 236- 247):  
+
+<|ref|>text<|/ref|><|det|>[[115, 690, 884, 888]]<|/det|>
+"Recently, AAVMYO2 and AAVMYO3 myotropic variants were identified to be more liver- detargeted while transducing skeletal and cardiac muscles less effectively than AAVMYO<sup>16</sup>. We then conducted a similar head- to- head comparison of these two variants with 9rh74_4um9 (6- week- old WT mice, dose: 1E13 vg/kg, n=4). All three capsids showed very low transduction in kidney, lung and brain (Fig S3F). Similar to previous AAVMYO comparison<sup>16</sup>, AAVMYO2 and AAVMYO3 both showed significantly lower VCN (3.24- 7.44/2.61- 6.16 times, respectively, \(p< 0.001\) ) and transgene activity (12.41- 18.42/11.1- 15.91 times, respectively, \(p< 0.05\) ) in all tested skeletal/cardiac muscles compared to LICA1 (Fig S3F- G). On another hand, the liver transduction of AAVMYO2/3 is not significantly different from that of LICA1, yet the transgene expression is significantly lower (Fig S3F- G). Altogether, LICA1 and AAVMYO2/3 showed
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 81, 883, 123]]<|/det|>
+highest ratio of muscle- to- liver transduction in all tested myotropic capsids (Fig S3H- I), yet LICA1 is considerably better in transgene delivery and expression in skeletal muscles than AAVMYO2/3."  
+
+<|ref|>text<|/ref|><|det|>[[114, 133, 884, 468]]<|/det|>
+An important question is whether the RGD- containing capsids can show improved transduction in the skeletal muscle across species, especially in non- human primate (NHP). MYOAAV family has been discovered and validated in NHP \(^{4}\) . In this revised manuscript, we also validated significantly enhanced transduction of LICA1 in NHP (please see the discussion with Reviewer #2 for the attention of reviewer). However, in the latest data presented at ASGCT 2024 (Abstract 214), researchers showed no improvement of either AAVMYO, AAVMYO2, AAVMYO3, all shared P1 peptide – RGDLGLS, compared to natural capsids, AAV8, AAV9. It is worth noting that while LICA1 and MYOAAV family both target \(\alpha \mathrm{V}\beta 6\) while P1 peptide from AAVMYO/AAVMYO- 2/3 potentially targets \(\alpha \mathrm{V}\beta 8\) or \(\beta 3 / \beta 5^{18}\) , which is lowly enriched in human skeletal myocyte (Figure R1 below for the attention of the reviewer). It suggests that the integrin subunits that RGD- containing capsid target is essentially important for cross- species transduction. Besides, an alternative explanation is a potential competition between AAV serotypes being pooled and co- injected, as seen in Fig S6D or by others \(^{15}\) . Since different AAV serotypes were identified by various methodologies (LICA1 – rational design, MYOAAVs – directed evolution which is known to perform very well in competing conditions such as library transduction, and AAVMYO/- 2/- 3 – seminational design), evaluation methods should be carefully taken into account.
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[120, 81, 880, 600]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 601, 880, 634]]<|/det|>
+<center>Figure R1: Tissue Cell Type enrichment score of different ITGB subunits' mRNA in human biopsies (Figures generated by The Human Protein Atlas). </center>  
+
+<|ref|>text<|/ref|><|det|>[[115, 645, 883, 754]]<|/det|>
+2) The therapeutic efficacy of the LICA1 variant in the DMD and LGMD mouse models was only compared to AAV9 (Fig. 4 & 5) instead of also including the known myotropic variants AAV MYO, MYOAAV1A and MYOAAV2A that were used in the current study in the wild-type mice. To demonstrate the superiority of LICA1 over the state of the art, it would have been more compelling to also include the AAVMYO2 and AAVMYO3 variants for comparison.  
+
+<|ref|>text<|/ref|><|det|>[[115, 766, 884, 896]]<|/det|>
+Response: We strongly believe that we have provided sufficient evidence to support the myotropic properties of LICA1 in various cellular and animal models. Besides, we only claimed the equivalent transduction level in murine skeletal muscle between LICA1, AAVMYO and MYOAAV- 1A/2A. The difference between myotropic serotypes was only observed in the level of liver detargeting, which aims to reduce the risk of hepatoxicity mainly reported at very high- dose AAV injection, in human patients 19, 20 or NHP 21. Therefore, we do not expect any difference in muscle transduction level as well as liver
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 82, 884, 257]]<|/det|>
+toxicity phenotypes (not observed in the present study) between LICA1 and other mAAVs at this low-dose treatment (5E12 vg/kg). Besides, we observed a considerably better transduction level of LICA1 than AAVMYO- 2/3 as added in the revised manuscript, while no improvement of AAVMYO- 2/3 has been observed in NHP as reported in ASGCT 2024 (Abstract 214). We therefore think that the comparison is unnecessary for the present manuscript because there would be no added value for translatability. However, we completely agree with the reviewer that the comparison of LICA1 with other myotropic AAVs at higher dose regarding adverse events, especially in the liver, is important for future studies.  
+
+<|ref|>text<|/ref|><|det|>[[115, 270, 883, 355]]<|/det|>
+3) It would appear that the increase in muscle transduction obtained with LICA1 (vs AAV9) based on vector copy number (VCN) in the dystrophic mdx model (at least in the quadriceps) is relatively modest (2-fold) (Fig. 4) compared to the relative increase transduction in wildtype mice (Fig. 3). What is the reason for this apparent discrepancy?  
+
+<|ref|>text<|/ref|><|det|>[[115, 367, 883, 407]]<|/det|>
+Response: We have two hypotheses for the discrepancy of VCN transduction level between WT and dystrophic muscle.  
+
+<|ref|>text<|/ref|><|det|>[[115, 419, 884, 617]]<|/det|>
+First, the VCN data was presented as vector genome found per nucleus. In dystrophic muscle compared to WT, number of nuclei significantly increases due to the infiltration of immune cells, newly formed myocytes, proliferated satellite cells and fibrotic cells. Similarly, the nuclei number differs also between effective or ineffective treated muscles, where cellular composition can alter according to immune response, fibrosis level, and muscle regeneration. The discrepancy of denominator between conditions therefore makes it difficult to compare between two conditions, in addition to other technical differences between protocols (age of animals: 6-week versus 4-week, duration of the treatment: 21-day versus 28- day, and the dose: 1E13 versus 5E12 vg/kg). Normalization of the viral entry in skeletal muscle should be more appropriate by dividing to number of myocyte nuclei only, but rather practically difficult.  
+
+<|ref|>text<|/ref|><|det|>[[115, 629, 884, 894]]<|/det|>
+Second, discrepancy of transduction efficiency between WT and dystrophic muscle or injury- induced muscle regeneration has been reported with AAV1<sup>22</sup>. It suggests that the muscle physiology plays an important role in viral transduction. Since LICA1 targets specifically \(\alpha \mathrm{V}\beta 6\) , which is shown to constantly trafficked actively participate in endocytic- exocytic pathways in cells<sup>23</sup>, we hypothesize that \(\alpha \mathrm{V}\beta 6\) - dependent intracellular trafficking might explain the global transduction improvement of LICA1, in addition to improved cellular internalization at the plasma membrane. Indeed, we and others have recently reported dysregulated endocytosis in multiple muscular dystrophies<sup>24, 25</sup> (Myology 2024 - Presentation S5L3). Concurrently, we have observed upregulation of ITGB6 in dystrophic myocytes (Fig S1A). This might also explain the discrepancy in the \(\mathrm{FC}_{\mathrm{LICA1 / AAV9}}\) between models. Nonetheless, the impact of dysregulated endocytosis on LICA1 transduction in dystrophic muscles remains inadequately understood and will be a focus of our future research. We added this point in the discussion of the revised manuscript.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[117, 82, 670, 99]]<|/det|>
+We added elements of discussion in the revised manuscript (line 500- 510):  
+
+<|ref|>text<|/ref|><|det|>[[115, 111, 885, 332]]<|/det|>
+"Mechanism of enhanced transduction by \(\alpha \mathrm{V}\beta 6\) binding remains unclear. In most cases, the improved transduction of AAV_ITGs and other mAAVs was evident at the VCN level, indicating better cell entry via \(\alpha \mathrm{V}\beta 6\) binding. In some cases, for example observed in Dia and Heart of dystrophic mice, the improvement is only seen at the transgene expression level, suggesting the role of \(\alpha \mathrm{V}\beta 6\) beyond cellular internalization. Indeed, integrins are constantly trafficked in cells, initially internalized by either clathrin- mediated or clathrin- independent endocytosis to enter the endocytic- exocytic pathway before being recycled \(^{58}\) . The foot- and- mouth disease virus, which contains \(\alpha \mathrm{V}\beta 6\) - binding RGD motif to infect cells, also requires EEA1/Rab5- positive vesicles for intracellular trafficking \(^{59,60}\) , suggesting a similar trafficking mechanism with \(\alpha \mathrm{V}\beta 6\) - targeting AAVs. Future studies are necessary to better understand the \(\alpha \mathrm{V}\beta 6\) 's role in intracellular myotropic AAV trafficking within endocytic system."  
+
+<|ref|>text<|/ref|><|det|>[[115, 344, 883, 407]]<|/det|>
+4) There appears to be no significant difference in vector copy number (VCN) in the diaphragm when comparing LICA1 with AAV9 in the mdx mice (Fig. 4). Why is there a significant difference in microDYS mRNA levels and DYS+ fibers despite the lack of any difference in VCN?  
+
+<|ref|>text<|/ref|><|det|>[[114, 417, 884, 729]]<|/det|>
+Response: The \(\mathrm{FC}_{\mathrm{LICA1 / AAV9}}\) of mRNA is much higher than that of VCN in all muscles tested ( \(\mathrm{FC}_{\mathrm{TA:VCN}} = 1.86\) versus \(\mathrm{FC}_{\mathrm{TA:mRNA}} = 4.56\) ; \(\mathrm{FC}_{\mathrm{Qua:VCN}} = 2.03\) versus \(\mathrm{FC}_{\mathrm{Qua:mRNA}} = 5.56\) ; \(\mathrm{FC}_{\mathrm{Dia:VCN}} = 1.06\) versus \(\mathrm{FC}_{\mathrm{Dia:mRNA}} = 7.57\) ). In the Fig S3B- E, there was also discrepancy between VCN and transgene expression of all tested myotropic AAVs in multiple cell lines. Similarly, in the library screening, no correlation between VCN and mRNA level of AAV variants was observed in multiple tissues \(^{4}\) . We hypothesize that, as discussed in point 3, the transduction improvement of LICA1 is not only from cell entry by better internalization, but also largely due to the \(\alpha \mathrm{V}\beta 6\) - dependent intracellular endocytic trafficking. The foot- and- mouth disease virus, which contains RGD motif to bind to \(\alpha \mathrm{V}\beta 6\) to infect cells \(^{26,27}\) , also requires EEA1/Rab5- positive vesicles for intracellular trafficking, suggesting a similar trafficking mechanism with \(\alpha \mathrm{V}\beta 6\) - targeting AAVs. Besides, it was also suggested that the role of AAV universal receptor, AAVR, might lie more within the endosome trafficking than virus uptake in the cell surface \(^{28}\) . Besides trafficking, another possible explanation is that the capsid can influence the epigenetic profile the vector genome, subsequently can impact the transgene expression \(^{29}\) . Further studies are therefore required to explore the mechanisms underlying this phenomenon.  
+
+<|ref|>text<|/ref|><|det|>[[115, 741, 883, 781]]<|/det|>
+5) Fig. 3C suggest increased cardiac transduction with the LICA1 variant. Does this translate into increased cardiac transduction and phenotypic correction in the DMDM and LGMD disease models?  
+
+<|ref|>text<|/ref|><|det|>[[115, 793, 883, 878]]<|/det|>
+Response: We want to clarify that we initially did not expect an improvement in heart transduction of LICA1 since its receptor, ITGB6, expresses relatively lowly in heart compared to skeletal muscle (Figure R2 below for the attention of reviewer), despite high enrichment score comparing to other cell types from other tissues (Fig S1C in the revised manuscript in a response to reviewer #1). It might
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 81, 881, 123]]<|/det|>
+explain a lower improvement of LICA1 in heart \((\mathrm{FC}_{\mathrm{Luc}|\mathrm{Heart:LICA1 / AAV9}} = 4.65)\) compared to what seen in skeletal muscle \((\mathrm{FC}_{\mathrm{Luc}|\mathrm{Qua:LICA1 / AAV9}} = 17.52)\) .  
+
+<|ref|>text<|/ref|><|det|>[[115, 133, 883, 219]]<|/det|>
+In response to the question of reviewer, we compared the VCN and mRNA level in the heart, and showed improvement in heart transduction of LICA1 over AAV9 in both models. Again, the improvement and expression level in heart is modest compared to what seen in skeletal muscles. We added the data in Fig 4A, 4B, 5B, 5C, and modified the text in the revised manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[115, 231, 733, 248]]<|/det|>
+We are reproducing Fig 4A- B and Fig 5B- C below for the reviewer's convenience.  
+
+<|ref|>image<|/ref|><|det|>[[120, 262, 878, 368]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 369, 881, 401]]<|/det|>
+<center>Fig 4. Comparison of transduction efficacy between AAV9 and LICA1 in three skeletal muscles and heart, in terms of VCN (A), and \(\mu\) Dys mRNA level (B). </center>  
+
+<|ref|>text<|/ref|><|det|>[[115, 405, 881, 469]]<|/det|>
+We added in the main text (line 284- 286): "In the heart, there were no difference in VCN but slightly significantly higher in mRNA level in LICA1- treated group compared to AAV9 (Fig 4A- B, \(FC_{VCN} = 0.72\) , \(p = 0.073\) ; \(FC_{mRNA} = 1.39\) , \(p = 0.021\) )."  
+
+<|ref|>image<|/ref|><|det|>[[120, 490, 875, 633]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 635, 881, 667]]<|/det|>
+<center>Fig 5. Comparison of transduction efficacy between AAV9 and LICA1 in three skeletal muscles and heart, in terms of VCN (A), and hSGCA mRNA level (B). </center>  
+
+<|ref|>text<|/ref|><|det|>[[115, 672, 881, 735]]<|/det|>
+We added in the main text (line 342- 344): "Besides, slight significant increase was observed in both VCN and mRNA level in LICA1- treated group compared to AAV9 in the heart (Fig 5B- C, \(FC_{VCN} = 2.01\) , \(p = 0.0069\) ; \(FC_{mRNA} = 5.11\) , \(p = 0.018\) )."  
+
+<|ref|>text<|/ref|><|det|>[[115, 747, 883, 831]]<|/det|>
+There is no cardiac pathology reported at 4- week of age in both mouse models \(^{30,31}\) . We therefore did not examine the cardiac phenotype after AAV injection in the present study. However, we agree that it is important to access cardiac phenotypes of different AAV treatment in more advanced pathological stages.
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[117, 81, 234, 280]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 287, 880, 320]]<|/det|>
+<center>Figure R2: The bulk mRNA expression of ITGB6 in skeletal muscle, heart, and liver from human biopsies (data was reanalyzed from GTEx.V8). </center>  
+
+<|ref|>text<|/ref|><|det|>[[116, 340, 247, 356]]<|/det|>
+Minor comments  
+
+<|ref|>text<|/ref|><|det|>[[116, 362, 883, 424]]<|/det|>
+1) Why is there only a partial inhibition of transduction (about \(30\%\) ) in the blocking experiment with recombinant aVb6 protein (Fig. 2E)? Were different concentrations tested? Were equimolar amounts of the rSGCA control used?  
+
+<|ref|>text<|/ref|><|det|>[[115, 435, 884, 611]]<|/det|>
+Response: We added a more comprehensive experiment in Fig 6C and Fig S6B, with different concentrations of recombinant \(\alpha \mathrm{V}\beta 6\) from different species in responding to Reviewer #2. In Fig 2E, \(2\mu \mathrm{g}\) of recombinant protein, both for SGCA control and \(\alpha \mathrm{V}\beta 6\) , were incubated with \(1\times 10^{10}\mathrm{vg}\) AAV. In Fig 6C, SCGA control was used at the same equimolar amount as the highest concentration tested with \(\alpha \mathrm{V}\beta 6\) ( \(120\mathrm{nM}\) ). Details about the concentration was added in the figure legends. The reason for partial inhibition in LICA1 in Fig 2E is due to the low concentration of recombinant protein, which of equivalent of \(\sim 1.5\mathrm{nM}\) of what has been presented in Fig 6C, S6B. For comparison, LICA1 transduction level was reduced by \(81.64\%\) at \(30\mathrm{nM}\) and by \(94.2\%\) at \(60\mathrm{nM}\) .  
+
+<|ref|>text<|/ref|><|det|>[[115, 623, 881, 664]]<|/det|>
+2) The term 'infection' should not be used in the context of replication-defective viral vectors. The correct term is 'transduction'.  
+
+<|ref|>text<|/ref|><|det|>[[115, 676, 535, 694]]<|/det|>
+Response: This was modified in the revised manuscript.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[115, 83, 242, 98]]<|/det|>
+## REFERENCES  
+
+<|ref|>text<|/ref|><|det|>[[110, 100, 885, 888]]<|/det|>
+1. Pillay, S. et al. An essential receptor for adeno-associated virus infection. Nature 530, 108-112 (2016).  
+2. Zhang, R. et al. Divergent engagements between adeno-associated viruses with their cellular receptor AAVR. Nature communications 10, 3760 (2019).  
+3. Xu, G. et al. Structural basis for the neurotropic AAV9 and the engineered AAVPHP.eB recognition with cellular receptors. Molecular therapy. Methods & clinical development 26, 52-60 (2022).  
+4. Tabebordbar, M. et al. Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell 184, 4919-4938 e4922 (2021).  
+5. Meyer, N.L. et al. Structure of the gene therapy vector, adeno-associated virus with its cell receptor, AAVR. Elife 8 (2019).  
+6. Jang, S., Shen, H.K., Ding, X., Miles, T.F. & Gradinaru, V. Structural basis of receptor usage by the engineered capsid AAV-PHP.eB. Molecular therapy. Methods & clinical development 26, 343-354 (2022).  
+7. Zhang, F. et al. Characterization of interactions between heparin/glycosaminoglycan and adeno-associated virus. Biochemistry 52, 6275-6285 (2013).  
+8. Dusart, P. et al. A tissue centric atlas of cell type transcriptome enrichment signatures. bioRxiv, 2023.2001.2010.520698 (2023).  
+9. Norreen-Thorsen, M. et al. A human adipose tissue cell-type transcriptome atlas. Cell Rep 40, 111046 (2022).  
+10. Hordeaux, J. et al. The GPI-Linked Protein LY6A Drives AAV-PHP.B Transport across the Blood-Brain Barrier. Molecular therapy : the journal of the American Society of Gene Therapy 27, 912-921 (2019).  
+11. Hordeaux, J. et al. The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL/6J Mice. Molecular therapy : the journal of the American Society of Gene Therapy 26, 664-668 (2018).  
+12. Liguore, W.A. et al. AAV-PHP.B Administration Results in a Differential Pattern of CNS Biodistribution in Non-human Primates Compared with Mice. Molecular therapy : the journal of the American Society of Gene Therapy 27, 2018-2037 (2019).  
+13. Huang, Q. et al. An AAV capsid reprogrammed to bind human Transferrin Receptor mediates brain-wide gene delivery. bioRxiv, 2023.2012.2020.572615 (2023).  
+14. Shay, T.F. et al. Primate-conserved carbonic anhydrase IV and murine-restricted LY6C1 enable blood-brain barrier crossing by engineered viral vectors. Science advances 9, eadg6618 (2023).  
+15. de Alencastro, G. et al. Tracking Adeno-Associated Virus Capsid Evolution by High-Throughput Sequencing. Human gene therapy 31, 553-564 (2020).  
+16. Manini, A., Abati, E., Nuredini, A., Corti, S. & Comi, G.P. Adeno-Associated Virus (AAV)-Mediated Gene Therapy for Duchenne Muscular Dystrophy: The Issue of Transgene Persistence. Frontiers in neurology 12, 814174 (2021).  
+17. El Andari, J. et al. Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders. Science advances 8, eabn4704 (2022).  
+18. Bauer, A. et al. Molecular Signature of Astrocytes for Gene Delivery by the Synthetic Adeno-Associated Viral Vector rAAV9P1. Adv Sci (Weinh) 9, e2104979 (2022).  
+19. Chand, D. et al. Hepatotoxicity following administration of onasemnogene abeparvovec (AVXS-101) for the treatment of spinal muscular atrophy. J Hepatol 74, 560-566 (2021).  
+20. Duan, D. Lethal immunotoxicity in high-dose systemic AAV therapy. Molecular therapy : the journal of the American Society of Gene Therapy 31, 3123-3126 (2023).  
+21. Hudry, E. et al. Liver injury in cynomolgus monkeys following intravenous and intrathecal scAAV9 gene therapy delivery. Molecular therapy : the journal of the American Society of Gene Therapy 31, 2999-3014 (2023).  
+22. Mollard, A. et al. Muscle regeneration affects Adeno Associated Virus 1 mediated transgene transcription. Sci Rep 12, 9674 (2022).  
+23. Moreno-Layseca, P., Icha, J., Hamidi, H. & Ivaska, J. Integrin trafficking in cells and tissues. Nat Cell Biol 21, 122-132 (2019).
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 82, 881, 108]]<|/det|>
+24. Amor, F. et al. Cholesterol metabolism is a potential therapeutic target in Duchenne muscular dystrophy. J Cachexia Sarcopenia Muscle 12, 677-693 (2021).  
+
+<|ref|>text<|/ref|><|det|>[[115, 108, 880, 140]]<|/det|>
+25. Yu, L. et al. Small-molecule activation of lysosomal TRP channels ameliorates Duchenne muscular dystrophy in mouse models. Science advances 6, eaaz2736 (2020).  
+
+<|ref|>text<|/ref|><|det|>[[115, 140, 880, 188]]<|/det|>
+26. Jackson, T., Sheppard, D., Denyer, M., Blakemore, W. & King, A.M. The epithelial integrin alphavbeta6 is a receptor for foot-and-mouth disease virus. Journal of virology 74, 4949-4956 (2000).  
+
+<|ref|>text<|/ref|><|det|>[[115, 188, 880, 219]]<|/det|>
+27. Kotecha, A. et al. Rules of engagement between alphavbeta6 integrin and foot-and-mouth disease virus. Nature communications 8, 15408 (2017).  
+
+<|ref|>text<|/ref|><|det|>[[115, 219, 880, 250]]<|/det|>
+28. Summerford, C., Johnson, J.S. & Samulski, R.J. AAVR: A Multi-Serotype Receptor for AAV. Molecular therapy: the journal of the American Society of Gene Therapy 24, 663-666 (2016).  
+
+<|ref|>text<|/ref|><|det|>[[115, 250, 880, 295]]<|/det|>
+29. Gonzalez-Sandoval, A. et al. The AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner. Nature communications 14, 2448 (2023).  
+
+<|ref|>text<|/ref|><|det|>[[115, 295, 880, 325]]<|/det|>
+30. Duclos, F. et al. Progressive muscular dystrophy in alpha-sarcoglycan-deficient mice. The Journal of cell biology 142, 1461-1471 (1998).  
+
+<|ref|>text<|/ref|><|det|>[[115, 325, 880, 356]]<|/det|>
+31. Stedman, H.H. et al. The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature 352, 536-539 (1991).
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[115, 90, 303, 107]]<|/det|>
+## REVIEWERS' COMMENTS  
+
+<|ref|>text<|/ref|><|det|>[[115, 127, 393, 144]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 163, 707, 180]]<|/det|>
+The authors have done an excellent job of addressing the issues that were raised.  
+
+<|ref|>text<|/ref|><|det|>[[115, 218, 393, 234]]<|/det|>
+Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 254, 780, 271]]<|/det|>
+The authors have carried out additional experiments and satisfactorily addressed concerns.  
+
+<|ref|>text<|/ref|><|det|>[[115, 309, 393, 325]]<|/det|>
+Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 345, 875, 473]]<|/det|>
+The authors have significantly improved the manuscript and addressed most of my comments. In particular, the authors have included non- human primate studies and extended upon the potential translatability of their work which is a very important addition to the paper that further strengthens the study. There were still a few issues that were not completely resolved but the authors provided possible explanations that could account for the observations and that were adequately discussed, without requiring further experimentation. This is an important and timely study that contributes to a better understanding of developing muscle- tropic vectors for gene therapy.  
+
+<|ref|>text<|/ref|><|det|>[[115, 492, 843, 527]]<|/det|>
+There are just a few remaining minor comments that the authors should address, which is relatively straightforward:  
+
+<|ref|>text<|/ref|><|det|>[[115, 547, 244, 563]]<|/det|>
+Minor comments  
+
+<|ref|>text<|/ref|><|det|>[[115, 583, 866, 619]]<|/det|>
+1. Some of the figures are too small and may be very difficult to read in the final version of the manuscript once it is printed. I would suggest to improve those and increase the font sizes significantly.  
+
+<|ref|>text<|/ref|><|det|>[[115, 638, 866, 729]]<|/det|>
+2. The exact nature of the capsid DNA sequence and the corresponding myotropic target peptide is not \(100\%\) clear. In particular, some amino acids from the AAV9 rh74 loop were deleted, whereas some additional amino acids were included as linker. It would appear that the precise sequencing conferring muscle-specificity was not clearly disclosed, only the "consensus motif" from the TGF-β1 and TGF-β3 domains.  
+
+<|ref|>text<|/ref|><|det|>[[115, 748, 870, 820]]<|/det|>
+To exclude any remaining ambiguity it is therefore important to include the actual DNA sequence of the relevant capsid sequence with the muscle-tropic targeting sequence, the deleted AAV9 rh74 sequence and the additional flanking sequences would be included in the paper along with the corresponding translated amino acid sequence.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[0, 0, 997, 997]]<|/det|>
+# 1. 1. 1. 1. 1. 1. 1. 2. 2. 2. 2. 2. 2. 2.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[117, 83, 425, 100]]<|/det|>
+## Authors' responses to the reviewers  
+
+<|ref|>sub_title<|/ref|><|det|>[[117, 118, 346, 133]]<|/det|>
+## Color code in this document:  
+
+<|ref|>text<|/ref|><|det|>[[117, 138, 304, 171]]<|/det|>
+Reviewers text in black  Author responses in blue  
+
+<|ref|>sub_title<|/ref|><|det|>[[117, 196, 421, 211]]<|/det|>
+## Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[117, 214, 712, 230]]<|/det|>
+The authors have done an excellent job of addressing the issues that were raised.  
+
+<|ref|>text<|/ref|><|det|>[[115, 240, 881, 275]]<|/det|>
+Response: We would like to thank the reviewer for your time and for recommending our manuscript for publication.  
+
+<|ref|>sub_title<|/ref|><|det|>[[117, 299, 421, 315]]<|/det|>
+## Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[117, 318, 785, 334]]<|/det|>
+The authors have carried out additional experiments and satisfactorily addressed concerns.  
+
+<|ref|>text<|/ref|><|det|>[[115, 344, 881, 378]]<|/det|>
+Response: We would like to thank the reviewer for your time and for recommending our manuscript for publication.  
+
+<|ref|>sub_title<|/ref|><|det|>[[117, 403, 421, 419]]<|/det|>
+## Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 421, 882, 550]]<|/det|>
+The authors have significantly improved the manuscript and addressed most of my comments. In particular, the authors have included non- human primate studies and extended upon the potential translatability of their work which is a very important addition to the paper that further strengthens the study. There were still a few issues that were not completely resolved but the authors provided possible explanations that could account for the observations and that were adequately discussed, without requiring further experimentation. This is an important and timely study that contributes to a better understanding of developing muscle- tropic vectors for gene therapy.  
+
+<|ref|>text<|/ref|><|det|>[[115, 560, 881, 594]]<|/det|>
+Response: We would like to thank the reviewer for your time and for recommending our manuscript for publication.  
+
+<|ref|>text<|/ref|><|det|>[[115, 618, 880, 653]]<|/det|>
+There are just a few remaining minor comments that the authors should address, which is relatively straightforward:  
+
+<|ref|>text<|/ref|><|det|>[[117, 657, 247, 671]]<|/det|>
+Minor comments  
+
+<|ref|>text<|/ref|><|det|>[[115, 675, 881, 748]]<|/det|>
+1. Some of the figures are too small and may be very difficult to read in the final version of the manuscript once it is printed. I would suggest to improve those and increase the font sizes significantly. Response: We have reformatted the figures to comply with Nature Communications' submission guidelines and enhance readability.  
+
+<|ref|>text<|/ref|><|det|>[[115, 757, 881, 848]]<|/det|>
+2. The exact nature of the capsid DNA sequence and the corresponding myotropic target peptide is not \(100\%\) clear. In particular, some amino acids from the AAV9 rh74 loop were deleted, whereas some additional amino acids were included as linker. It would appear that the precise sequencing conferring muscle-specificity was not clearly disclosed, only the "consensus motif" from the TGF-β1 and TGF-β3 domains.  
+
+<|ref|>text<|/ref|><|det|>[[115, 851, 881, 886]]<|/det|>
+To exclude any remaining ambiguity it is therefore important to include the actual DNA sequence of the relevant capsid sequence with the muscle-tropic targeting sequence, the deleted AAV9 rh74 sequence
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[113, 82, 883, 117]]<|/det|>
+and the additional flanking sequences would be included in the paper along with the corresponding translated amino acid sequence.  
+
+<|ref|>text<|/ref|><|det|>[[113, 119, 883, 155]]<|/det|>
+Response: We have added Data File S3, which includes the DNA and amino acid sequences of all capsid variants generated in this study, with sequence differences between constructs highlighted.  
+
+<|ref|>text<|/ref|><|det|>[[115, 157, 475, 173]]<|/det|>
+In the Data Availability section, we have added:  
+
+<|ref|>text<|/ref|><|det|>[[113, 176, 883, 211]]<|/det|>
+"The DNA and amino acid sequences of AAV capsid variants used in this study are provided in the data file S3".
+
+<--- Page Split --->

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+
+# nature portfolio  
+
+Peer Review File  
+
+Low- field onset of Wannier- Stark localization in a polycrystalline hybrid organic inorganic perovskite  
+
+![PLACEHOLDER_0_0]
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+## REVIEWER COMMENTS  
+
+Reviewer #1 (Remarks to the Author):  
+
+Berghoff et al report Wannier- Stark localization in a thin film of hybrid perovskite MAPbI3 achieved via non- resonant excitation with a 20THz electric field. This is achieved in a self- assembled material and at low electric field intensities compared to previous results in GaAs. Given the potential applications in ultrafast optically controlled switching of material properties, the presented results are not only novel but also have a broad interest. The experiment involves ultrafast THz pump - optical probe spectroscopy and supported by extensive and well- crafted calculations, comparing the effect of static and transient electric fields and accounting for the effect of the sample polycrystallinity. This work represents an exciting contribution, and is suitable for publication in Nature Communications. A few minor points to be addressed are as follows: 1. It would be helpful to clearly note that in the data presented in the main manuscript (Fig.2(a)), the low- field regime refers only to the early delay times before the THz field peak. A separate measurement, with an overall lower THz field, could also be shown for comparison, providing a reference where no localization occurs.  
+
+2. The discussion of figure 2, which shows the main experimental finding of the paper and its interpretation, should be expanded. In particular, panel d is not sufficiently explained in the main text: how are the separate peaks appearing in the absorbance attributed to \(\mathrm{n = 0}\) , \(+ / - 1\) ? The possibility of optically controlled switching between 3D materials and QWs is fascinating and could be explored more at length.  
+
+3. Since the low-field onset of Wannier-Stark localization is a key claim of this work, is it possible to provide a confidence interval to the estimation of the THz electric field amplitude? Can it be absolutely calibrated e.g. using a thin (smaller than the coherence length between the EOS probe and all relevant THz wavelengths) electro-optic crystal?  
+
+4. Considering the in-depth discussion of orientations in both real space and Brillouin space in pages 12-13, a diagram not only of MAPI structure in general but of the specific details being addressed would facilitate understanding (e.g. what Pb-I bond angles? Which direction is \Gamma Gamma M?)  
+
+5. Is there a specific reason to consider MAPbI rather than other hybrid perovskites? Could one expect to easily replicate these results in other materials in the same family? Would a softer lattice, e.g. by selecting different cations or substituting a lighter metal for Pb, result in localization at even lower fields?  
+
+A few typos and mistakes in figure presentation should also be corrected:  
+
+\* In the section "Experimental observation of Wannier-Stark Localization", units are incorrect in the sentence: "relatively weak fields, \(\mathrm{E}< 3 \mathrm{MV}\) , for \(\mathrm{T}< - 100 \mathrm{fs}\) ". It should read \(\mathrm{MV / cm}\) .  
+
+\*Is all the data presented (e.g. in fig. S2, S3) measured with a 6MV/cm THz field? Please specify so in captions.  
+
+\* The x-axis of figure S1 is labeled incorrectly, it should probably be in eV and not nm  
+
+\* Figures S4/S5 are not referred to in the main text  
+
+## Reviewer #2 (Remarks to the Author):  
+
+This work provided an experimental observation of transient Wannier- Stark (WS) localization in a polycrystalline MAPbI3 perovskite based on similar previous work performed in the GaAs/AlGaAs superlattice (Ref. 8 in this manuscript). As the authors emphasized, the main difference is that the field amplitude needed here (3MV/cm) is far less than that in GaAs/AlGaAs (which exceeds 10 MV/cm) due to the large relevant lattice constant, the small width of electronic energy bands, and the coincidence of these two along the same high- symmetry direction. The results and the method discussed here are interesting and the manuscript is well written. However, since similar work has been reported in literature and I did not find enough novelty of current work, I cannot accept the present paper for publication in high level journals like Nat Comm. The authors may want to show and discuss about the novelty and new highlights of their work should they try to submit to similar journals again. For instance:  
+
+1) Further analysis and emphasis of the peculiarity unique to MAPbI3 (such as low field strengths required for WS), and in particular the applications of this feature.  
+
+2) Any other typical physical phenomena not limited to WS localization that are based on the
+
+<--- Page Split --->
+
+transient spectral analysis? The authors developed a direct method to show WS localization in real natural solids. Since such localization has already been extensively studied in previous work, it will be interesting if this method can also be used for the study of other physical phenomena. 3) MAPbI3 perovskite exhibits wide light absorption range and excellent photo- electronic properties and is considered as a prominent light harvester. In this manuscript, the description and discussion of novel properties of MAPbI3, especially related to this work, seems absent.  
+
+Other questions and suggestions:  
+
+1. In this work, the WS localization is created by applying the bias field from the phase-stable THz pulse and can be detected by optical absorption spectra. Is this method generally applicable for single-crystalline and polycrystalline materials? 
+2. The observed WS step is slightly lower than the expected value. Is it possible that the step is higher than the theoretical value? Can the disorder of the sample be controlled here, and what is the influence of disorder on the experimental results? 
+3. For MAPbI3, transient optical response is in fact dominated by the least dispersive direction of the band structure. Do other polycrystalline materials also have the same feature? 
+4. WS ladders and associated phenomena predicted and observed in biased semiconductor superlattices have intrigued scientists for decades (it seems the work of J. Bleuse et al., Phys. Rev. Lett. 60, 220, 1988, should be cited together with [4, 5]). The concept was later introduced and explored also in ultracold atoms (Phys. Rev. Lett. 76, 4512, 1996) and optical waveguide arrays superimposed with a linear optical potential (Opt. Lett. 23, 1701, 1998; Opt. Lett. 39, 1065, 2014). Since the paper is intended for an interdisciplinary journal like NC, the authors should discuss the broader impact of their work and possible connection to other fields.  
+
+Reviewer #3 (Remarks to the Author):  
+
+In this manuscript, the authors synthesized a MAPbI3 polycrystalline film and studied ultrafast nonlinear optical responses of the MAPbI3 film by performing THz- pump/NIR to visible- probe spectroscopy and theoretical calculations. They observed a transient absorption change induced by THz pump with a center frequency of 20 THz and assigned its origin to the Wannier- Stark localization. They found that the transient Wannier- Stark localization in a MAPbI3 film can be observed at lower THz field strength than that in a single crystal GaAs, which results from a narrow electronic bandwidth and a large relevant lattice constant of MAPbI3. The finding that its narrow electric bandwidth and large lattice constant make a MAPbI3 film a unique optical nonlinear material would be important for developing novel photonic devices based on halide perovskites. However, the discussions are not enough to guarantee that the observed transient absorption change originates from the Wannier- Stark localization, on which I commented below. Therefore, my opinion is that this manuscript is not suitable for publication in Nature Communications as it stands.  
+
+The following points are the reasons why I doubt the interpretation of the data as the Wannier- Stark localization:  
+
+On page 4, the authors mentioned that the measured transient absorption change is well explained by a two- band model. However, I question why the higher energy states, such as light and heavy electron states, do not contribute to the optical nonlinear processes. In fact, the previous study [Z. Wei et al., Nat. Commun. 10, 5342 (2019).] reported that light and heavy electron states exist around 2.25 eV and the optical transitions between those states and the band- edge conduction band states significantly contribute to the two- photon absorption processes in a MAPbI3 film. Therefore, I suspect that the observed transient absorption change originates from the Bloch- Siegert shift [E. J. Sie et al., Science 355, 1066 (2017).] in multiple states consisting of the band- edge valence and conduction band states and the higher energy conduction band states. Is it possible for the authors to comment on this?  
+
+How much is the spectral width of the THz pump pulses? As the authors stated on page 6, the phonon modes of halide perovskites fall in the low frequency range around several THz. Therefore, if the spectral width is larger than the phonon frequencies, impulsive stimulated Raman scattering
+
+<--- Page Split --->
+
+should occur. As some of the authors reported, driving the phonon modes leads to the similar transient absorption change albeit in a different crystal structure [Fig. 4a in H. Kim et al., Nat. Commun. 8, 687 (2017).]. Is it possible to discuss the contributions of phonons to the observed signals?  
+
+On page 13, the authors mentioned that the crystallites are arbitrarily oriented and the theoretical calculations were performed based on this assumption. Is there any possibility that some preferential orientations of the crystallites exist? Do the authors experimentally verify the assumption from, for example, XRD spectra? The detailed sample properties should be described in the Support Information, because the sample is not single crystal but complicated polycrystalline film.  
+
+On page 15, the authors claimed that the calculation result shown as a black curve in Fig. 4a is in good agreement with experimental results at high field amplitudes in Fig. 4b. However, the spectral oscillations around 2 eV can be seen only in the experimental results, not in the calculation. In addition, a bleaching signal around 2.2 eV which is expected from the calculation does not appear in the experimental results. What are the reasons for such disagreements?  
+
+I suggest some more points for improving manuscript:  
+
+On page 8, why did the authors consider the band gap energy of 2D perovskites with \(I = 2\) , not \(I = 1\) , 3 or 4?  
+
+With regard to Ref. 25, the authors should cite the published version [J.- C. Blancon et al., Nat. Commun. 9, 2254 (2018).], not that in arXiv.
+
+<--- Page Split --->
+
+Reviewer #1 (Remarks to the Author):  
+
+Berghoff et al report Wannier- Stark localization in a thin film of hybrid perovskite MAPbI3 achieved via non- resonant excitation with a 20THz electric field. This is achieved in a self- assembled material and at low electric field intensities compared to previous results in GaAs. Given the potential applications in ultrafast optically controlled switching of material properties, the presented results are not only novel but also have a broad interest. The experiment involves ultrafast THz pump - optical probe spectroscopy and supported by extensive and well- crafted calculations, comparing the effect of static and transient electric fields and accounting for the effect of the sample polycrystallinity. This work represents an exciting contribution, and is suitable for publication in Nature Communications. A few minor points to be addressed are as follows:  
+
+1. It would be helpful to clearly note that in the data presented in the main manuscript (Fig.2(a)), the low-field regime refers only to the early delay times before the THz field peak. A separate measurement, with an overall lower THz field, could also be shown for comparison, providing a reference where no localization occurs.  
+
+\(\Rightarrow\) First of all, we appreciate the reviewer for very encouraging and constructive comments. We agree with the reviewer's first suggestion and thus clarified the first regime as follows in the main text:  
+
+"More importantly, two distinct regimes can be identified in the time- resolved transient spectrum (Fig. 2(a)). The first regime appears at delay times \(\tau < - 100\) fs, where the field strength is relatively weak \((E < 3 \text{MV/cm})\) , as an induced absorption (blue, \(\Delta T / T < 0\) ) right below and an induced transmission (red, \(\Delta T / T > 0\) ) right above the bandgap of \(E_{\text{gap}} = 1.62 \text{eV}\) . The second regime is apparent for field strengths \(E > 3 \text{MV/cm}\) , occurring between delay times \(- 100 < \tau < 100\) fs (Fig. 2(b))."  
+
+Regarding the separate measurement, the low- field regime \((< 2 \text{MV/cm})\) has been analyzed in our previous work using a narrower- band optical probe [Nat. Commun. 8, 687] and in another reported work using \(\sim \text{kHz}\) field [ACS Photonics 2016, 3, 1060- 1068]. There, one could find the electroabsorption spectra for comparison, where no localization occurs (Franz Keldysh effect).  
+
+Nonetheless, we have performed a separate measurement at a slightly lower peak field strength (4 MV/cm) with a different center frequency of 30 THz. In this case, we reproduce the observed transient Wannier Stark localization with lower peak field strength and different THz center frequency. We have added this result and the THz field characteristics in the supplementary figure S5.  
+
+2. The discussion of figure 2, which shows the main experimental finding of the paper and its interpretation, should be expanded. In particular, panel d is not sufficiently explained in the main text: how are the separate peaks appearing in the absorbance attributed to \(n = 0\) , \(+/- 1\) ? The possibility of optically controlled switching between 3D materials and QWs is fascinating and could be explored more at length.  
+
+\(\Rightarrow\) Yes, we agree that the detailed explanation of Figure 2 (c, d) would be necessary not only for clarifying the mechanism, but also for strengthening our interpretation. Therefore, we have added the following paragraph in the main text (experimental results):
+
+<--- Page Split --->
+
+"The extended structure of the transient spectral response can be understood with the assistance of Figure 2 (c, d). The localized Wannier- Stark states, equally spaced in energy by an amount \(eE_{THZ}D\) , are depicted in the real- space along the field direction, z, in Fig 2 (c). D is the lattice period unit length and n the index. This space- dependent energy shift results in differentiating the electronic transition energies within the same site (arrow with \(n = 0\) , Fig 2 (c)) from those between different sites (arrows with \(n = \pm 1\) , Fig 2 (c)). As the difference in the transition energy with respect to the central spatially- direct ( \(n = 0\) ) transition is \(nE_{THZ}D\) , one could assign the induced absorption below the band gap and above 1.9 eV to be \(n = - 1\) and \(n = 0\) transitions, respectively (Fig 2 (d)). The reduced absorption right above the band gap stems from the spectral transfer from non- perturbed optical transition to red- ( \(n = - 1\) ) and blue- ( \(n = 0\) ) shifted transitions (Fig 2 (d)). Depending on the strength of \(E_{THZ}\) and the degree of localization, \(Inl > 1\) transitions could, in principle, also be observed. In this case (Figure 2(a)), the observed single central step from reduced to increased absorption near the center of the band \(E_{pr} = 1.9\) eV, is a noticeable signature of Stark localization, where the Wannier- Stark states are localized onto one unit cell."  
+
+The possibility of optical switching between 3D delocalized and effective- 2D localized electronic states is emphasized as follows:  
+
+"The large unit cell and the small bandwidths along one direction of this material allows for optical switching with up to \(\sim 40\%\) transmission modulation depth using relatively moderate biasing fields. Also, the optical modulation of the material is extremely fast (sub- 20 fs), as demonstrated directly by the quasi- instantaneous response to an electric field oscillating at mid- infrared frequency."  
+
+"The polycrystallinity of this material does not impede the optical switching performance of the material, since the least dispersive direction of the band structure dominates the contribution to the optical response, which favors low- cost fabrications. Together with the outstanding photophysical properties of MAPbl3, this finding highlights the potential of this material in novel photonic applications."  
+
+3. Since the low-field onset of Wannier-Stark localization is a key claim of this work, is it possible to provide a confidence interval to the estimation of the THz electric field amplitude? Can it be absolutely calibrated e.g. using a thin (smaller than the coherence length between the EOS probe and all relevant THz wavelengths) electro-optic crystal?  
+
+\(\Rightarrow\) The reviewer raises an excellent point. In response, we have added the following statements to describe the accuracy of the THz electric field amplitude in the main text. What we would like to emphasize here is that the estimated field amplitude inside the material was even slightly overestimated because we use the reported refractive index of the material at near-infrared frequency (2.2, by which the estimated peak field amplitude was 6 MV/cm as we report in the manuscript), which it is likely slightly higher in the measured frequency (20 THz). We note that the refractive index at 3 THz is reported to be \(\sim 3\) (by which the estimated peak field amplitude is \(\sim 5\) MV/cm). Therefore, we mention that we report the upper limit of the actual field strength.  
+
+"The phase- stable THz biasing fields are generated using a difference- frequency generation scheme in GaSe and characterized by ultrabroadband electro- optic sampling. The accuracy for determination of the absolute electric field strength in the center of the pump spot is estimated to be \(\pm 15\%\) . The peak field strength at the interior of the MAPbl3 perovskite sample is obtained using the Fresnel transmission coefficient with the reported refractive index \(n = 2.2\) at near- infrared frequency, which sets the upper limit of the actual field strength."
+
+<--- Page Split --->
+
+The crystal we have used exhibits a rather flat response function in the relavant frequency range. Consequently, it does not distort the waveform significantly in the time domain. The crystal would be thinner than the coherence length for all relevant THz input frequencies. A detailed description of the procedure for determination of the field is found in the Methods section:  
+
+"The electric field transient is characterized by ultrabroadband electro- optic sampling at a \(30 - \mu m\) thick GaSe crystal using balanced detection of an 8- fs probe pulse centered at a wavelength of \(1.2 \mu m\) as the gating pulse. The quantitative value of the field amplitude is obtained by measuring the THz average power, pulse repetition rate and focal spot size. Then, the value at the interior of the MAPbI3 perovskite sample is estimated using the Fresnel transmission coefficient for the THz field at the air- MAPbI3 interface."  
+
+4. Considering the in-depth discussion of orientations in both real space and Brillouin space in pages 12-13, a diagram not only of MAPI structure in general but of the specific details being addressed would facilitate understanding (e.g. what Pb-I bond angles? Which direction is \GammaGamma M?)  
+
+\(\Rightarrow\) We thank the reviewer for the helpful suggestion. Accordingly, we modified Fig 1a to indicate the exact Pb-I-Pb angle, the Pb-I bond length, and the most relevant direction (Gamma Z) mentioned in the main text on pages 12-13. Also, we refer to the figure when these details are mentioned (highlighted in the main text).  
+
+5. Is there a specific reason to consider MAPbI rather than other hybrid perovskites? Could one expect to easily replicate these results in other materials in the same family? Would a softer lattice, e.g. by selecting different cations or substituting a lighter metal for Pb, result in localization at even lower fields?  
+
+\(\Rightarrow\) We appreciate the question highlighting the uniqueness of the MAPbI3. In general, Wannier- Stark localization is very challenging to observe in natural crystals due to the small lattice constants, the rapid scattering, and the dielectric breakdown conditions. One of most important messages of our work is that MAPbI3, in particular, has the combination of the narrow bandwidth, the large periodicity, and the coincidence of the two directions. These unique features of its band structure promote this material to the Wannier Stark regime under relatively modest field amplitudes. Therefore, if one could find any other material (including other perovskites) where these conditions are met, we could expect similar results, provided other material- specific disturbances such as interband tunnelings are absent. Regarding the softness of lattice, if it means the lower nuclear vibration energy, there is not necessarily nor always a correlation between the electronic band dispersion near the band gap and a certain phonon frequency.  
+
+In the main text, we have tried to emphasize the unique electronic properties of MAPbI3 that enable our observation, including the above- cited (comment #2) sentences:  
+
+"The large unit cell and the small bandwidth along one direction of this material allows for optical switching with up to \(\sim 40\%\) transmission modulation depth using relatively moderate biasing fields. Also, the optical modulation of the material is extremely fast (sub- 20 fs), as demonstrated directly by the quasi- instantaneous response to an electric field oscillating at mid- infrared frequency."
+
+<--- Page Split --->
+
+A few typos and mistakes in figure presentation should also be corrected:  
+
+\* In the section "Experimental observation of Wannier- Stark Localization", units are incorrect in the sentence: "relatively weak fields, \(E < 3 \text{MV}\) , for \(\tau < - 100 \text{fs}\) ". It should read MV/cm. \*Is all the data presented (e.g. in fig. S2, S3) measured with a 6MV/cm THz field? Please specify so in captions. \* The x- axis of figure S1 is labeled incorrectly, it should probably be in eV and not nm \* Figures S4/S5 are not referred to in the main text  
+
+\(\Rightarrow\) We thank the reviewer for thoroughly reviewing our materials and kindly raising the points that we overlooked. We corrected all of them accordingly (highlighted).
+
+<--- Page Split --->
+
+Reviewer #2 (Remarks to the Author):  
+
+This work provided an experimental observation of transient Wannier- Stark (WS) localization in a polycrystalline MAPbI3 perovskite based on similar previous work performed in the GaAs/AlGaAs superlattice (Ref. 8 in this manuscript). As the authors emphasized, the main difference is that the field amplitude needed here (3MV/cm) is far less than that in GaAs/AlGaAs (which exceeds 10 MV/cm) due to the large relevant lattice constant, the small width of electronic energy bands, and the coincidence of these two along the same high- symmetry direction. The results and the method discussed here are interesting and the manuscript is well written. However, since similar work has been reported in literature and I did not find enough novelty of current work, I cannot accept the present paper for publication in high level journals like Nat Comm. The authors may want to show and discuss about the novelty and new highlights of their work should they try to submit to similar journals again. For instance:  
+
+1) Further analysis and emphasis of the peculiarity unique to MAPbI3 (such as low field strengths required for WS), and in particular the applications of this feature.  
+
+\(\Rightarrow\) First of all, we appreciate the reviewer's critical yet very helpful comments. For highlighting the first point, we have added the following paragraphs to the introduction, and have rewritten the conclusion and abstract:  
+
+[Introduction] "Besides their use in solar cells and light- emitting diodes, in this work, we demonstrate that MAPbI3 also has a great potential in photonic applications, including optical modulators, optical switches, and optical signal processing. For any optical amplitude modulator, one of the essential properties is a substantial change of the absorption edge with relatively low required energies in general. We demonstrate that solution- processed, polycrystalline MAPbI3 shows drastic changes in optical properties via Wannier Stark localization, at weak biasing fields. Whereas conventional semiconductors constituting photo- detectors, e.g. Si or InGaAs, require costly manufacturing processes and are limited to traditional rigid type devices, perovskites with distinct crystal structures exhibit ultrafast response (sub- 20 fs), while simultaneously supporting cheap and flexible polycrystalline film fabrication.  
+
+The large unit cell and the small bandwidths along one direction of this material allows for optical switching with up to \(\sim 40\%\) transmission modulation depth using relatively moderate biasing fields. Also, the optical modulation of the material is extremely fast (sub- 20 fs), as demonstrated directly by the quasi- instantaneous response to an electric field oscillating at mid- infrared frequency."  
+
+[Conclusion] "we have demonstrated that solution- processed, polycrystalline MAPbI3 shows optical transmission change by tens of percent at relatively modest field strengths via transient Wannier Stark localization. The large lattice periodicity, the narrow electronic energy bandwidths, and the coincidence of these two along the same high- symmetry direction promotes this material to the Wannier Stark regime under relatively moderate biasing fields. Polycrystallinity of this material turns out not to hinder the Wannier Stark localization effect as observed, due to the dominant contribution from the least dispersive direction of the band structure, which favors low- cost fabrications with this material as optical modulators. The degree of disorder and relative orientation among crystallites may influence the modulation spectral shape slightly, e.g. the position of the photon energy where the induced transmission to induced absorption happens, which could be finely tuned depending on the desired device performance by further systematic studies."
+
+<--- Page Split --->
+
+[Abstract] "Methylammonium lead iodide perovskite \((MAPbI_3)\) , renowned for an impressive power conversion efficiency rise and cost- effective fabrication for photovoltaics, exhibits a huge potential for optical modulation- type applications, in this work. We demonstrate that polycrystalline \(MAPbI_3\) s undergo drastic changes in optical properties with the modulation depth to be tens of percent at moderate field strengths, via transient Wannier Stark localization with an ultrafast response time. The distinct band structure of this material - the large lattice periodicity, the narrow electronic energy bandwidths, and the coincidence of these two along the same high- symmetry direction - enables relatively weak fields to bring this material into the Wannier Stark regime. Its polycrystalline nature is not detrimental to the optical switching performance of the material, since the least dispersive direction of the band structure dominates the contribution to the optical response, which favors low- cost fabrication. Together with the outstanding photophysical properties of \(MAPbI_3\) , this finding highlights the potential of this material in novel photonic applications."  
+
+2) Any other typical physical phenomena not limited to WS localization that are based on the transient spectral analysis? The authors developed a direct method to show WS localization in real natural solids. Since such localization has already been extensively studied in previous work, it will be interesting if this method can also be used for the study of other physical phenomena.  
+
+\(\Rightarrow\) We thank the reviewer for the suggestion to emphasize the advantage and potential use of our experimental technique and approach. Accordingly, we have added a dedicated paragraph in the conclusion as follows:  
+
+"Moreover, the phase stable THz field transients and the ultra- broadband optical pulses of 7 fs duration revealed that the optical modulation of this material has an extremely fast, quasi- instantaneous (sub- 20fs) temporal response in visible/near- IR spectral region. This technique could be generalized for realizing transient Wannier Stark localization in other semiconductor solids in a carefully prepared single- crystalline or a polycrystalline form. More generally, this method enables to analyze any ultrafast changes in optical properties induced by the phase- locked and intense electromagnetic field transients, be it resonantly or non- resonantly. While here we used only the electric field of the transients, one could also exploit the magnetic component for exploring ultrafast magneto- optic effects, by enhancing the magnetic field with respect to the electric field with, e.g., a specially designed plasmonic nanoaperture."  
+
+3) MAPbI3 perovskite exhibits wide light absorption range and excellent photo-electronic properties and is considered as a prominent light harvester. In this manuscript, the description and discussion of novel properties of MAPbI3, especially related to this work, seems absent.  
+
+\(\Rightarrow\) We thank the reviewer for this suggestion for improving our work. We have added the following paragraph to the introduction to better connect to the cited part of the conclusion:  
+
+[Introduction] "Methylammonium lead iodide perovskite \((MAPbI_3)\) has become a remarkable material for photovoltaic applications due to the dramatic increase of the power conversion efficiency and the cost- effective fabrication processes. The success of this material has been attributed to large absorption coefficient and the exceptional transport properties such as long carrier diffusion length, high carrier mobilities and defect tolerance."
+
+<--- Page Split --->
+
+[Conclusion] "Together with the renowned photophysical properties of MAPbl3, such as the long carrier diffusion length, low mid- gap trap density, and large absorption coefficient, this finding of high modulation depth, ultrafast response, and low onset field for Wannier- Stark localization highlights the potential of this material in photonic applications."  
+
+Other questions and suggestions:  
+
+1. In this work, the WS localization is created by applying the bias field from the phase-stable THz pulse and can be detected by optical absorption spectra. Is this method generally applicable for single-crystalline and polycrystalline materials?  
+
+\(\Rightarrow\) Yes, it could be generally applicable for single-crystalline and polycrystalline materials. Thus, we added this sentence in the conclusion:  
+
+"This technique could be generalized for realizing transient Wannier Stark localization in other semiconductor solids in a carefully prepared single-crystalline or a polycrystalline form."  
+
+2. The observed WS step is slightly lower than the expected value. Is it possible that the step is higher than the theoretical value? Can the disorder of the sample be controlled here, and what is the influence of disorder on the experimental results?  
+
+\(\Rightarrow\) First, we would like to point out that the predicted position where the WS step occurs depends on the level of theory. If we compare the Fig 3 and Fig 4(c, d), the step occurs at slightly below 2 and \(1.9 \text{eV}\) , respectively, and the experimentally observed position was \(1.9 \text{eV}\) . The difference in the theoretical approaches to obtain Fig 3 and Fig 4(c,d) is whether the other direction is taken into account (i.e., with (Fig 4(c,d)) and without (Fig 3) the averaging over the disorder). The disorder of the sample could not be controlled experimentally here, but from this theoretical comparison, what one could learn about the influence of disorder on the experimental results would be indeed the position of the WS step (with \(\sim 100 \text{meV}\) range). We strengthened this discussion by adding the following to the discussion and conclusion sections:  
+
+[Discussion] "This finding is remarkable because it means the two extreme cases – the completely random orientation of a polycrystalline sample and the perfectly oriented single crystal – are expected to produce very similar optical responses. The only slight difference between the two extremes would be a small shift in the photon energy (\~100 meV) where the induced transmission turns to the induced absorption and in the transient spectral shape. The two extremes include partial preferential orientations. We also note that the averaging process using only two extreme directions does not contain any material-specific information, which means that one could expect other polycrystalline materials to behave similarly."  
+
+[Conclusion] "The degree of disorder and relative orientation among crystallites may influence the modulation spectral shape slightly, e.g. the position of the photon energy where the induced transmission to induced absorption happens, which might be finely tuned depending on the desired device performance by further systematic studies."  
+
+3. For MAPbl3, transient optical response is in fact dominated by the least dispersive direction of the band structure. Do other polycrystalline materials also have the same feature?  
+
+\(\Rightarrow\) Yes, we believe so, because for the larger bandwidth, the field required for WS localization will be larger. This means that the direction with the smallest dispersion will always be where WS
+
+<--- Page Split --->
+
+localizations occur for the smallest field. While we observe the localization along the least dispersive direction, the contributions from the other directions are negligible, since the transmission changes are smaller for fields below the WS threshold. It is indeed what we observe from the theoretical model, including averaging over the arbitrarily oriented crystallites. Regarding this point, we added this sentence in the main text (discussion section):  
+
+"We also note that the averaging process using only two extreme directions does not contain any material- specific information, which means that one could expect other polycrystalline materials to behave similarly."  
+
+4. WS ladders and associated phenomena predicted and observed in biased semiconductor superlattices have intrigued scientists for decades (it seems the work of J. Bleuse et al, Phys. Rev. Lett. 60, 220, 1988, should be cited together with [4, 5]). The concept was later introduced and explored also in ultracold atoms (Phys. Rev. Lett. 76, 4512, 1996) and optical waveguide arrays superimposed with a linear optical potential (Opt. Lett. 23, 1701, 1998; Opt. Lett. 39, 1065, 2014). Since the paper is intended for an interdisciplinary journal like NC, the authors should discuss the broader impact of their work and possible connection to other fields.  
+
+\(\Rightarrow\) We thank the reviewer for this very insightful suggestion to position our work in a broader context and for the listed literature. We have, in our revised manuscript, cited and discussed the suggested literature and make a connection in the introduction as follows:  
+
+"Following the initial observations in semiconductor superlattices under static bias fields \(^{8 - 10}\) Wannier- Stark ladders have been proposed and realized in various physical systems featuring wave propagation in the presence of periodic potentials and a homogeneous force. Examples include ultracold atoms in an accelerating 1D standing wave \(^{5}\) , waveguide arrays with linearly varying propagation constants \(^{6}\) , and self- accelerating optical beams in 1D photonic lattice \(^{7}\) . Several fundamental observations and device applications from the Wannier- Stark localization have been focused on statically biased artificial semiconductor superlattices \(^{8 - 12}\) . However, in natural homogeneous solids, where the periodicity is dictated by the atomic structure, such an extreme state of matter has never been achieved using static biasing. To resolve optical transitions to individual Wannier- Stark states in, e.g., absorption spectra, their energetic spacing needs to be larger than the (total) linewidth \(\Gamma\) , i.e., \(eED > \Gamma^{9,10,13}\) Due to the small lattice constant of bulk crystals and the large linewidth which results from the scattering of electrons with lattice vibrations and other electrons, the requirement \(eED > \Gamma\) can typically not be fulfilled under stationary external fields below the strength where the dielectric breakdown occurs \(^{11,12}\) ."
+
+<--- Page Split --->
+
+Reviewer #3 (Remarks to the Author):  
+
+In this manuscript, the authors synthesized a MAPbl3 polycrystalline film and studied ultrafast nonlinear optical responses of the MAPbl3 film by performing THz- pump/NIR to visible- probe spectroscopy and theoretical calculations. They observed a transient absorption change induced by THz pump with a center frequency of 20 THz and assigned its origin to the Wannier- Stark localization. They found that the transient Wannier- Stark localization in a MAPbl3 film can be observed at lower THz field strength than that in a single crystal GaAs, which results from a narrow electronic bandwidth and a large relevant lattice constant of MAPbl3. The finding that its narrow electric bandwidth and large lattice constant make a MAPbl3 film a unique optical nonlinear material would be important for developing novel photonic devices based on halide perovskites. However, the discussions are not enough to guarantee that the observed transient absorption change originates from the Wannier- Stark localization, on which I commented below. Therefore, my opinion is that this manuscript is not suitable for publication in Nature Communications as it stands.  
+
+The following points are the reasons why I doubt the interpretation of the data as the Wannier- Stark localization:  
+
+On page 4, the authors mentioned that the measured transient absorption change is well explained by a two- band model. However, I question why the higher energy states, such as light and heavy electron states, do not contribute to the optical nonlinear processes. In fact, the previous study [Z. Wei et al., Nat. Commun. 10, 5342 (2019).] reported that light and heavy electron states exist around \(2.25 \text{eV}\) and the optical transitions between those states and the band- edge conduction band states significantly contribute to the two- photon absorption processes in a MAPbl3 film. Therefore, I suspect that the observed transient absorption change originates from the Bloch- Siegert shift [E. J. Sie et al., Science 355, 1066 (2017).] in multiple states consisting of the band- edge valence and conduction band states and the higher energy conduction band states. Is it possible for the authors to comment on this?  
+
+\(\Rightarrow\) First of all, we appreciate the reviewer's critical, yet very constructive comments. We also thank the reviewer for raising this possible alternative explanation, based on which we were able to improve our manuscript substantially. Here we clarify the detailed assignment of each spectral component (in our optical response) based on our mechanism (WS localization), which seem inconsistent with other nonlinear optical processes, and strengthen our interpretation as follows:  
+
+"The extended structure of the transient spectral response can be understood with the assistance of Figure 2 (c, d). The localized Wannier- Stark states, equally spaced in energy by an amount \(eE_{THZ}D\) , are depicted in the real- space along the field direction, z, in Fig 2 (c). D is the lattice period unit length and n the index. This space- dependent energy shift results in differentiating the electronic transition energies within the same site (arrow with \(n = 0\) , Fig 2 (c)) from between different sites (arrows with \(n = \pm 1\) , Fig 2 (c)). As the difference in the transition energy with respect to the central spatially- direct ( \(n = 0\) ) transition is \(nE_{THZ}D\) , one could assign the induced absorption below the band gap and above \(1.9 \text{eV}\) to be \(n = - 1\) and \(n = 0\) transitions, respectively (Fig 2 (d)). The reduced absorption right above the band gap stems from the spectral transfer from non- perturbed optical transition to red- ( \(n = - 1\) ) and blue- ( \(n = 0\) ) shifted transitions (Fig 2 (d)). Depending on the strength of \(E_{THZ}\) and the degree of localization, \(Inl > 1\) transitions could, in principle, also be observed. In this case (Figure 2(a)), the observed single central step from reduced to increased
+
+<--- Page Split --->
+
+absorption near the center of the band \(E_{pr} = 1.9 \text{eV}\) , is a noticeable signature of Stark localization, where the Wannier- Stark states are localized onto one unit cell.  
+
+This observed transient response is clearly distinct from the shift of transition energy due to the presence of photon- dressed states<sup>28</sup>. Photon- dressed states can shift energies in only one direction (mostly blue- shift) with energy orders of magnitude smaller than the bandwidth. Also, the contribution from the possible higher energy bands within our probe photon energy range<sup>29</sup> is negligible. Indeed, we neither observe any additional Franz- Keldysh and/or Wannier- Stark response within our probe energy, nor find any decay of the entire signal as a function of the field strength due to the tunneling to higher energy bands at intense field regime. Therefore, we could consider simple two- band systems to understand our experimental demonstration of Wannier- Stark localization in further detail. As will be shown below, the two- band model explains our observations."  
+
+Regarding the first point (in slightly more detail), given the presence of the higher band with the energy difference of around \(2.25 \text{eV}\) , there are two possibilities of experimental features one could observe in our result.  
+
+The first possibility is the case of tunneling to a higher energy band in the presence of strong THz fields. In that case, the photoexcited electrons and holes could tunnel into an energetically higher band and would then not contribute to WS localization in the lowest bands. Also, the signal could possibly decay as a function of the field amplitude for strong fields (since the tunneling rate has an exponential dependence on the field amplitude). However, we do not see indications in the experiment that this is happening to a significant degree.  
+
+In the other possibility, where the tunneling does not occur, if WS localization is also not the origin of our experimental observation, then each band would have shown at least Franz Keldysh effect at one around the primary bandgap of \(1.6 \text{eV}\) and the other one at around the secondary gap above \(2.25 \text{eV}\) (as the TPA paper reports). Since the Franz Keldysh effect appears as an oscillatory feature only near the gap (within \(100 \text{meV}\) in probe phonon energy), in that case one would expect two separate Franz Keldysh features - one in the \(1.5 \sim 1.7 \text{eV}\) range and another at \(2.1 \sim 2.3 \text{eV}\) , with no response for energies in between \(1.7 \sim 2.1 \text{eV}\) . In contrast, we find an induced transparency from \(1.6 \text{eV}\) all the way up to \(1.9 \text{eV}\) without interruption. The reason we did not observe any response in \(2.2 \text{eV}\) (even in FKE) could be understood from the different transition probability in the one- photon and two- photon transition processes. Our optical probing scheme involves one- photon transition where the response around \(2.2 \text{eV}\) is much weaker than the case of two- photon transition. Therefore, we could safely rule out the other scenario, too.  
+
+Regarding the second point, the Bloch- Siegert shift is typically very small in magnitude \(< 1 \text{ueV}\) and, in exceptional cases at best comparable to the optical Stark shift ( \(\sim 10 \text{meV}\) in the mentioned literature). Also, such a shift happens in only one direction: typically only blue shift (because this effect originates from the state repulsion). However, in our case, we have induced absorption below and above the band gap, which means that both red- shifted and blue- shifted transitions occur simultaneously, and reduced absorption at around the band gap due to the spectral transfer. These features are unique to Wannier Stark localization.
+
+<--- Page Split --->
+
+How much is the spectral width of the THz pump pulses? As the authors stated on page 6, the phonon modes of halide perovskites fall in the low frequency range around several THz. Therefore, if the spectral width is larger than the phonon frequencies, impulsive stimulated Raman scattering should occur. As some of the authors reported, driving the phonon modes leads to the similar transient absorption change albeit in a different crystal structure [Fig. 4a in H. Kim et al., Nat. Commun. 8, 687 (2017).]. Is it possible to discuss the contributions of phonons to the observed signals?  
+
+\(\Rightarrow\) The spectral width of the THz pump pulses is \(\sim 4\) THz. We added the spectrum of the THz pulses of the main result (fig 2a) in the supplementary information (fig. S1). For comparison, we also added the Fourier transform of the time profile in fig 2a in the SI (fig. S4).  
+
+As evident in this spectrum (fig S4), the main oscillation around time zero is 40 THz, and there is no distinct oscillation below 4 THz except for the slow envelope of the pulse. More importantly, if there is any phonon coherence, we know that the dephasing times of at least 1 and 2 THz modes are 0.3 and 1 ps, respectively, and both are much longer than the duration of the IR pulse and our experimental window. So if that was the case, we should have seen responses after the overlap of IR pulses and the visible probe (after \(\mathrm{t} = 200\) fs). However, we do not see any oscillation immediately after that, which indicates no contribution from phonons within the bandwidth. Thus, the impulsive stimulated Raman scattering could be safely ruled out. We added this argument om the main text as follows:  
+
+"It is noteworthy that the bandwidth of the THz pulse is \(\sim 4\) THz (< 40 THz modulation, Fig. S4), so that in principle impulsive stimulated Raman excitation of sub- 4 THz modes be possible. However, no oscillatory signal was observed after 150 fs, which is much shorter than the dephasing times of reported phonon modes with frequency up to 4 THz. Therefore, any possibility of coherent phonon contribution to the temporal modulation can be ruled out."  
+
+On page 13, the authors mentioned that the crystallites are arbitrarily oriented and the theoretical calculations were performed based on this assumption. Is there any possibility that some preferential orientations of the crystallites exist? Do the authors experimentally verify the assumption from, for example, XRD spectra? The detailed sample properties should be described in the Support Information, because the sample is not single crystal but complicated polycrystalline film.  
+
+\(\Rightarrow\) We understand and appreciate the reviewer's concern. However, any possibility of preferential orientations of the crystallites does not make any critical effect, as the two extreme cases - completely arbitrary orientation and perfectly single orientation - produce more or less the same THz induced optical spectra. From this theoretical approach, what we learn is that, no matter how much preferential orientation we have (from zero to complete), the contribution from the least dispersive direction dominates the optical response. Although we indeed can not completely rule out the possibility of partial preferential orientation, this possibility does not affect our conclusion. We highlight the point by adding these sentences in the main text:  
+
+"The results of Fig. 4(a, b) suggest that, for the randomly oriented crystallites in the film, the overall response is dominated by the response originating from the band dispersion in the \(\Gamma \bar{Z}\) direction. This reasoning is substantiated by the averaged field- dependent absorption changes calculated for both a static and a THz field shown in Figs. 4(c) and (d), respectively. This finding is remarkable because it means the two extreme cases - the completely random orientation of a polycrystalline
+
+<--- Page Split --->
+
+sample and the perfectly oriented single crystal – are expected to produce very similar optical responses. The only slight difference between the two extremes would be a small shift in the photon energy ( \(\sim 100\) meV) where the induced transmission turns to the induced absorption and in the transient spectral shape. The two extremes include partial preferential orientations."  
+
+Regarding our sample, we added the SEM image of the polycrystalline perovskite MAPbl3 film spin- coated on TOPAS® substrate in the supplementary information (fig S2). We did not measure the XRD data of the measured sample, but we do have the XRD spectra of the perovskite polycrystalline film prepared in the exactly same way [fig S15 of J. Phys. Chem. Lett. 2015, 6, 4991]. There, one could find not only (hh0) but also other Bragg peaks. Besides, even in the case of a complete preferential orientation of a surface, still the most critical orientation is the relative angle between the [001] direction of each crystallite and the THz field polarization, which is not possible to define. The orientations of [001] direction of each crystallite on top of a completely disordered substrate can still be assumed to be arbitrarily oriented and difficult to measure with the currently available resolution of X- ray microscopy ( \(\sim 500\) nm in J. Phys. Chem. C. 2017, 121, 7596).  
+
+On page 15, the authors claimed that the calculation result shown as a black curve in Fig. 4a is in good agreement with experimental results at high field amplitudes in Fig. 4b. However, the spectral oscillations around \(2 \text{eV}\) can be seen only in the experimental results, not in the calculation. In addition, a bleaching signal around \(2.2 \text{eV}\) which is expected from the calculation does not appear in the experimental results. What are the reasons for such disagreements?  
+
+\(\Rightarrow\) We thank the reviewer for thoroughly examining our results. Yes, the first point, the spectral oscillations around \(2 \text{eV}\) , is indeed something we have tried to understand. The oscillation (or repeated peaks) has an interval of \(80 \sim 100 \text{meV}\) regardless of the THz field strength. Interestingly this interval matches well with our THz photon energy (83 meV). The oscillations are quite stable as function of the field amplitude, however, they do not correspond to Franz- Keldysh and neither to WS. To analyze the possible origin of these oscillations, we did perform additional model calculations. To that end, we, in particular, modified the k/energy dependence of the interband dipole and also considered a complex interband dipole with a symmetric real and an antisymmetric imaginary part (it was recently shown that such an interband dipole with such a k- dependence gives rise to SHG in two- band models). With such phenomenological model extensions, we do obtain signatures having similarities with THz sidebands in the optical spectra, see Fig. S9 in the supplementary information. However, the obtained results are quite different from the oscillations observed in the experiment and the model assumptions required to obtain them are rather unrealistic.  
+
+Regarding the second point, in a 1D model with WS localization, we remove absorption from the band edges, and this concentrates in the band center. Since we average over systems of different bandwidths and lattice constants, the critical field where the transition to WS takes places and also the position of the band center is not fixed but depends on the individual case, i.e., differs for the different f's. As a result, in Fig. 4(c) and 4(d) the transitions from blue- to red- shift appear at higher energies with increasing field amplitude, since with increasing amplitude also systems with larger bandwidth, i.e., larger f, enter the WS regime. Therefore, with any two- band model one should always see some bleaching near the upper band edge due to the concentration, or in other words, a shift of the oscillator strength towards the center of the band.
+
+<--- Page Split --->
+
+In any case, the precision of our simple modeling involving some fitting of DFT results with few parameters gets more inaccurate with increasing photon energies.  
+
+To clarify this, we add an explanation in the main text as follows:  
+
+"The averaged graph is in good agreement with the differential spectra at high field amplitudes given very few parameters to describe the entire contributions from complicated actual band structure (i.e. two extreme bandwidths, lattice constants, and linear interpolation of them). The prediction of 1.9 eV, almost exactly where the change from bleaching to induced absorption is observed in the experiment (Fig 4(b), upper curves) is remarkable. A few minor features including the shape at higher photon energy could be improved by modifying the model band curve and oscillations around 2 eV in Fig 4(b) need further studies (Fig S9). More importantly, this position of 1.9eV is very close to the center of the band structure for the \(\overline{\Gamma Z}\) direction, 2 eV as the single direction model indicated above."  
+
+I suggest some more points for improving manuscript:  
+
+On page 8, why did the authors consider the band gap energy of 2D perovskites with \(I = 2\) , not \(I = 1\) , 3 or 4?  
+
+\(\Rightarrow\) It is because of the doubling of the unit cell in the tetragonal phase compared to the cubic phase. It means the double layers \((I = 2)\) Pb- I octahedra is the repeating unit, and in turn the spatial range of confinement. That was the reason we particularly compared the probe energy where one could observe the abrupt change of transmittance and the band gap energy of 2D perovskite with \(I = 2\) . We realized it could be confusing without further clarification. As such, we modified the corresponding sentence in the discussion section as following:  
+
+"In the case of \((BA)_2(MA)_{1 - 1}PbI_{3l + 1}\) perovskites, where the \(PbI_{6}\) octahedral network forms a double layer \((I = 2)\) , the same periodicity of the sample along the \(c\) axis, the optical band gap is \(\sim 2.1\) eV, which is comparable to the observed 1.9 eV."  
+
+With regard to Ref. 25, the authors should cite the published version [J.- C. Blancon et al., Nat. Commun. 9, 2254 (2018).], not that in arXiv.  
+
+\(\Rightarrow\) We cited this reference accordingly (highlighted).
+
+<--- Page Split --->
+
+Reviewers' comments:  
+
+Reviewer #1 (Remarks to the Author):  
+
+The authors have addressed all my concerns/comments in the revised manuscript. However, there are two important concerns from other reviewers which are very important. One is the role of the polycrystallinity of the sample. The only way to address this is to perform measurements on single crystals or two have extensive structural characterization of the sample, which are out of the scope of current work. Publication of this manuscript will inspire future work in that direction, I reckon.  
+
+The second concern, as reviewer 3 rightly points out, is with regards to possible alternative explanations to the presented experimental results. The authors provide a convincing arguments to discount other possibilities. Some of the explanations provided in the response to reviewer 3 were not included in the manuscript, for example, on why Bloch- Siegert is not a viable explanation. It may be very instructional to expand on this discussion in the main manuscript.  
+
+Apart from these very minor comments, the manuscript is suitable for publication in Nature Communications.  
+
+Reviewer #2 (Remarks to the Author):  
+
+I have read through authors' response, and the revised manuscript. I'm okay with authors' response to my comments, and thus I don't have further objection to accept this manuscript for publication in NC.  
+
+Reviewer #3 (Remarks to the Author):  
+
+I confirmed the replies from the authors, the revised manuscript, and the supporting information. I appreciate the efforts of the authors to answer my questions and comments. However, I still do not think that it is convincing that the origin of the observed nonlinear responses can be attributed to the Wannier- Stark localization. As the authors replied, the theoretical curves shown in Fig. 4a do not fully reproduce the experimental results in Fig. 4b partly because the theoretical model is too simple. Therefore, to make the interpretation as the Wannier- Stark localization solid, it is important to rule out the other possible origins. Although the other possibilities are discussed in the reply letter and the revised manuscript, I am not satisfied with some of those discussions. Thus, I do not recommend publication of the manuscript for Nature Communications as it stands. I suggest that the following points should be considered.  
+
+1. On page 9 in the main manuscript, the authors mentioned that the two induced absorption signals shown in Fig. 2d can be assign to \(n = -1\) and \(n = 0\) transitions, whose difference in the transition energy is \(\mathrm{eE}< \mathrm{sub} > \mathrm{THz}< /\mathrm{sub} > \mathrm{D}\) . How much is the value of \(\mathrm{eE}< \mathrm{sub} > \mathrm{THz}< /\mathrm{sub} > \mathrm{D}\) ? In addition, I cannot read from Fig. 2d what the \(n = -1\) and \(n = 0\) transition energies are.  
+
+2. On page 9 in the main manuscript, the authors stated "Photon-dressed states can shift energies in only one direction (mostly blue-shift) ..." However, I do not agree with the statement. If the higher energy levels exist, the Autler-Townes effect can shift the band-edge transitions in both directions, i.e. blue- and red- shifts, as reported in [C.-K. Yong et al. Nat. Mater. 18, 1065-1070 (2019).]. In addition, [G. Yumoto et al. Nat. Commun. 12, 3026 (2021).] recently reported that three-level Autler-Townes effect indeed occurs in halide perovskites. How do the authors comment on the possibility that the Autler-Townes effect can be the origin of the observed optical nonlinear responses?  
+
+3. On page 10 in the main manuscript, the authors stated "... nor find any decay of the entire signal as a function of the field strength due to the tunneling to higher energy bands at intense field regime." I agree to the authors that the real excitation of carriers cannot explain the data due to the absence of the signal decay. However, the virtual population generated only under the THz-field irradiation should induce additional transitions for probe pulses, which would result in induced absorption signals. Is it possible to comment on this?
+
+<--- Page Split --->
+
+Reviewer #1 (Remarks to the Author):  
+
+The authors have addressed all my concerns/comments in the revised manuscript. However, there are two important concerns from other reviewers which are very important. One is the role of the polycrystallinity of the sample. The only way to address this is to perform measurements on single crystals or two have extensive structural characterization of the sample, which are out of the scope of current work. Publication of this manuscript will inspire future work in that direction, I reckon.  
+
+The second concern, as reviewer 3 rightly points out, is with regards to possible alternative explanations to the presented experimental results. The authors provide a convincing arguments to discount other possibilities. Some of the explanations provided in the response to reviewer 3 were not included in the manuscript, for example, on why Bloch- Siegert is not a viable explanation. It may be very instructional to expand on this discussion in the main manuscript.  
+
+Apart from these very minor comments, the manuscript is suitable for publication in Nature Communications.  
+
+\(\Rightarrow\) First of all, we appreciate the reviewer for very encouraging and constructive comments. We agree with the reviewer's suggestion, and thus, we explicitly include a discussion explaining why these scenarios can be ruled out in this revised manuscript as follows:  
+
+"It is important to distinguish this transient Wannier Stark localization from the optical Stark- type effects such as the Autler- Townes effect and the Bloch- Siegert shift. In general, an external electric field affects the optical properties of a semiconductor in two ways: there are spectral and kinetic aspects. Spectral aspects refer to energy shifts and broadenings that arise from mixing two states by the external optical field. The mixing of the wavefunctions results in dressed states and leads to the Stark- type shifts. The magnitude of such shifts increases with the amplitude of the incident field and the interband dipole matrix, but decreases with increasing detuning between the light frequency and the transition frequency. On the other hand, kinetic aspects represent the evolution of the particle distributions in the renormalized states driven by the external field, which is called "intraband acceleration". This intraband acceleration leads to the Franz Keldysh effect at a moderate field strength and eventually Wannier- Stark localization in the strong- field regime.  
+
+Each of these two contributions can be straightforwardly separated in the semiconductor Bloch equations (SBE). Specifically, the third and second terms on the right- hand side of the Eq (1) represent the spectral (optical Stark- type effects) and the kinetic (Wannier Stark localization) aspects, respectively. One can thus directly compare each contribution to the differential optical response. As evident from Supplementary Fig. 6, the THz- induced optical Stark effect is shown to be much weaker (on the order of a few meV) compared to the shifts arising from the Wannier Stark localization which corresponds to approximately half the band width (several 100 meV). Therefore, we conclude that the observed transient response is mainly contributed from the Wannier Stark localization."
+
+<--- Page Split --->
+
+I have read through authors' response, and the revised manuscript. I'm okay with authors' response to my comments, and thus I don't have further objection to accept this manuscript for publication in NC.  
+
+\(\Rightarrow\) We thank the reviewer very much for the positive comments.  
+
+Reviewer #3 (Remarks to the Author):  
+
+I confirmed the replies from the authors, the revised manuscript, and the supporting information. I appreciate the efforts of the authors to answer my questions and comments. However, I still do not think that it is convincing that the origin of the observed nonlinear responses can be attributed to the Wannier- Stark localization. As the authors replied, the theoretical curves shown in Fig. 4a do not fully reproduce the experimental results in Fig. 4b partly because the theoretical model is too simple. Therefore, to make the interpretation as the Wannier- Stark localization solid, it is important to rule out the other possible origins. Although the other possibilities are discussed in the reply letter and the revised manuscript, I am not satisfied with some of those discussions. Thus, I do not recommend publication of the manuscript for Nature Communications as it stands. I suggest that the following points should be considered.  
+
+\(\Rightarrow\) We appreciate the reviewer for the thorough review and for giving us the opportunity to improve our manuscript even further. We apologize for apparently underestimating this point: we mistakenly felt it inappropriate to devote a substantial part of the discussion in the manuscript to this point. Rather, we tried to convince the reviewer in the rebuttal. However, we take this concern more carefully into account this time, and explicitly include a discussion explaining what are the similarities and differences between these optical Stark- type effects (i.e. Autler- Townes effects and Bloch- Siegert effects) in a theoretical context and how exactly each effect contributes to the experimentally observed optical response. This was also suggested by reviewer #1.  
+
+In the strong field regime (like our experiment), the Wannier- Stark localization (WSL) shifts the onset of the energy gap by approximately half the bandwidth, i.e., several hundred meV. Since the THz frequency is much lower than the interband transition frequencies, the optical Stark shift can be described perturbatively and is determined by the square of the Rabi frequency divided by the detuning. For a maximal field amplitude of 10 MV/cm and the interband dipole matrix element, the resulting Stark shifts are on the order of just a few meV, which is orders of magnitude smaller than the shifts arising from the WSL. These arguments are in agreement with and supported by new simulations in which we artificially neglected the intraband acceleration in order to study Stark- type shifts separately (see Supplementary Figure 6 and Supplementary Method).  
+
+Therefore, we could further confirm that the Wannier- Stark localization (originating from the intraband acceleration driven by the field) dominates the observed differential transmission. In contrast, the optical Stark- type effects (which leads to the spectral shift of each k- state of the band) have a negligible amplitude compared to the WSL.  
+
+We thank the reviewer for the opportunity to revisit the fundamentals and strengthen our interpretation. Further details are covered in our reply to comment #2 below.
+
+<--- Page Split --->
+
+1. On page 9 in the main manuscript, the authors mentioned that the two induced absorption signals shown in Fig. 2d can be assign to \(n = -1\) and \(n = 0\) transitions, whose difference in the transition energy is \(eE_{THZ}D\) . How much is the value of \(eE_{THZ}D\) ? In addition, I cannot read from Fig. 2d what the \(n = -1\) and \(n = 0\) transition energies are.  
+
+\(\Rightarrow\) We agree with the reviewer that the value of energy distance \(eE_{THZ}D\) is only inferred, and the necessary numbers appear in a rather scattered way (e.g. \(E_{THZ} = 6 \text{MV/cm}\) in page 11, and \(D =\) the largest lattice constant along the c axis, 12.5 Å, in page 15). Therefore, we added the figure caption of figure 2d the following sentence:  
+
+"In case of \(E_{THZ} = 6 \text{MV/cm}\) in considering the lattice constant \(D\) of 12.5 Å, \(\Delta E_{WSL} = eE_{THZ}D\) is estimated to be 750 meV, consistent with the spectrum showing that the absorption band of \(n = - 1\) and \(n = 0\) are approximately 750 meV apart."  
+
+Indeed, the \(n = - 1\) and \(n = 0\) transitions have certain energy ranges rather than a single transition energy (i.e. two- level systems) since these are "interband" transitions among mini- bands. So the range of transition energies for \(n = - 1\) is 1.2\~1.62eV, and that for \(n = 0\) is 1.95\~2.38 eV, and these transition energy ranges are approximately 750 meV apart (figure 2d).  
+
+2. On page 9 in the main manuscript, the authors stated "Photon-dressed states can shift energies in only one direction (mostly blue-shift) ..." However, I do not agree with the statement. If the higher energy levels exist, the Autler-Townes effect can shift the band-edge transitions in both directions, i.e. blue- and red- shifts, as reported in [C.-K. Yong et al. Nat. Mater. 18, 1065-1070 (2019).]. In addition, [G. Yumoto et al. Nat. Commun. 12, 3026 (2021).] recently reported that three-level Autler-Townes effect indeed occurs in halide perovskites. How do the authors comment on the possibility that the Autler-Townes effect can be the origin of the observed optical nonlinear responses?  
+
+\(\Rightarrow\) We agree with the reviewer that the optical Stark effect (i.e. the Autler- Townes effect) in fact leads to energy splitting. This initially two- level system with energies of \(\epsilon_{1}\) and \(\epsilon_{2}\) undergoes light- field induced level splitting to \(\epsilon_{1} + \frac{\nu}{2}\pm \frac{1}{2}\sqrt{\nu^{2} + \hbar^{2}\omega_{R}^{2}}\) and \(\epsilon_{2} - \frac{\nu}{2}\pm \frac{1}{2}\sqrt{\nu^{2} + \hbar^{2}\omega_{R}^{2}}\) respectively, where \(\nu\) is detuning \((\epsilon_{2} - \epsilon_{1} - \hbar \omega_{laser})\) and \(\omega_{R}\) is the Rabi frequency. The transitions among these split states are observed from singlet (without optical field) to triplet ("Mollow triplet", with optical field). In case of weak excitation and finite detuning (here we make a Taylor expansion of the square roots, so it's a perturbative result), the upper sideband \(\epsilon_{2} - \epsilon_{1} + \frac{1}{2}\frac{\omega_{R}^{2}}{\nu}\) is closest to the original resonance. This transition is the one that is observed in the experiment as a small blueshift depending on the intensity of the light field (and the interband dipole matrix element), if the frequency of the exciting light is smaller than the transition frequency. Although by changing the detuning (frequency difference between the light frequency and the transition frequency) it is possible to observe the peak splitting as the reviewer has pointed out, the blue shift is the common signature for large detuning (such as our experiment - for THz field the detuning to an optical transition is very large). [H. Haug & S. W. Koch, "Quantum Theory of the Optical and Electronic Properties of Semiconductors"]
+
+<--- Page Split --->
+
+However, more fundamentally, in a semiconductor, unlike two- level systems (or multi- levels without k- space dispersion), an external field has pronounced influence on the relative motion of optically generated electron- hole pair, going well beyond the field- induced shifts of excitonic resonance. And this is what we demonstrate by separating the contribution of the optical Stark effect and the intraband acceleration: WSL is by far the dominating contribution to the observed differential optical response and produces the spectral shape in very good agreement. In contrast, the optical Stark- type contributions (Autler- Townes effect from the corotating field and Bloch Siegert shift from the counterrotating field) are almost negligible and, moreover, do not agree with the observed spectral shape. Therefore, we added the following paragraph and the supplementary figure 6 showing this additional result:  
+
+"It is important to distinguish this transient Wannier Stark localization from the optical Stark- type effects such as the Autler- Townes effect and the Bloch- Siegert shift. In general, an external electric field affects the optical properties of a semiconductor in two ways: there are spectral and kinetic aspects. Spectral aspects refer to energy shifts and broadenings that arise from mixing two states by the external optical field. The mixing of the wavefunctions results in dressed states and leads to the Stark- type shifts. The magnitude of such shifts increases with the amplitude of the incident field and the interband dipole matrix, but decreases with increasing detuning between the light frequency and the transition frequency. On the other hand, kinetic aspects represent the evolution of the particle distributions in the renormalized states driven by the external field, which is called "intraband acceleration". This intraband acceleration leads to the Franz Keldysh effect at a moderate field strength and eventually Wannier- Stark localization in the strong- field regime.  
+
+Each of these two contributions can be straightforwardly separated in the semiconductor Bloch equations (SBE). Specifically, the third and second terms on the right- hand side of the Eq (1) represent the spectral (optical Stark- type effects) and the kinetic (Wannier Stark localization) aspects, respectively. One can thus directly compare each contribution to the differential optical response. As evident from Supplementary Fig. 6, the THz- induced optical Stark effect is shown to be much weaker (on the order of a few meV) compared to the shifts arising from the Wannier Stark localization which corresponds to approximately half the band width (several 100 meV). Therefore, we conclude that the observed transient response is mainly contributed from the Wannier Stark localization."  
+
+3. On page 10 in the main manuscript, the authors stated "... nor find any decay of the entire signal as a function of the field strength due to the tunneling to higher energy bands at intense field regime." I agree to the authors that the real excitation of carriers cannot explain the data due to the absence of the signal decay. However, the virtual population generated only under the THz-field irradiation should induce additional transitions for probe pulses, which would result in induced absorption signals. Is it possible to comment on this?  
+
+\(\rightarrow\) The original manuscript contained a paragraph on the possible carrier generation by THz (multiphoton processes and impact ionization) in our method section and also the Supplementary figure:  
+
+"In this linear- optical regime, we thus neglect carrier generation by multi- photon processes and impact ionization, which does not seem to play a dominant role in the measured transient spectra. Interband tunneling by the THz field could lead to bleaching at later delay times and the slightly
+
+<--- Page Split --->
+
+asymmetric spectral evolution with respect to \(\tau = 0\) (Fig. 2(A)) (corresponding to the trailing edge of the THz transient in the Supplementary Material of ref [20]). However, significant carrier multiplication does not occur within this experimental window, as shown in Supplementary Fig. 10."  
+
+However, in addition to that, we have performed new simulations that include the possible effects of THz- induced generation of virtual and real carriers that may arise from multi- photon transitions by solving a full set of semiconductor Bloch equations, in which the THz field is treated non- perturbatively, and the weak optical probe pulse is considered linearly. The result is shown in the supplementary fig 6, indicating that for the perovskite, the high- order/multi- photon interband effects are basically negligible. Therefore, our simplified model defined by Eq. (1) in the main text captures all the relevant physics. Accordingly, we have added the following statement in the main text:  
+
+"Furthermore, to analyze the possible effects of THz- induced generation of virtual and real carriers that may arise from multi- photon transitions, we solve a full set of SBE (Supplementary Methods) in which the THz field is included non- perturbatively and the weak optical probe pulse is considered linearly. The results (Supplementary Figure 6) show that for the considered field amplitudes, such higher- order interband effects arising from the THz fields are negligible, as the results from the full equations are very close to the ones obtained from the simplified Eq. (1)."
+
+<--- Page Split --->
+
+## REVIEWERS' COMMENTS  
+
+Reviewer #3 (Remarks to the Author):  
+
+I appreciate the efforts of the authors to answer my questions and comments. The authors have cleared up my concerns about the interpretation of the observed nonlinear responses by performing new calculations (Supplementary Figure 6) and ruling out the possible interpretations other than the Wannier- Stark localization. Therefore, now I think the manuscript is suitable to publish in Nature Communications.
+
+<--- Page Split --->
+
+Reviewer #3 (Remarks to the Author):  
+
+I appreciate the efforts of the authors to answer my questions and comments. The authors have cleared up my concerns about the interpretation of the observed nonlinear responses by performing new calculations (Supplementary Figure 6) and ruling out the possible interpretations other than the Wannier- Stark localization. Therefore, now I think the manuscript is suitable to publish in Nature Communications.  
+
+\(\Rightarrow\) We thank the reviewer very much for the thorough reviews and constructive comments.
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__00eb7cf6559fe9e55f1f5595a3abcbbf42befb8fdab5e70d9108735caa725b65/supplementary_0_Peer Review File__00eb7cf6559fe9e55f1f5595a3abcbbf42befb8fdab5e70d9108735caa725b65_det.mmd

@@ -0,0 +1,546 @@
+<|ref|>title<|/ref|><|det|>[[61, 40, 506, 90]]<|/det|>
+# nature portfolio  
+
+<|ref|>text<|/ref|><|det|>[[68, 110, 361, 139]]<|/det|>
+Peer Review File  
+
+<|ref|>text<|/ref|><|det|>[[71, 162, 761, 219]]<|/det|>
+Low- field onset of Wannier- Stark localization in a polycrystalline hybrid organic inorganic perovskite  
+
+<|ref|>image<|/ref|><|det|>[[56, 732, 239, 780]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[250, 732, 911, 784]]<|/det|>
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[119, 85, 295, 98]]<|/det|>
+## REVIEWER COMMENTS  
+
+<|ref|>text<|/ref|><|det|>[[120, 112, 415, 126]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[117, 140, 876, 308]]<|/det|>
+Berghoff et al report Wannier- Stark localization in a thin film of hybrid perovskite MAPbI3 achieved via non- resonant excitation with a 20THz electric field. This is achieved in a self- assembled material and at low electric field intensities compared to previous results in GaAs. Given the potential applications in ultrafast optically controlled switching of material properties, the presented results are not only novel but also have a broad interest. The experiment involves ultrafast THz pump - optical probe spectroscopy and supported by extensive and well- crafted calculations, comparing the effect of static and transient electric fields and accounting for the effect of the sample polycrystallinity. This work represents an exciting contribution, and is suitable for publication in Nature Communications. A few minor points to be addressed are as follows: 1. It would be helpful to clearly note that in the data presented in the main manuscript (Fig.2(a)), the low- field regime refers only to the early delay times before the THz field peak. A separate measurement, with an overall lower THz field, could also be shown for comparison, providing a reference where no localization occurs.  
+
+<|ref|>text<|/ref|><|det|>[[117, 323, 870, 392]]<|/det|>
+2. The discussion of figure 2, which shows the main experimental finding of the paper and its interpretation, should be expanded. In particular, panel d is not sufficiently explained in the main text: how are the separate peaks appearing in the absorbance attributed to \(\mathrm{n = 0}\) , \(+ / - 1\) ? The possibility of optically controlled switching between 3D materials and QWs is fascinating and could be explored more at length.  
+
+<|ref|>text<|/ref|><|det|>[[117, 393, 870, 448]]<|/det|>
+3. Since the low-field onset of Wannier-Stark localization is a key claim of this work, is it possible to provide a confidence interval to the estimation of the THz electric field amplitude? Can it be absolutely calibrated e.g. using a thin (smaller than the coherence length between the EOS probe and all relevant THz wavelengths) electro-optic crystal?  
+
+<|ref|>text<|/ref|><|det|>[[117, 448, 872, 504]]<|/det|>
+4. Considering the in-depth discussion of orientations in both real space and Brillouin space in pages 12-13, a diagram not only of MAPI structure in general but of the specific details being addressed would facilitate understanding (e.g. what Pb-I bond angles? Which direction is \Gamma Gamma M?)  
+
+<|ref|>text<|/ref|><|det|>[[117, 504, 876, 560]]<|/det|>
+5. Is there a specific reason to consider MAPbI rather than other hybrid perovskites? Could one expect to easily replicate these results in other materials in the same family? Would a softer lattice, e.g. by selecting different cations or substituting a lighter metal for Pb, result in localization at even lower fields?  
+
+<|ref|>text<|/ref|><|det|>[[120, 561, 682, 574]]<|/det|>
+A few typos and mistakes in figure presentation should also be corrected:  
+
+<|ref|>text<|/ref|><|det|>[[117, 575, 860, 616]]<|/det|>
+\* In the section "Experimental observation of Wannier-Stark Localization", units are incorrect in the sentence: "relatively weak fields, \(\mathrm{E}< 3 \mathrm{MV}\) , for \(\mathrm{T}< - 100 \mathrm{fs}\) ". It should read \(\mathrm{MV / cm}\) .  
+
+<|ref|>text<|/ref|><|det|>[[117, 606, 860, 633]]<|/det|>
+\*Is all the data presented (e.g. in fig. S2, S3) measured with a 6MV/cm THz field? Please specify so in captions.  
+
+<|ref|>text<|/ref|><|det|>[[120, 633, 780, 647]]<|/det|>
+\* The x-axis of figure S1 is labeled incorrectly, it should probably be in eV and not nm  
+
+<|ref|>text<|/ref|><|det|>[[120, 647, 520, 660]]<|/det|>
+\* Figures S4/S5 are not referred to in the main text  
+
+<|ref|>sub_title<|/ref|><|det|>[[120, 688, 415, 701]]<|/det|>
+## Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[117, 715, 876, 867]]<|/det|>
+This work provided an experimental observation of transient Wannier- Stark (WS) localization in a polycrystalline MAPbI3 perovskite based on similar previous work performed in the GaAs/AlGaAs superlattice (Ref. 8 in this manuscript). As the authors emphasized, the main difference is that the field amplitude needed here (3MV/cm) is far less than that in GaAs/AlGaAs (which exceeds 10 MV/cm) due to the large relevant lattice constant, the small width of electronic energy bands, and the coincidence of these two along the same high- symmetry direction. The results and the method discussed here are interesting and the manuscript is well written. However, since similar work has been reported in literature and I did not find enough novelty of current work, I cannot accept the present paper for publication in high level journals like Nat Comm. The authors may want to show and discuss about the novelty and new highlights of their work should they try to submit to similar journals again. For instance:  
+
+<|ref|>text<|/ref|><|det|>[[117, 868, 864, 896]]<|/det|>
+1) Further analysis and emphasis of the peculiarity unique to MAPbI3 (such as low field strengths required for WS), and in particular the applications of this feature.  
+
+<|ref|>text<|/ref|><|det|>[[117, 897, 832, 910]]<|/det|>
+2) Any other typical physical phenomena not limited to WS localization that are based on the
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[117, 83, 878, 168]]<|/det|>
+transient spectral analysis? The authors developed a direct method to show WS localization in real natural solids. Since such localization has already been extensively studied in previous work, it will be interesting if this method can also be used for the study of other physical phenomena. 3) MAPbI3 perovskite exhibits wide light absorption range and excellent photo- electronic properties and is considered as a prominent light harvester. In this manuscript, the description and discussion of novel properties of MAPbI3, especially related to this work, seems absent.  
+
+<|ref|>text<|/ref|><|det|>[[119, 183, 376, 196]]<|/det|>
+Other questions and suggestions:  
+
+<|ref|>text<|/ref|><|det|>[[116, 197, 876, 405]]<|/det|>
+1. In this work, the WS localization is created by applying the bias field from the phase-stable THz pulse and can be detected by optical absorption spectra. Is this method generally applicable for single-crystalline and polycrystalline materials? 
+2. The observed WS step is slightly lower than the expected value. Is it possible that the step is higher than the theoretical value? Can the disorder of the sample be controlled here, and what is the influence of disorder on the experimental results? 
+3. For MAPbI3, transient optical response is in fact dominated by the least dispersive direction of the band structure. Do other polycrystalline materials also have the same feature? 
+4. WS ladders and associated phenomena predicted and observed in biased semiconductor superlattices have intrigued scientists for decades (it seems the work of J. Bleuse et al., Phys. Rev. Lett. 60, 220, 1988, should be cited together with [4, 5]). The concept was later introduced and explored also in ultracold atoms (Phys. Rev. Lett. 76, 4512, 1996) and optical waveguide arrays superimposed with a linear optical potential (Opt. Lett. 23, 1701, 1998; Opt. Lett. 39, 1065, 2014). Since the paper is intended for an interdisciplinary journal like NC, the authors should discuss the broader impact of their work and possible connection to other fields.  
+
+<|ref|>text<|/ref|><|det|>[[119, 448, 415, 461]]<|/det|>
+Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[117, 476, 878, 659]]<|/det|>
+In this manuscript, the authors synthesized a MAPbI3 polycrystalline film and studied ultrafast nonlinear optical responses of the MAPbI3 film by performing THz- pump/NIR to visible- probe spectroscopy and theoretical calculations. They observed a transient absorption change induced by THz pump with a center frequency of 20 THz and assigned its origin to the Wannier- Stark localization. They found that the transient Wannier- Stark localization in a MAPbI3 film can be observed at lower THz field strength than that in a single crystal GaAs, which results from a narrow electronic bandwidth and a large relevant lattice constant of MAPbI3. The finding that its narrow electric bandwidth and large lattice constant make a MAPbI3 film a unique optical nonlinear material would be important for developing novel photonic devices based on halide perovskites. However, the discussions are not enough to guarantee that the observed transient absorption change originates from the Wannier- Stark localization, on which I commented below. Therefore, my opinion is that this manuscript is not suitable for publication in Nature Communications as it stands.  
+
+<|ref|>text<|/ref|><|det|>[[118, 673, 848, 700]]<|/det|>
+The following points are the reasons why I doubt the interpretation of the data as the Wannier- Stark localization:  
+
+<|ref|>text<|/ref|><|det|>[[117, 714, 878, 854]]<|/det|>
+On page 4, the authors mentioned that the measured transient absorption change is well explained by a two- band model. However, I question why the higher energy states, such as light and heavy electron states, do not contribute to the optical nonlinear processes. In fact, the previous study [Z. Wei et al., Nat. Commun. 10, 5342 (2019).] reported that light and heavy electron states exist around 2.25 eV and the optical transitions between those states and the band- edge conduction band states significantly contribute to the two- photon absorption processes in a MAPbI3 film. Therefore, I suspect that the observed transient absorption change originates from the Bloch- Siegert shift [E. J. Sie et al., Science 355, 1066 (2017).] in multiple states consisting of the band- edge valence and conduction band states and the higher energy conduction band states. Is it possible for the authors to comment on this?  
+
+<|ref|>text<|/ref|><|det|>[[118, 869, 875, 910]]<|/det|>
+How much is the spectral width of the THz pump pulses? As the authors stated on page 6, the phonon modes of halide perovskites fall in the low frequency range around several THz. Therefore, if the spectral width is larger than the phonon frequencies, impulsive stimulated Raman scattering
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 84, 857, 141]]<|/det|>
+should occur. As some of the authors reported, driving the phonon modes leads to the similar transient absorption change albeit in a different crystal structure [Fig. 4a in H. Kim et al., Nat. Commun. 8, 687 (2017).]. Is it possible to discuss the contributions of phonons to the observed signals?  
+
+<|ref|>text<|/ref|><|det|>[[118, 154, 872, 239]]<|/det|>
+On page 13, the authors mentioned that the crystallites are arbitrarily oriented and the theoretical calculations were performed based on this assumption. Is there any possibility that some preferential orientations of the crystallites exist? Do the authors experimentally verify the assumption from, for example, XRD spectra? The detailed sample properties should be described in the Support Information, because the sample is not single crystal but complicated polycrystalline film.  
+
+<|ref|>text<|/ref|><|det|>[[118, 252, 866, 321]]<|/det|>
+On page 15, the authors claimed that the calculation result shown as a black curve in Fig. 4a is in good agreement with experimental results at high field amplitudes in Fig. 4b. However, the spectral oscillations around 2 eV can be seen only in the experimental results, not in the calculation. In addition, a bleaching signal around 2.2 eV which is expected from the calculation does not appear in the experimental results. What are the reasons for such disagreements?  
+
+<|ref|>text<|/ref|><|det|>[[118, 336, 540, 350]]<|/det|>
+I suggest some more points for improving manuscript:  
+
+<|ref|>text<|/ref|><|det|>[[118, 350, 855, 378]]<|/det|>
+On page 8, why did the authors consider the band gap energy of 2D perovskites with \(I = 2\) , not \(I = 1\) , 3 or 4?  
+
+<|ref|>text<|/ref|><|det|>[[118, 392, 845, 420]]<|/det|>
+With regard to Ref. 25, the authors should cite the published version [J.- C. Blancon et al., Nat. Commun. 9, 2254 (2018).], not that in arXiv.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 90, 420, 107]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 122, 883, 275]]<|/det|>
+Berghoff et al report Wannier- Stark localization in a thin film of hybrid perovskite MAPbI3 achieved via non- resonant excitation with a 20THz electric field. This is achieved in a self- assembled material and at low electric field intensities compared to previous results in GaAs. Given the potential applications in ultrafast optically controlled switching of material properties, the presented results are not only novel but also have a broad interest. The experiment involves ultrafast THz pump - optical probe spectroscopy and supported by extensive and well- crafted calculations, comparing the effect of static and transient electric fields and accounting for the effect of the sample polycrystallinity. This work represents an exciting contribution, and is suitable for publication in Nature Communications. A few minor points to be addressed are as follows:  
+
+<|ref|>text<|/ref|><|det|>[[115, 289, 882, 357]]<|/det|>
+1. It would be helpful to clearly note that in the data presented in the main manuscript (Fig.2(a)), the low-field regime refers only to the early delay times before the THz field peak. A separate measurement, with an overall lower THz field, could also be shown for comparison, providing a reference where no localization occurs.  
+
+<|ref|>text<|/ref|><|det|>[[115, 372, 882, 423]]<|/det|>
+\(\Rightarrow\) First of all, we appreciate the reviewer for very encouraging and constructive comments. We agree with the reviewer's first suggestion and thus clarified the first regime as follows in the main text:  
+
+<|ref|>text<|/ref|><|det|>[[115, 438, 883, 541]]<|/det|>
+"More importantly, two distinct regimes can be identified in the time- resolved transient spectrum (Fig. 2(a)). The first regime appears at delay times \(\tau < - 100\) fs, where the field strength is relatively weak \((E < 3 \text{MV/cm})\) , as an induced absorption (blue, \(\Delta T / T < 0\) ) right below and an induced transmission (red, \(\Delta T / T > 0\) ) right above the bandgap of \(E_{\text{gap}} = 1.62 \text{eV}\) . The second regime is apparent for field strengths \(E > 3 \text{MV/cm}\) , occurring between delay times \(- 100 < \tau < 100\) fs (Fig. 2(b))."  
+
+<|ref|>text<|/ref|><|det|>[[115, 555, 882, 623]]<|/det|>
+Regarding the separate measurement, the low- field regime \((< 2 \text{MV/cm})\) has been analyzed in our previous work using a narrower- band optical probe [Nat. Commun. 8, 687] and in another reported work using \(\sim \text{kHz}\) field [ACS Photonics 2016, 3, 1060- 1068]. There, one could find the electroabsorption spectra for comparison, where no localization occurs (Franz Keldysh effect).  
+
+<|ref|>text<|/ref|><|det|>[[115, 638, 882, 722]]<|/det|>
+Nonetheless, we have performed a separate measurement at a slightly lower peak field strength (4 MV/cm) with a different center frequency of 30 THz. In this case, we reproduce the observed transient Wannier Stark localization with lower peak field strength and different THz center frequency. We have added this result and the THz field characteristics in the supplementary figure S5.  
+
+<|ref|>text<|/ref|><|det|>[[115, 739, 882, 823]]<|/det|>
+2. The discussion of figure 2, which shows the main experimental finding of the paper and its interpretation, should be expanded. In particular, panel d is not sufficiently explained in the main text: how are the separate peaks appearing in the absorbance attributed to \(n = 0\) , \(+/- 1\) ? The possibility of optically controlled switching between 3D materials and QWs is fascinating and could be explored more at length.  
+
+<|ref|>text<|/ref|><|det|>[[115, 840, 882, 890]]<|/det|>
+\(\Rightarrow\) Yes, we agree that the detailed explanation of Figure 2 (c, d) would be necessary not only for clarifying the mechanism, but also for strengthening our interpretation. Therefore, we have added the following paragraph in the main text (experimental results):
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 89, 884, 323]]<|/det|>
+"The extended structure of the transient spectral response can be understood with the assistance of Figure 2 (c, d). The localized Wannier- Stark states, equally spaced in energy by an amount \(eE_{THZ}D\) , are depicted in the real- space along the field direction, z, in Fig 2 (c). D is the lattice period unit length and n the index. This space- dependent energy shift results in differentiating the electronic transition energies within the same site (arrow with \(n = 0\) , Fig 2 (c)) from those between different sites (arrows with \(n = \pm 1\) , Fig 2 (c)). As the difference in the transition energy with respect to the central spatially- direct ( \(n = 0\) ) transition is \(nE_{THZ}D\) , one could assign the induced absorption below the band gap and above 1.9 eV to be \(n = - 1\) and \(n = 0\) transitions, respectively (Fig 2 (d)). The reduced absorption right above the band gap stems from the spectral transfer from non- perturbed optical transition to red- ( \(n = - 1\) ) and blue- ( \(n = 0\) ) shifted transitions (Fig 2 (d)). Depending on the strength of \(E_{THZ}\) and the degree of localization, \(Inl > 1\) transitions could, in principle, also be observed. In this case (Figure 2(a)), the observed single central step from reduced to increased absorption near the center of the band \(E_{pr} = 1.9\) eV, is a noticeable signature of Stark localization, where the Wannier- Stark states are localized onto one unit cell."  
+
+<|ref|>text<|/ref|><|det|>[[115, 339, 884, 374]]<|/det|>
+The possibility of optical switching between 3D delocalized and effective- 2D localized electronic states is emphasized as follows:  
+
+<|ref|>text<|/ref|><|det|>[[115, 388, 884, 457]]<|/det|>
+"The large unit cell and the small bandwidths along one direction of this material allows for optical switching with up to \(\sim 40\%\) transmission modulation depth using relatively moderate biasing fields. Also, the optical modulation of the material is extremely fast (sub- 20 fs), as demonstrated directly by the quasi- instantaneous response to an electric field oscillating at mid- infrared frequency."  
+
+<|ref|>text<|/ref|><|det|>[[115, 472, 884, 557]]<|/det|>
+"The polycrystallinity of this material does not impede the optical switching performance of the material, since the least dispersive direction of the band structure dominates the contribution to the optical response, which favors low- cost fabrications. Together with the outstanding photophysical properties of MAPbl3, this finding highlights the potential of this material in novel photonic applications."  
+
+<|ref|>text<|/ref|><|det|>[[115, 572, 883, 640]]<|/det|>
+3. Since the low-field onset of Wannier-Stark localization is a key claim of this work, is it possible to provide a confidence interval to the estimation of the THz electric field amplitude? Can it be absolutely calibrated e.g. using a thin (smaller than the coherence length between the EOS probe and all relevant THz wavelengths) electro-optic crystal?  
+
+<|ref|>text<|/ref|><|det|>[[115, 656, 883, 790]]<|/det|>
+\(\Rightarrow\) The reviewer raises an excellent point. In response, we have added the following statements to describe the accuracy of the THz electric field amplitude in the main text. What we would like to emphasize here is that the estimated field amplitude inside the material was even slightly overestimated because we use the reported refractive index of the material at near-infrared frequency (2.2, by which the estimated peak field amplitude was 6 MV/cm as we report in the manuscript), which it is likely slightly higher in the measured frequency (20 THz). We note that the refractive index at 3 THz is reported to be \(\sim 3\) (by which the estimated peak field amplitude is \(\sim 5\) MV/cm). Therefore, we mention that we report the upper limit of the actual field strength.  
+
+<|ref|>text<|/ref|><|det|>[[115, 805, 884, 907]]<|/det|>
+"The phase- stable THz biasing fields are generated using a difference- frequency generation scheme in GaSe and characterized by ultrabroadband electro- optic sampling. The accuracy for determination of the absolute electric field strength in the center of the pump spot is estimated to be \(\pm 15\%\) . The peak field strength at the interior of the MAPbl3 perovskite sample is obtained using the Fresnel transmission coefficient with the reported refractive index \(n = 2.2\) at near- infrared frequency, which sets the upper limit of the actual field strength."
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 883, 156]]<|/det|>
+The crystal we have used exhibits a rather flat response function in the relavant frequency range. Consequently, it does not distort the waveform significantly in the time domain. The crystal would be thinner than the coherence length for all relevant THz input frequencies. A detailed description of the procedure for determination of the field is found in the Methods section:  
+
+<|ref|>text<|/ref|><|det|>[[115, 173, 884, 274]]<|/det|>
+"The electric field transient is characterized by ultrabroadband electro- optic sampling at a \(30 - \mu m\) thick GaSe crystal using balanced detection of an 8- fs probe pulse centered at a wavelength of \(1.2 \mu m\) as the gating pulse. The quantitative value of the field amplitude is obtained by measuring the THz average power, pulse repetition rate and focal spot size. Then, the value at the interior of the MAPbI3 perovskite sample is estimated using the Fresnel transmission coefficient for the THz field at the air- MAPbI3 interface."  
+
+<|ref|>text<|/ref|><|det|>[[115, 289, 883, 356]]<|/det|>
+4. Considering the in-depth discussion of orientations in both real space and Brillouin space in pages 12-13, a diagram not only of MAPI structure in general but of the specific details being addressed would facilitate understanding (e.g. what Pb-I bond angles? Which direction is \GammaGamma M?)  
+
+<|ref|>text<|/ref|><|det|>[[115, 372, 883, 439]]<|/det|>
+\(\Rightarrow\) We thank the reviewer for the helpful suggestion. Accordingly, we modified Fig 1a to indicate the exact Pb-I-Pb angle, the Pb-I bond length, and the most relevant direction (Gamma Z) mentioned in the main text on pages 12-13. Also, we refer to the figure when these details are mentioned (highlighted in the main text).  
+
+<|ref|>text<|/ref|><|det|>[[115, 453, 883, 519]]<|/det|>
+5. Is there a specific reason to consider MAPbI rather than other hybrid perovskites? Could one expect to easily replicate these results in other materials in the same family? Would a softer lattice, e.g. by selecting different cations or substituting a lighter metal for Pb, result in localization at even lower fields?  
+
+<|ref|>text<|/ref|><|det|>[[115, 535, 883, 720]]<|/det|>
+\(\Rightarrow\) We appreciate the question highlighting the uniqueness of the MAPbI3. In general, Wannier- Stark localization is very challenging to observe in natural crystals due to the small lattice constants, the rapid scattering, and the dielectric breakdown conditions. One of most important messages of our work is that MAPbI3, in particular, has the combination of the narrow bandwidth, the large periodicity, and the coincidence of the two directions. These unique features of its band structure promote this material to the Wannier Stark regime under relatively modest field amplitudes. Therefore, if one could find any other material (including other perovskites) where these conditions are met, we could expect similar results, provided other material- specific disturbances such as interband tunnelings are absent. Regarding the softness of lattice, if it means the lower nuclear vibration energy, there is not necessarily nor always a correlation between the electronic band dispersion near the band gap and a certain phonon frequency.  
+
+<|ref|>text<|/ref|><|det|>[[115, 735, 883, 770]]<|/det|>
+In the main text, we have tried to emphasize the unique electronic properties of MAPbI3 that enable our observation, including the above- cited (comment #2) sentences:  
+
+<|ref|>text<|/ref|><|det|>[[116, 786, 884, 853]]<|/det|>
+"The large unit cell and the small bandwidth along one direction of this material allows for optical switching with up to \(\sim 40\%\) transmission modulation depth using relatively moderate biasing fields. Also, the optical modulation of the material is extremely fast (sub- 20 fs), as demonstrated directly by the quasi- instantaneous response to an electric field oscillating at mid- infrared frequency."
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 105, 700, 123]]<|/det|>
+A few typos and mistakes in figure presentation should also be corrected:  
+
+<|ref|>text<|/ref|><|det|>[[112, 123, 886, 225]]<|/det|>
+\* In the section "Experimental observation of Wannier- Stark Localization", units are incorrect in the sentence: "relatively weak fields, \(E < 3 \text{MV}\) , for \(\tau < - 100 \text{fs}\) ". It should read MV/cm. \*Is all the data presented (e.g. in fig. S2, S3) measured with a 6MV/cm THz field? Please specify so in captions. \* The x- axis of figure S1 is labeled incorrectly, it should probably be in eV and not nm \* Figures S4/S5 are not referred to in the main text  
+
+<|ref|>text<|/ref|><|det|>[[115, 240, 886, 275]]<|/det|>
+\(\Rightarrow\) We thank the reviewer for thoroughly reviewing our materials and kindly raising the points that we overlooked. We corrected all of them accordingly (highlighted).
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 90, 419, 107]]<|/det|>
+Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 122, 883, 308]]<|/det|>
+This work provided an experimental observation of transient Wannier- Stark (WS) localization in a polycrystalline MAPbI3 perovskite based on similar previous work performed in the GaAs/AlGaAs superlattice (Ref. 8 in this manuscript). As the authors emphasized, the main difference is that the field amplitude needed here (3MV/cm) is far less than that in GaAs/AlGaAs (which exceeds 10 MV/cm) due to the large relevant lattice constant, the small width of electronic energy bands, and the coincidence of these two along the same high- symmetry direction. The results and the method discussed here are interesting and the manuscript is well written. However, since similar work has been reported in literature and I did not find enough novelty of current work, I cannot accept the present paper for publication in high level journals like Nat Comm. The authors may want to show and discuss about the novelty and new highlights of their work should they try to submit to similar journals again. For instance:  
+
+<|ref|>text<|/ref|><|det|>[[115, 323, 883, 358]]<|/det|>
+1) Further analysis and emphasis of the peculiarity unique to MAPbI3 (such as low field strengths required for WS), and in particular the applications of this feature.  
+
+<|ref|>text<|/ref|><|det|>[[116, 372, 883, 424]]<|/det|>
+\(\Rightarrow\) First of all, we appreciate the reviewer's critical yet very helpful comments. For highlighting the first point, we have added the following paragraphs to the introduction, and have rewritten the conclusion and abstract:  
+
+<|ref|>text<|/ref|><|det|>[[115, 439, 884, 608]]<|/det|>
+[Introduction] "Besides their use in solar cells and light- emitting diodes, in this work, we demonstrate that MAPbI3 also has a great potential in photonic applications, including optical modulators, optical switches, and optical signal processing. For any optical amplitude modulator, one of the essential properties is a substantial change of the absorption edge with relatively low required energies in general. We demonstrate that solution- processed, polycrystalline MAPbI3 shows drastic changes in optical properties via Wannier Stark localization, at weak biasing fields. Whereas conventional semiconductors constituting photo- detectors, e.g. Si or InGaAs, require costly manufacturing processes and are limited to traditional rigid type devices, perovskites with distinct crystal structures exhibit ultrafast response (sub- 20 fs), while simultaneously supporting cheap and flexible polycrystalline film fabrication.  
+
+<|ref|>text<|/ref|><|det|>[[116, 624, 884, 690]]<|/det|>
+The large unit cell and the small bandwidths along one direction of this material allows for optical switching with up to \(\sim 40\%\) transmission modulation depth using relatively moderate biasing fields. Also, the optical modulation of the material is extremely fast (sub- 20 fs), as demonstrated directly by the quasi- instantaneous response to an electric field oscillating at mid- infrared frequency."  
+
+<|ref|>text<|/ref|><|det|>[[115, 706, 884, 906]]<|/det|>
+[Conclusion] "we have demonstrated that solution- processed, polycrystalline MAPbI3 shows optical transmission change by tens of percent at relatively modest field strengths via transient Wannier Stark localization. The large lattice periodicity, the narrow electronic energy bandwidths, and the coincidence of these two along the same high- symmetry direction promotes this material to the Wannier Stark regime under relatively moderate biasing fields. Polycrystallinity of this material turns out not to hinder the Wannier Stark localization effect as observed, due to the dominant contribution from the least dispersive direction of the band structure, which favors low- cost fabrications with this material as optical modulators. The degree of disorder and relative orientation among crystallites may influence the modulation spectral shape slightly, e.g. the position of the photon energy where the induced transmission to induced absorption happens, which could be finely tuned depending on the desired device performance by further systematic studies."
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 105, 884, 323]]<|/det|>
+[Abstract] "Methylammonium lead iodide perovskite \((MAPbI_3)\) , renowned for an impressive power conversion efficiency rise and cost- effective fabrication for photovoltaics, exhibits a huge potential for optical modulation- type applications, in this work. We demonstrate that polycrystalline \(MAPbI_3\) s undergo drastic changes in optical properties with the modulation depth to be tens of percent at moderate field strengths, via transient Wannier Stark localization with an ultrafast response time. The distinct band structure of this material - the large lattice periodicity, the narrow electronic energy bandwidths, and the coincidence of these two along the same high- symmetry direction - enables relatively weak fields to bring this material into the Wannier Stark regime. Its polycrystalline nature is not detrimental to the optical switching performance of the material, since the least dispersive direction of the band structure dominates the contribution to the optical response, which favors low- cost fabrication. Together with the outstanding photophysical properties of \(MAPbI_3\) , this finding highlights the potential of this material in novel photonic applications."  
+
+<|ref|>text<|/ref|><|det|>[[115, 339, 883, 407]]<|/det|>
+2) Any other typical physical phenomena not limited to WS localization that are based on the transient spectral analysis? The authors developed a direct method to show WS localization in real natural solids. Since such localization has already been extensively studied in previous work, it will be interesting if this method can also be used for the study of other physical phenomena.  
+
+<|ref|>text<|/ref|><|det|>[[115, 422, 883, 474]]<|/det|>
+\(\Rightarrow\) We thank the reviewer for the suggestion to emphasize the advantage and potential use of our experimental technique and approach. Accordingly, we have added a dedicated paragraph in the conclusion as follows:  
+
+<|ref|>text<|/ref|><|det|>[[115, 489, 884, 658]]<|/det|>
+"Moreover, the phase stable THz field transients and the ultra- broadband optical pulses of 7 fs duration revealed that the optical modulation of this material has an extremely fast, quasi- instantaneous (sub- 20fs) temporal response in visible/near- IR spectral region. This technique could be generalized for realizing transient Wannier Stark localization in other semiconductor solids in a carefully prepared single- crystalline or a polycrystalline form. More generally, this method enables to analyze any ultrafast changes in optical properties induced by the phase- locked and intense electromagnetic field transients, be it resonantly or non- resonantly. While here we used only the electric field of the transients, one could also exploit the magnetic component for exploring ultrafast magneto- optic effects, by enhancing the magnetic field with respect to the electric field with, e.g., a specially designed plasmonic nanoaperture."  
+
+<|ref|>text<|/ref|><|det|>[[115, 673, 883, 724]]<|/det|>
+3) MAPbI3 perovskite exhibits wide light absorption range and excellent photo-electronic properties and is considered as a prominent light harvester. In this manuscript, the description and discussion of novel properties of MAPbI3, especially related to this work, seems absent.  
+
+<|ref|>text<|/ref|><|det|>[[115, 740, 883, 773]]<|/det|>
+\(\Rightarrow\) We thank the reviewer for this suggestion for improving our work. We have added the following paragraph to the introduction to better connect to the cited part of the conclusion:  
+
+<|ref|>text<|/ref|><|det|>[[116, 789, 884, 872]]<|/det|>
+[Introduction] "Methylammonium lead iodide perovskite \((MAPbI_3)\) has become a remarkable material for photovoltaic applications due to the dramatic increase of the power conversion efficiency and the cost- effective fabrication processes. The success of this material has been attributed to large absorption coefficient and the exceptional transport properties such as long carrier diffusion length, high carrier mobilities and defect tolerance."
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 90, 884, 157]]<|/det|>
+[Conclusion] "Together with the renowned photophysical properties of MAPbl3, such as the long carrier diffusion length, low mid- gap trap density, and large absorption coefficient, this finding of high modulation depth, ultrafast response, and low onset field for Wannier- Stark localization highlights the potential of this material in photonic applications."  
+
+<|ref|>text<|/ref|><|det|>[[116, 174, 385, 191]]<|/det|>
+Other questions and suggestions:  
+
+<|ref|>text<|/ref|><|det|>[[116, 191, 883, 241]]<|/det|>
+1. In this work, the WS localization is created by applying the bias field from the phase-stable THz pulse and can be detected by optical absorption spectra. Is this method generally applicable for single-crystalline and polycrystalline materials?  
+
+<|ref|>text<|/ref|><|det|>[[116, 257, 881, 291]]<|/det|>
+\(\Rightarrow\) Yes, it could be generally applicable for single-crystalline and polycrystalline materials. Thus, we added this sentence in the conclusion:  
+
+<|ref|>text<|/ref|><|det|>[[116, 306, 883, 340]]<|/det|>
+"This technique could be generalized for realizing transient Wannier Stark localization in other semiconductor solids in a carefully prepared single-crystalline or a polycrystalline form."  
+
+<|ref|>text<|/ref|><|det|>[[116, 356, 883, 406]]<|/det|>
+2. The observed WS step is slightly lower than the expected value. Is it possible that the step is higher than the theoretical value? Can the disorder of the sample be controlled here, and what is the influence of disorder on the experimental results?  
+
+<|ref|>text<|/ref|><|det|>[[115, 422, 883, 574]]<|/det|>
+\(\Rightarrow\) First, we would like to point out that the predicted position where the WS step occurs depends on the level of theory. If we compare the Fig 3 and Fig 4(c, d), the step occurs at slightly below 2 and \(1.9 \text{eV}\) , respectively, and the experimentally observed position was \(1.9 \text{eV}\) . The difference in the theoretical approaches to obtain Fig 3 and Fig 4(c,d) is whether the other direction is taken into account (i.e., with (Fig 4(c,d)) and without (Fig 3) the averaging over the disorder). The disorder of the sample could not be controlled experimentally here, but from this theoretical comparison, what one could learn about the influence of disorder on the experimental results would be indeed the position of the WS step (with \(\sim 100 \text{meV}\) range). We strengthened this discussion by adding the following to the discussion and conclusion sections:  
+
+<|ref|>text<|/ref|><|det|>[[115, 589, 884, 723]]<|/det|>
+[Discussion] "This finding is remarkable because it means the two extreme cases – the completely random orientation of a polycrystalline sample and the perfectly oriented single crystal – are expected to produce very similar optical responses. The only slight difference between the two extremes would be a small shift in the photon energy (\~100 meV) where the induced transmission turns to the induced absorption and in the transient spectral shape. The two extremes include partial preferential orientations. We also note that the averaging process using only two extreme directions does not contain any material-specific information, which means that one could expect other polycrystalline materials to behave similarly."  
+
+<|ref|>text<|/ref|><|det|>[[115, 739, 883, 806]]<|/det|>
+[Conclusion] "The degree of disorder and relative orientation among crystallites may influence the modulation spectral shape slightly, e.g. the position of the photon energy where the induced transmission to induced absorption happens, which might be finely tuned depending on the desired device performance by further systematic studies."  
+
+<|ref|>text<|/ref|><|det|>[[112, 822, 883, 857]]<|/det|>
+3. For MAPbl3, transient optical response is in fact dominated by the least dispersive direction of the band structure. Do other polycrystalline materials also have the same feature?  
+
+<|ref|>text<|/ref|><|det|>[[112, 872, 883, 907]]<|/det|>
+\(\Rightarrow\) Yes, we believe so, because for the larger bandwidth, the field required for WS localization will be larger. This means that the direction with the smallest dispersion will always be where WS
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 89, 883, 174]]<|/det|>
+localizations occur for the smallest field. While we observe the localization along the least dispersive direction, the contributions from the other directions are negligible, since the transmission changes are smaller for fields below the WS threshold. It is indeed what we observe from the theoretical model, including averaging over the arbitrarily oriented crystallites. Regarding this point, we added this sentence in the main text (discussion section):  
+
+<|ref|>text<|/ref|><|det|>[[115, 190, 883, 241]]<|/det|>
+"We also note that the averaging process using only two extreme directions does not contain any material- specific information, which means that one could expect other polycrystalline materials to behave similarly."  
+
+<|ref|>text<|/ref|><|det|>[[115, 256, 883, 374]]<|/det|>
+4. WS ladders and associated phenomena predicted and observed in biased semiconductor superlattices have intrigued scientists for decades (it seems the work of J. Bleuse et al, Phys. Rev. Lett. 60, 220, 1988, should be cited together with [4, 5]). The concept was later introduced and explored also in ultracold atoms (Phys. Rev. Lett. 76, 4512, 1996) and optical waveguide arrays superimposed with a linear optical potential (Opt. Lett. 23, 1701, 1998; Opt. Lett. 39, 1065, 2014). Since the paper is intended for an interdisciplinary journal like NC, the authors should discuss the broader impact of their work and possible connection to other fields.  
+
+<|ref|>text<|/ref|><|det|>[[115, 389, 883, 441]]<|/det|>
+\(\Rightarrow\) We thank the reviewer for this very insightful suggestion to position our work in a broader context and for the listed literature. We have, in our revised manuscript, cited and discussed the suggested literature and make a connection in the introduction as follows:  
+
+<|ref|>text<|/ref|><|det|>[[115, 456, 884, 690]]<|/det|>
+"Following the initial observations in semiconductor superlattices under static bias fields \(^{8 - 10}\) Wannier- Stark ladders have been proposed and realized in various physical systems featuring wave propagation in the presence of periodic potentials and a homogeneous force. Examples include ultracold atoms in an accelerating 1D standing wave \(^{5}\) , waveguide arrays with linearly varying propagation constants \(^{6}\) , and self- accelerating optical beams in 1D photonic lattice \(^{7}\) . Several fundamental observations and device applications from the Wannier- Stark localization have been focused on statically biased artificial semiconductor superlattices \(^{8 - 12}\) . However, in natural homogeneous solids, where the periodicity is dictated by the atomic structure, such an extreme state of matter has never been achieved using static biasing. To resolve optical transitions to individual Wannier- Stark states in, e.g., absorption spectra, their energetic spacing needs to be larger than the (total) linewidth \(\Gamma\) , i.e., \(eED > \Gamma^{9,10,13}\) Due to the small lattice constant of bulk crystals and the large linewidth which results from the scattering of electrons with lattice vibrations and other electrons, the requirement \(eED > \Gamma\) can typically not be fulfilled under stationary external fields below the strength where the dielectric breakdown occurs \(^{11,12}\) ."
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 90, 420, 107]]<|/det|>
+Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 122, 883, 340]]<|/det|>
+In this manuscript, the authors synthesized a MAPbl3 polycrystalline film and studied ultrafast nonlinear optical responses of the MAPbl3 film by performing THz- pump/NIR to visible- probe spectroscopy and theoretical calculations. They observed a transient absorption change induced by THz pump with a center frequency of 20 THz and assigned its origin to the Wannier- Stark localization. They found that the transient Wannier- Stark localization in a MAPbl3 film can be observed at lower THz field strength than that in a single crystal GaAs, which results from a narrow electronic bandwidth and a large relevant lattice constant of MAPbl3. The finding that its narrow electric bandwidth and large lattice constant make a MAPbl3 film a unique optical nonlinear material would be important for developing novel photonic devices based on halide perovskites. However, the discussions are not enough to guarantee that the observed transient absorption change originates from the Wannier- Stark localization, on which I commented below. Therefore, my opinion is that this manuscript is not suitable for publication in Nature Communications as it stands.  
+
+<|ref|>text<|/ref|><|det|>[[116, 356, 882, 390]]<|/det|>
+The following points are the reasons why I doubt the interpretation of the data as the Wannier- Stark localization:  
+
+<|ref|>text<|/ref|><|det|>[[115, 406, 883, 575]]<|/det|>
+On page 4, the authors mentioned that the measured transient absorption change is well explained by a two- band model. However, I question why the higher energy states, such as light and heavy electron states, do not contribute to the optical nonlinear processes. In fact, the previous study [Z. Wei et al., Nat. Commun. 10, 5342 (2019).] reported that light and heavy electron states exist around \(2.25 \text{eV}\) and the optical transitions between those states and the band- edge conduction band states significantly contribute to the two- photon absorption processes in a MAPbl3 film. Therefore, I suspect that the observed transient absorption change originates from the Bloch- Siegert shift [E. J. Sie et al., Science 355, 1066 (2017).] in multiple states consisting of the band- edge valence and conduction band states and the higher energy conduction band states. Is it possible for the authors to comment on this?  
+
+<|ref|>text<|/ref|><|det|>[[116, 590, 883, 674]]<|/det|>
+\(\Rightarrow\) First of all, we appreciate the reviewer's critical, yet very constructive comments. We also thank the reviewer for raising this possible alternative explanation, based on which we were able to improve our manuscript substantially. Here we clarify the detailed assignment of each spectral component (in our optical response) based on our mechanism (WS localization), which seem inconsistent with other nonlinear optical processes, and strengthen our interpretation as follows:  
+
+<|ref|>text<|/ref|><|det|>[[115, 689, 884, 890]]<|/det|>
+"The extended structure of the transient spectral response can be understood with the assistance of Figure 2 (c, d). The localized Wannier- Stark states, equally spaced in energy by an amount \(eE_{THZ}D\) , are depicted in the real- space along the field direction, z, in Fig 2 (c). D is the lattice period unit length and n the index. This space- dependent energy shift results in differentiating the electronic transition energies within the same site (arrow with \(n = 0\) , Fig 2 (c)) from between different sites (arrows with \(n = \pm 1\) , Fig 2 (c)). As the difference in the transition energy with respect to the central spatially- direct ( \(n = 0\) ) transition is \(nE_{THZ}D\) , one could assign the induced absorption below the band gap and above \(1.9 \text{eV}\) to be \(n = - 1\) and \(n = 0\) transitions, respectively (Fig 2 (d)). The reduced absorption right above the band gap stems from the spectral transfer from non- perturbed optical transition to red- ( \(n = - 1\) ) and blue- ( \(n = 0\) ) shifted transitions (Fig 2 (d)). Depending on the strength of \(E_{THZ}\) and the degree of localization, \(Inl > 1\) transitions could, in principle, also be observed. In this case (Figure 2(a)), the observed single central step from reduced to increased
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 90, 883, 123]]<|/det|>
+absorption near the center of the band \(E_{pr} = 1.9 \text{eV}\) , is a noticeable signature of Stark localization, where the Wannier- Stark states are localized onto one unit cell.  
+
+<|ref|>text<|/ref|><|det|>[[115, 139, 884, 305]]<|/det|>
+This observed transient response is clearly distinct from the shift of transition energy due to the presence of photon- dressed states<sup>28</sup>. Photon- dressed states can shift energies in only one direction (mostly blue- shift) with energy orders of magnitude smaller than the bandwidth. Also, the contribution from the possible higher energy bands within our probe photon energy range<sup>29</sup> is negligible. Indeed, we neither observe any additional Franz- Keldysh and/or Wannier- Stark response within our probe energy, nor find any decay of the entire signal as a function of the field strength due to the tunneling to higher energy bands at intense field regime. Therefore, we could consider simple two- band systems to understand our experimental demonstration of Wannier- Stark localization in further detail. As will be shown below, the two- band model explains our observations."  
+
+<|ref|>text<|/ref|><|det|>[[115, 322, 883, 373]]<|/det|>
+Regarding the first point (in slightly more detail), given the presence of the higher band with the energy difference of around \(2.25 \text{eV}\) , there are two possibilities of experimental features one could observe in our result.  
+
+<|ref|>text<|/ref|><|det|>[[115, 389, 883, 490]]<|/det|>
+The first possibility is the case of tunneling to a higher energy band in the presence of strong THz fields. In that case, the photoexcited electrons and holes could tunnel into an energetically higher band and would then not contribute to WS localization in the lowest bands. Also, the signal could possibly decay as a function of the field amplitude for strong fields (since the tunneling rate has an exponential dependence on the field amplitude). However, we do not see indications in the experiment that this is happening to a significant degree.  
+
+<|ref|>text<|/ref|><|det|>[[114, 506, 883, 707]]<|/det|>
+In the other possibility, where the tunneling does not occur, if WS localization is also not the origin of our experimental observation, then each band would have shown at least Franz Keldysh effect at one around the primary bandgap of \(1.6 \text{eV}\) and the other one at around the secondary gap above \(2.25 \text{eV}\) (as the TPA paper reports). Since the Franz Keldysh effect appears as an oscillatory feature only near the gap (within \(100 \text{meV}\) in probe phonon energy), in that case one would expect two separate Franz Keldysh features - one in the \(1.5 \sim 1.7 \text{eV}\) range and another at \(2.1 \sim 2.3 \text{eV}\) , with no response for energies in between \(1.7 \sim 2.1 \text{eV}\) . In contrast, we find an induced transparency from \(1.6 \text{eV}\) all the way up to \(1.9 \text{eV}\) without interruption. The reason we did not observe any response in \(2.2 \text{eV}\) (even in FKE) could be understood from the different transition probability in the one- photon and two- photon transition processes. Our optical probing scheme involves one- photon transition where the response around \(2.2 \text{eV}\) is much weaker than the case of two- photon transition. Therefore, we could safely rule out the other scenario, too.  
+
+<|ref|>text<|/ref|><|det|>[[115, 723, 883, 840]]<|/det|>
+Regarding the second point, the Bloch- Siegert shift is typically very small in magnitude \(< 1 \text{ueV}\) and, in exceptional cases at best comparable to the optical Stark shift ( \(\sim 10 \text{meV}\) in the mentioned literature). Also, such a shift happens in only one direction: typically only blue shift (because this effect originates from the state repulsion). However, in our case, we have induced absorption below and above the band gap, which means that both red- shifted and blue- shifted transitions occur simultaneously, and reduced absorption at around the band gap due to the spectral transfer. These features are unique to Wannier Stark localization.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 89, 886, 208]]<|/det|>
+How much is the spectral width of the THz pump pulses? As the authors stated on page 6, the phonon modes of halide perovskites fall in the low frequency range around several THz. Therefore, if the spectral width is larger than the phonon frequencies, impulsive stimulated Raman scattering should occur. As some of the authors reported, driving the phonon modes leads to the similar transient absorption change albeit in a different crystal structure [Fig. 4a in H. Kim et al., Nat. Commun. 8, 687 (2017).]. Is it possible to discuss the contributions of phonons to the observed signals?  
+
+<|ref|>text<|/ref|><|det|>[[116, 224, 884, 275]]<|/det|>
+\(\Rightarrow\) The spectral width of the THz pump pulses is \(\sim 4\) THz. We added the spectrum of the THz pulses of the main result (fig 2a) in the supplementary information (fig. S1). For comparison, we also added the Fourier transform of the time profile in fig 2a in the SI (fig. S4).  
+
+<|ref|>text<|/ref|><|det|>[[115, 290, 884, 441]]<|/det|>
+As evident in this spectrum (fig S4), the main oscillation around time zero is 40 THz, and there is no distinct oscillation below 4 THz except for the slow envelope of the pulse. More importantly, if there is any phonon coherence, we know that the dephasing times of at least 1 and 2 THz modes are 0.3 and 1 ps, respectively, and both are much longer than the duration of the IR pulse and our experimental window. So if that was the case, we should have seen responses after the overlap of IR pulses and the visible probe (after \(\mathrm{t} = 200\) fs). However, we do not see any oscillation immediately after that, which indicates no contribution from phonons within the bandwidth. Thus, the impulsive stimulated Raman scattering could be safely ruled out. We added this argument om the main text as follows:  
+
+<|ref|>text<|/ref|><|det|>[[116, 456, 884, 540]]<|/det|>
+"It is noteworthy that the bandwidth of the THz pulse is \(\sim 4\) THz (< 40 THz modulation, Fig. S4), so that in principle impulsive stimulated Raman excitation of sub- 4 THz modes be possible. However, no oscillatory signal was observed after 150 fs, which is much shorter than the dephasing times of reported phonon modes with frequency up to 4 THz. Therefore, any possibility of coherent phonon contribution to the temporal modulation can be ruled out."  
+
+<|ref|>text<|/ref|><|det|>[[115, 556, 883, 658]]<|/det|>
+On page 13, the authors mentioned that the crystallites are arbitrarily oriented and the theoretical calculations were performed based on this assumption. Is there any possibility that some preferential orientations of the crystallites exist? Do the authors experimentally verify the assumption from, for example, XRD spectra? The detailed sample properties should be described in the Support Information, because the sample is not single crystal but complicated polycrystalline film.  
+
+<|ref|>text<|/ref|><|det|>[[115, 673, 883, 807]]<|/det|>
+\(\Rightarrow\) We understand and appreciate the reviewer's concern. However, any possibility of preferential orientations of the crystallites does not make any critical effect, as the two extreme cases - completely arbitrary orientation and perfectly single orientation - produce more or less the same THz induced optical spectra. From this theoretical approach, what we learn is that, no matter how much preferential orientation we have (from zero to complete), the contribution from the least dispersive direction dominates the optical response. Although we indeed can not completely rule out the possibility of partial preferential orientation, this possibility does not affect our conclusion. We highlight the point by adding these sentences in the main text:  
+
+<|ref|>text<|/ref|><|det|>[[115, 823, 884, 908]]<|/det|>
+"The results of Fig. 4(a, b) suggest that, for the randomly oriented crystallites in the film, the overall response is dominated by the response originating from the band dispersion in the \(\Gamma \bar{Z}\) direction. This reasoning is substantiated by the averaged field- dependent absorption changes calculated for both a static and a THz field shown in Figs. 4(c) and (d), respectively. This finding is remarkable because it means the two extreme cases - the completely random orientation of a polycrystalline
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 89, 884, 157]]<|/det|>
+sample and the perfectly oriented single crystal – are expected to produce very similar optical responses. The only slight difference between the two extremes would be a small shift in the photon energy ( \(\sim 100\) meV) where the induced transmission turns to the induced absorption and in the transient spectral shape. The two extremes include partial preferential orientations."  
+
+<|ref|>text<|/ref|><|det|>[[115, 173, 883, 358]]<|/det|>
+Regarding our sample, we added the SEM image of the polycrystalline perovskite MAPbl3 film spin- coated on TOPAS® substrate in the supplementary information (fig S2). We did not measure the XRD data of the measured sample, but we do have the XRD spectra of the perovskite polycrystalline film prepared in the exactly same way [fig S15 of J. Phys. Chem. Lett. 2015, 6, 4991]. There, one could find not only (hh0) but also other Bragg peaks. Besides, even in the case of a complete preferential orientation of a surface, still the most critical orientation is the relative angle between the [001] direction of each crystallite and the THz field polarization, which is not possible to define. The orientations of [001] direction of each crystallite on top of a completely disordered substrate can still be assumed to be arbitrarily oriented and difficult to measure with the currently available resolution of X- ray microscopy ( \(\sim 500\) nm in J. Phys. Chem. C. 2017, 121, 7596).  
+
+<|ref|>text<|/ref|><|det|>[[115, 372, 883, 457]]<|/det|>
+On page 15, the authors claimed that the calculation result shown as a black curve in Fig. 4a is in good agreement with experimental results at high field amplitudes in Fig. 4b. However, the spectral oscillations around \(2 \text{eV}\) can be seen only in the experimental results, not in the calculation. In addition, a bleaching signal around \(2.2 \text{eV}\) which is expected from the calculation does not appear in the experimental results. What are the reasons for such disagreements?  
+
+<|ref|>text<|/ref|><|det|>[[115, 473, 883, 707]]<|/det|>
+\(\Rightarrow\) We thank the reviewer for thoroughly examining our results. Yes, the first point, the spectral oscillations around \(2 \text{eV}\) , is indeed something we have tried to understand. The oscillation (or repeated peaks) has an interval of \(80 \sim 100 \text{meV}\) regardless of the THz field strength. Interestingly this interval matches well with our THz photon energy (83 meV). The oscillations are quite stable as function of the field amplitude, however, they do not correspond to Franz- Keldysh and neither to WS. To analyze the possible origin of these oscillations, we did perform additional model calculations. To that end, we, in particular, modified the k/energy dependence of the interband dipole and also considered a complex interband dipole with a symmetric real and an antisymmetric imaginary part (it was recently shown that such an interband dipole with such a k- dependence gives rise to SHG in two- band models). With such phenomenological model extensions, we do obtain signatures having similarities with THz sidebands in the optical spectra, see Fig. S9 in the supplementary information. However, the obtained results are quite different from the oscillations observed in the experiment and the model assumptions required to obtain them are rather unrealistic.  
+
+<|ref|>text<|/ref|><|det|>[[115, 723, 883, 874]]<|/det|>
+Regarding the second point, in a 1D model with WS localization, we remove absorption from the band edges, and this concentrates in the band center. Since we average over systems of different bandwidths and lattice constants, the critical field where the transition to WS takes places and also the position of the band center is not fixed but depends on the individual case, i.e., differs for the different f's. As a result, in Fig. 4(c) and 4(d) the transitions from blue- to red- shift appear at higher energies with increasing field amplitude, since with increasing amplitude also systems with larger bandwidth, i.e., larger f, enter the WS regime. Therefore, with any two- band model one should always see some bleaching near the upper band edge due to the concentration, or in other words, a shift of the oscillator strength towards the center of the band.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 883, 125]]<|/det|>
+In any case, the precision of our simple modeling involving some fitting of DFT results with few parameters gets more inaccurate with increasing photon energies.  
+
+<|ref|>text<|/ref|><|det|>[[115, 140, 630, 157]]<|/det|>
+To clarify this, we add an explanation in the main text as follows:  
+
+<|ref|>text<|/ref|><|det|>[[115, 173, 884, 323]]<|/det|>
+"The averaged graph is in good agreement with the differential spectra at high field amplitudes given very few parameters to describe the entire contributions from complicated actual band structure (i.e. two extreme bandwidths, lattice constants, and linear interpolation of them). The prediction of 1.9 eV, almost exactly where the change from bleaching to induced absorption is observed in the experiment (Fig 4(b), upper curves) is remarkable. A few minor features including the shape at higher photon energy could be improved by modifying the model band curve and oscillations around 2 eV in Fig 4(b) need further studies (Fig S9). More importantly, this position of 1.9eV is very close to the center of the band structure for the \(\overline{\Gamma Z}\) direction, 2 eV as the single direction model indicated above."  
+
+<|ref|>text<|/ref|><|det|>[[115, 356, 546, 373]]<|/det|>
+I suggest some more points for improving manuscript:  
+
+<|ref|>text<|/ref|><|det|>[[115, 373, 872, 406]]<|/det|>
+On page 8, why did the authors consider the band gap energy of 2D perovskites with \(I = 2\) , not \(I = 1\) , 3 or 4?  
+
+<|ref|>text<|/ref|><|det|>[[115, 422, 883, 523]]<|/det|>
+\(\Rightarrow\) It is because of the doubling of the unit cell in the tetragonal phase compared to the cubic phase. It means the double layers \((I = 2)\) Pb- I octahedra is the repeating unit, and in turn the spatial range of confinement. That was the reason we particularly compared the probe energy where one could observe the abrupt change of transmittance and the band gap energy of 2D perovskite with \(I = 2\) . We realized it could be confusing without further clarification. As such, we modified the corresponding sentence in the discussion section as following:  
+
+<|ref|>text<|/ref|><|det|>[[115, 538, 883, 589]]<|/det|>
+"In the case of \((BA)_2(MA)_{1 - 1}PbI_{3l + 1}\) perovskites, where the \(PbI_{6}\) octahedral network forms a double layer \((I = 2)\) , the same periodicity of the sample along the \(c\) axis, the optical band gap is \(\sim 2.1\) eV, which is comparable to the observed 1.9 eV."  
+
+<|ref|>text<|/ref|><|det|>[[115, 621, 866, 656]]<|/det|>
+With regard to Ref. 25, the authors should cite the published version [J.- C. Blancon et al., Nat. Commun. 9, 2254 (2018).], not that in arXiv.  
+
+<|ref|>text<|/ref|><|det|>[[115, 671, 528, 689]]<|/det|>
+\(\Rightarrow\) We cited this reference accordingly (highlighted).
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[105, 75, 279, 88]]<|/det|>
+Reviewers' comments:  
+
+<|ref|>text<|/ref|><|det|>[[105, 102, 402, 116]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[105, 130, 889, 201]]<|/det|>
+The authors have addressed all my concerns/comments in the revised manuscript. However, there are two important concerns from other reviewers which are very important. One is the role of the polycrystallinity of the sample. The only way to address this is to perform measurements on single crystals or two have extensive structural characterization of the sample, which are out of the scope of current work. Publication of this manuscript will inspire future work in that direction, I reckon.  
+
+<|ref|>text<|/ref|><|det|>[[105, 215, 888, 286]]<|/det|>
+The second concern, as reviewer 3 rightly points out, is with regards to possible alternative explanations to the presented experimental results. The authors provide a convincing arguments to discount other possibilities. Some of the explanations provided in the response to reviewer 3 were not included in the manuscript, for example, on why Bloch- Siegert is not a viable explanation. It may be very instructional to expand on this discussion in the main manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[105, 299, 805, 327]]<|/det|>
+Apart from these very minor comments, the manuscript is suitable for publication in Nature Communications.  
+
+<|ref|>text<|/ref|><|det|>[[105, 355, 402, 369]]<|/det|>
+Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[105, 383, 870, 425]]<|/det|>
+I have read through authors' response, and the revised manuscript. I'm okay with authors' response to my comments, and thus I don't have further objection to accept this manuscript for publication in NC.  
+
+<|ref|>text<|/ref|><|det|>[[105, 452, 402, 466]]<|/det|>
+Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[104, 480, 886, 620]]<|/det|>
+I confirmed the replies from the authors, the revised manuscript, and the supporting information. I appreciate the efforts of the authors to answer my questions and comments. However, I still do not think that it is convincing that the origin of the observed nonlinear responses can be attributed to the Wannier- Stark localization. As the authors replied, the theoretical curves shown in Fig. 4a do not fully reproduce the experimental results in Fig. 4b partly because the theoretical model is too simple. Therefore, to make the interpretation as the Wannier- Stark localization solid, it is important to rule out the other possible origins. Although the other possibilities are discussed in the reply letter and the revised manuscript, I am not satisfied with some of those discussions. Thus, I do not recommend publication of the manuscript for Nature Communications as it stands. I suggest that the following points should be considered.  
+
+<|ref|>text<|/ref|><|det|>[[105, 634, 880, 691]]<|/det|>
+1. On page 9 in the main manuscript, the authors mentioned that the two induced absorption signals shown in Fig. 2d can be assign to \(n = -1\) and \(n = 0\) transitions, whose difference in the transition energy is \(\mathrm{eE}< \mathrm{sub} > \mathrm{THz}< /\mathrm{sub} > \mathrm{D}\) . How much is the value of \(\mathrm{eE}< \mathrm{sub} > \mathrm{THz}< /\mathrm{sub} > \mathrm{D}\) ? In addition, I cannot read from Fig. 2d what the \(n = -1\) and \(n = 0\) transition energies are.  
+
+<|ref|>text<|/ref|><|det|>[[104, 705, 891, 804]]<|/det|>
+2. On page 9 in the main manuscript, the authors stated "Photon-dressed states can shift energies in only one direction (mostly blue-shift) ..." However, I do not agree with the statement. If the higher energy levels exist, the Autler-Townes effect can shift the band-edge transitions in both directions, i.e. blue- and red- shifts, as reported in [C.-K. Yong et al. Nat. Mater. 18, 1065-1070 (2019).]. In addition, [G. Yumoto et al. Nat. Commun. 12, 3026 (2021).] recently reported that three-level Autler-Townes effect indeed occurs in halide perovskites. How do the authors comment on the possibility that the Autler-Townes effect can be the origin of the observed optical nonlinear responses?  
+
+<|ref|>text<|/ref|><|det|>[[104, 817, 894, 900]]<|/det|>
+3. On page 10 in the main manuscript, the authors stated "... nor find any decay of the entire signal as a function of the field strength due to the tunneling to higher energy bands at intense field regime." I agree to the authors that the real excitation of carriers cannot explain the data due to the absence of the signal decay. However, the virtual population generated only under the THz-field irradiation should induce additional transitions for probe pulses, which would result in induced absorption signals. Is it possible to comment on this?
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 90, 420, 107]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 123, 883, 224]]<|/det|>
+The authors have addressed all my concerns/comments in the revised manuscript. However, there are two important concerns from other reviewers which are very important. One is the role of the polycrystallinity of the sample. The only way to address this is to perform measurements on single crystals or two have extensive structural characterization of the sample, which are out of the scope of current work. Publication of this manuscript will inspire future work in that direction, I reckon.  
+
+<|ref|>text<|/ref|><|det|>[[116, 239, 883, 324]]<|/det|>
+The second concern, as reviewer 3 rightly points out, is with regards to possible alternative explanations to the presented experimental results. The authors provide a convincing arguments to discount other possibilities. Some of the explanations provided in the response to reviewer 3 were not included in the manuscript, for example, on why Bloch- Siegert is not a viable explanation. It may be very instructional to expand on this discussion in the main manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[116, 339, 883, 374]]<|/det|>
+Apart from these very minor comments, the manuscript is suitable for publication in Nature Communications.  
+
+<|ref|>text<|/ref|><|det|>[[116, 389, 883, 441]]<|/det|>
+\(\Rightarrow\) First of all, we appreciate the reviewer for very encouraging and constructive comments. We agree with the reviewer's suggestion, and thus, we explicitly include a discussion explaining why these scenarios can be ruled out in this revised manuscript as follows:  
+
+<|ref|>text<|/ref|><|det|>[[115, 456, 884, 641]]<|/det|>
+"It is important to distinguish this transient Wannier Stark localization from the optical Stark- type effects such as the Autler- Townes effect and the Bloch- Siegert shift. In general, an external electric field affects the optical properties of a semiconductor in two ways: there are spectral and kinetic aspects. Spectral aspects refer to energy shifts and broadenings that arise from mixing two states by the external optical field. The mixing of the wavefunctions results in dressed states and leads to the Stark- type shifts. The magnitude of such shifts increases with the amplitude of the incident field and the interband dipole matrix, but decreases with increasing detuning between the light frequency and the transition frequency. On the other hand, kinetic aspects represent the evolution of the particle distributions in the renormalized states driven by the external field, which is called "intraband acceleration". This intraband acceleration leads to the Franz Keldysh effect at a moderate field strength and eventually Wannier- Stark localization in the strong- field regime.  
+
+<|ref|>text<|/ref|><|det|>[[116, 655, 884, 807]]<|/det|>
+Each of these two contributions can be straightforwardly separated in the semiconductor Bloch equations (SBE). Specifically, the third and second terms on the right- hand side of the Eq (1) represent the spectral (optical Stark- type effects) and the kinetic (Wannier Stark localization) aspects, respectively. One can thus directly compare each contribution to the differential optical response. As evident from Supplementary Fig. 6, the THz- induced optical Stark effect is shown to be much weaker (on the order of a few meV) compared to the shifts arising from the Wannier Stark localization which corresponds to approximately half the band width (several 100 meV). Therefore, we conclude that the observed transient response is mainly contributed from the Wannier Stark localization."
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 106, 883, 157]]<|/det|>
+I have read through authors' response, and the revised manuscript. I'm okay with authors' response to my comments, and thus I don't have further objection to accept this manuscript for publication in NC.  
+
+<|ref|>text<|/ref|><|det|>[[118, 174, 620, 191]]<|/det|>
+\(\Rightarrow\) We thank the reviewer very much for the positive comments.  
+
+<|ref|>text<|/ref|><|det|>[[116, 240, 420, 256]]<|/det|>
+Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 272, 883, 441]]<|/det|>
+I confirmed the replies from the authors, the revised manuscript, and the supporting information. I appreciate the efforts of the authors to answer my questions and comments. However, I still do not think that it is convincing that the origin of the observed nonlinear responses can be attributed to the Wannier- Stark localization. As the authors replied, the theoretical curves shown in Fig. 4a do not fully reproduce the experimental results in Fig. 4b partly because the theoretical model is too simple. Therefore, to make the interpretation as the Wannier- Stark localization solid, it is important to rule out the other possible origins. Although the other possibilities are discussed in the reply letter and the revised manuscript, I am not satisfied with some of those discussions. Thus, I do not recommend publication of the manuscript for Nature Communications as it stands. I suggest that the following points should be considered.  
+
+<|ref|>text<|/ref|><|det|>[[115, 456, 883, 590]]<|/det|>
+\(\Rightarrow\) We appreciate the reviewer for the thorough review and for giving us the opportunity to improve our manuscript even further. We apologize for apparently underestimating this point: we mistakenly felt it inappropriate to devote a substantial part of the discussion in the manuscript to this point. Rather, we tried to convince the reviewer in the rebuttal. However, we take this concern more carefully into account this time, and explicitly include a discussion explaining what are the similarities and differences between these optical Stark- type effects (i.e. Autler- Townes effects and Bloch- Siegert effects) in a theoretical context and how exactly each effect contributes to the experimentally observed optical response. This was also suggested by reviewer #1.  
+
+<|ref|>text<|/ref|><|det|>[[115, 606, 883, 757]]<|/det|>
+In the strong field regime (like our experiment), the Wannier- Stark localization (WSL) shifts the onset of the energy gap by approximately half the bandwidth, i.e., several hundred meV. Since the THz frequency is much lower than the interband transition frequencies, the optical Stark shift can be described perturbatively and is determined by the square of the Rabi frequency divided by the detuning. For a maximal field amplitude of 10 MV/cm and the interband dipole matrix element, the resulting Stark shifts are on the order of just a few meV, which is orders of magnitude smaller than the shifts arising from the WSL. These arguments are in agreement with and supported by new simulations in which we artificially neglected the intraband acceleration in order to study Stark- type shifts separately (see Supplementary Figure 6 and Supplementary Method).  
+
+<|ref|>text<|/ref|><|det|>[[116, 773, 888, 840]]<|/det|>
+Therefore, we could further confirm that the Wannier- Stark localization (originating from the intraband acceleration driven by the field) dominates the observed differential transmission. In contrast, the optical Stark- type effects (which leads to the spectral shift of each k- state of the band) have a negligible amplitude compared to the WSL.  
+
+<|ref|>text<|/ref|><|det|>[[115, 857, 883, 890]]<|/det|>
+We thank the reviewer for the opportunity to revisit the fundamentals and strengthen our interpretation. Further details are covered in our reply to comment #2 below.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 105, 883, 175]]<|/det|>
+1. On page 9 in the main manuscript, the authors mentioned that the two induced absorption signals shown in Fig. 2d can be assign to \(n = -1\) and \(n = 0\) transitions, whose difference in the transition energy is \(eE_{THZ}D\) . How much is the value of \(eE_{THZ}D\) ? In addition, I cannot read from Fig. 2d what the \(n = -1\) and \(n = 0\) transition energies are.  
+
+<|ref|>text<|/ref|><|det|>[[115, 190, 883, 258]]<|/det|>
+\(\Rightarrow\) We agree with the reviewer that the value of energy distance \(eE_{THZ}D\) is only inferred, and the necessary numbers appear in a rather scattered way (e.g. \(E_{THZ} = 6 \text{MV/cm}\) in page 11, and \(D =\) the largest lattice constant along the c axis, 12.5 Å, in page 15). Therefore, we added the figure caption of figure 2d the following sentence:  
+
+<|ref|>text<|/ref|><|det|>[[117, 272, 884, 325]]<|/det|>
+"In case of \(E_{THZ} = 6 \text{MV/cm}\) in considering the lattice constant \(D\) of 12.5 Å, \(\Delta E_{WSL} = eE_{THZ}D\) is estimated to be 750 meV, consistent with the spectrum showing that the absorption band of \(n = - 1\) and \(n = 0\) are approximately 750 meV apart."  
+
+<|ref|>text<|/ref|><|det|>[[115, 339, 883, 409]]<|/det|>
+Indeed, the \(n = - 1\) and \(n = 0\) transitions have certain energy ranges rather than a single transition energy (i.e. two- level systems) since these are "interband" transitions among mini- bands. So the range of transition energies for \(n = - 1\) is 1.2\~1.62eV, and that for \(n = 0\) is 1.95\~2.38 eV, and these transition energy ranges are approximately 750 meV apart (figure 2d).  
+
+<|ref|>text<|/ref|><|det|>[[115, 456, 883, 592]]<|/det|>
+2. On page 9 in the main manuscript, the authors stated "Photon-dressed states can shift energies in only one direction (mostly blue-shift) ..." However, I do not agree with the statement. If the higher energy levels exist, the Autler-Townes effect can shift the band-edge transitions in both directions, i.e. blue- and red- shifts, as reported in [C.-K. Yong et al. Nat. Mater. 18, 1065-1070 (2019).]. In addition, [G. Yumoto et al. Nat. Commun. 12, 3026 (2021).] recently reported that three-level Autler-Townes effect indeed occurs in halide perovskites. How do the authors comment on the possibility that the Autler-Townes effect can be the origin of the observed optical nonlinear responses?  
+
+<|ref|>text<|/ref|><|det|>[[115, 621, 884, 891]]<|/det|>
+\(\Rightarrow\) We agree with the reviewer that the optical Stark effect (i.e. the Autler- Townes effect) in fact leads to energy splitting. This initially two- level system with energies of \(\epsilon_{1}\) and \(\epsilon_{2}\) undergoes light- field induced level splitting to \(\epsilon_{1} + \frac{\nu}{2}\pm \frac{1}{2}\sqrt{\nu^{2} + \hbar^{2}\omega_{R}^{2}}\) and \(\epsilon_{2} - \frac{\nu}{2}\pm \frac{1}{2}\sqrt{\nu^{2} + \hbar^{2}\omega_{R}^{2}}\) respectively, where \(\nu\) is detuning \((\epsilon_{2} - \epsilon_{1} - \hbar \omega_{laser})\) and \(\omega_{R}\) is the Rabi frequency. The transitions among these split states are observed from singlet (without optical field) to triplet ("Mollow triplet", with optical field). In case of weak excitation and finite detuning (here we make a Taylor expansion of the square roots, so it's a perturbative result), the upper sideband \(\epsilon_{2} - \epsilon_{1} + \frac{1}{2}\frac{\omega_{R}^{2}}{\nu}\) is closest to the original resonance. This transition is the one that is observed in the experiment as a small blueshift depending on the intensity of the light field (and the interband dipole matrix element), if the frequency of the exciting light is smaller than the transition frequency. Although by changing the detuning (frequency difference between the light frequency and the transition frequency) it is possible to observe the peak splitting as the reviewer has pointed out, the blue shift is the common signature for large detuning (such as our experiment - for THz field the detuning to an optical transition is very large). [H. Haug & S. W. Koch, "Quantum Theory of the Optical and Electronic Properties of Semiconductors"]
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 89, 883, 258]]<|/det|>
+However, more fundamentally, in a semiconductor, unlike two- level systems (or multi- levels without k- space dispersion), an external field has pronounced influence on the relative motion of optically generated electron- hole pair, going well beyond the field- induced shifts of excitonic resonance. And this is what we demonstrate by separating the contribution of the optical Stark effect and the intraband acceleration: WSL is by far the dominating contribution to the observed differential optical response and produces the spectral shape in very good agreement. In contrast, the optical Stark- type contributions (Autler- Townes effect from the corotating field and Bloch Siegert shift from the counterrotating field) are almost negligible and, moreover, do not agree with the observed spectral shape. Therefore, we added the following paragraph and the supplementary figure 6 showing this additional result:  
+
+<|ref|>text<|/ref|><|det|>[[115, 289, 884, 474]]<|/det|>
+"It is important to distinguish this transient Wannier Stark localization from the optical Stark- type effects such as the Autler- Townes effect and the Bloch- Siegert shift. In general, an external electric field affects the optical properties of a semiconductor in two ways: there are spectral and kinetic aspects. Spectral aspects refer to energy shifts and broadenings that arise from mixing two states by the external optical field. The mixing of the wavefunctions results in dressed states and leads to the Stark- type shifts. The magnitude of such shifts increases with the amplitude of the incident field and the interband dipole matrix, but decreases with increasing detuning between the light frequency and the transition frequency. On the other hand, kinetic aspects represent the evolution of the particle distributions in the renormalized states driven by the external field, which is called "intraband acceleration". This intraband acceleration leads to the Franz Keldysh effect at a moderate field strength and eventually Wannier- Stark localization in the strong- field regime.  
+
+<|ref|>text<|/ref|><|det|>[[115, 490, 884, 641]]<|/det|>
+Each of these two contributions can be straightforwardly separated in the semiconductor Bloch equations (SBE). Specifically, the third and second terms on the right- hand side of the Eq (1) represent the spectral (optical Stark- type effects) and the kinetic (Wannier Stark localization) aspects, respectively. One can thus directly compare each contribution to the differential optical response. As evident from Supplementary Fig. 6, the THz- induced optical Stark effect is shown to be much weaker (on the order of a few meV) compared to the shifts arising from the Wannier Stark localization which corresponds to approximately half the band width (several 100 meV). Therefore, we conclude that the observed transient response is mainly contributed from the Wannier Stark localization."  
+
+<|ref|>text<|/ref|><|det|>[[115, 689, 883, 790]]<|/det|>
+3. On page 10 in the main manuscript, the authors stated "... nor find any decay of the entire signal as a function of the field strength due to the tunneling to higher energy bands at intense field regime." I agree to the authors that the real excitation of carriers cannot explain the data due to the absence of the signal decay. However, the virtual population generated only under the THz-field irradiation should induce additional transitions for probe pulses, which would result in induced absorption signals. Is it possible to comment on this?  
+
+<|ref|>text<|/ref|><|det|>[[115, 807, 886, 841]]<|/det|>
+\(\rightarrow\) The original manuscript contained a paragraph on the possible carrier generation by THz (multiphoton processes and impact ionization) in our method section and also the Supplementary figure:  
+
+<|ref|>text<|/ref|><|det|>[[117, 857, 884, 907]]<|/det|>
+"In this linear- optical regime, we thus neglect carrier generation by multi- photon processes and impact ionization, which does not seem to play a dominant role in the measured transient spectra. Interband tunneling by the THz field could lead to bleaching at later delay times and the slightly
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 90, 884, 157]]<|/det|>
+asymmetric spectral evolution with respect to \(\tau = 0\) (Fig. 2(A)) (corresponding to the trailing edge of the THz transient in the Supplementary Material of ref [20]). However, significant carrier multiplication does not occur within this experimental window, as shown in Supplementary Fig. 10."  
+
+<|ref|>text<|/ref|><|det|>[[115, 173, 884, 307]]<|/det|>
+However, in addition to that, we have performed new simulations that include the possible effects of THz- induced generation of virtual and real carriers that may arise from multi- photon transitions by solving a full set of semiconductor Bloch equations, in which the THz field is treated non- perturbatively, and the weak optical probe pulse is considered linearly. The result is shown in the supplementary fig 6, indicating that for the perovskite, the high- order/multi- photon interband effects are basically negligible. Therefore, our simplified model defined by Eq. (1) in the main text captures all the relevant physics. Accordingly, we have added the following statement in the main text:  
+
+<|ref|>text<|/ref|><|det|>[[115, 322, 884, 424]]<|/det|>
+"Furthermore, to analyze the possible effects of THz- induced generation of virtual and real carriers that may arise from multi- photon transitions, we solve a full set of SBE (Supplementary Methods) in which the THz field is included non- perturbatively and the weak optical probe pulse is considered linearly. The results (Supplementary Figure 6) show that for the considered field amplitudes, such higher- order interband effects arising from the THz fields are negligible, as the results from the full equations are very close to the ones obtained from the simplified Eq. (1)."
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[119, 84, 310, 97]]<|/det|>
+## REVIEWERS' COMMENTS  
+
+<|ref|>text<|/ref|><|det|>[[120, 112, 416, 126]]<|/det|>
+Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 140, 863, 210]]<|/det|>
+I appreciate the efforts of the authors to answer my questions and comments. The authors have cleared up my concerns about the interpretation of the observed nonlinear responses by performing new calculations (Supplementary Figure 6) and ruling out the possible interpretations other than the Wannier- Stark localization. Therefore, now I think the manuscript is suitable to publish in Nature Communications.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 420, 107]]<|/det|>
+Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 122, 877, 207]]<|/det|>
+I appreciate the efforts of the authors to answer my questions and comments. The authors have cleared up my concerns about the interpretation of the observed nonlinear responses by performing new calculations (Supplementary Figure 6) and ruling out the possible interpretations other than the Wannier- Stark localization. Therefore, now I think the manuscript is suitable to publish in Nature Communications.  
+
+<|ref|>text<|/ref|><|det|>[[120, 223, 833, 241]]<|/det|>
+\(\Rightarrow\) We thank the reviewer very much for the thorough reviews and constructive comments.
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__011f1f7cdec2740845fc5c2f410ff02c63329260c767801a3ae4c3d8ae57e6f6/images_list.json

@@ -0,0 +1,10 @@
+[
+  {
+    "type": "image",
+    "img_path": "images/Figure_1.jpg",
+    "caption": "Figure 1: Reduced endothelial cells in the bone fraction of mutant mice. Absolute numbers of the indicated stroma cell subsets from bone fractions were determined by flow cytometry in WT, \\(+ / 1013\\) and 1013/1013 mice. Data (means + SEM) are from three independent experiments with 6 mice in total per group. \\*, P < 0.05; and \\*\\*, P < 0.005 compared with WT cells. §§, P < 0.005 compared with \\(+ / 1013\\) cells. (as determined using the two-tailed Student's t test).",
+    "footnote": [],
+    "bbox": [],
+    "page_idx": 0
+  }
+]

peer_reviews/supplementary_0_Peer Review File__011f1f7cdec2740845fc5c2f410ff02c63329260c767801a3ae4c3d8ae57e6f6/supplementary_0_Peer Review File__011f1f7cdec2740845fc5c2f410ff02c63329260c767801a3ae4c3d8ae57e6f6.mmd

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+
+# nature portfolio  
+
+Peer Review File  
+
+WHIM Syndrome- linked CXCR4 mutations drive osteoporosis  
+
+![](images/Figure_1.jpg)
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+## REVIEWER COMMENTS  
+
+## Reviewer #1 (Remarks to the Author):  
+
+Anginot et al. report on the importance of CXCR4 desensitization in skeletal stem cells (SSCs) in order to allow SSCs to proliferate adequately and differentiate into the osteogenic lineage, whereas chondrogenic and adipogenic differentiation seem not to be affected by gain of function mutation of CXCR4. These novel data certainly increase our understanding on CXCR4 signalling in osteogenic lineage cells. In addition, the authors combined numerous well- designed in vivo and in vitro experiments to elucidate the cellular mechanisms. However, several inconsistencies between the findings are present, especially concerning the effect of CXCR4 desensitization on SSC properties and their osteogenic differentiation potential.  
+
+Figure 2B. The decrease in stroma cell number in mutant mice ( \(60 \times 10^{3}\) versus \(100 \times 10^{3}\) in WT mice) cannot be explained by the combined decrease in SSC ( \(3 \times 10^{3}\) versus \(5 \times 10^{3}\) ) and OPC ( \(7 \times 10^{3}\) versus \(13 \times 10^{3}\) ). The question is therefore which other bone cell types are decreased in mutant mice as these other cell types might also contribute to the observed decrease in bone mass. Are endothelial cells decreased (H- type and L- type) in mutant mice as they express CXCR4 and might provide a vascular niche for the SSC?  
+
+Figure 2N and 2S. Parameters of TBV and cortical bone should be quantified, preferable by \(\mu \mathrm{CT}\) (or quantitative histological analysis). At this moment, only 1 image per condition is shown and this is an Opn staining, which is not considered to be the appropriate approach for quantitative bone measurements. This quantification of bone parameters is especially necessary to verify the bone loss that occurs when recipient mice are WT (Figure 2S), as the bone loss that is induced by transplantation of mutant donor cells in WT recipient mice is hard to be explained only by a reduced number of SSC and OPC, as is now suggested.  
+
+Figure 3. The authors suggest that the in vivo observed increase in osteoclasts in mutant mice is linked to an altered BM environment. To strengthen this statement, coculture experiments of osteogenic cells and osteoclast- precursors, in different combinations of WT vs mutant cells (treated with PTH, \(\mathrm{Pg}\) or 1,25- vitamin D) should be performed. At this moment, the data only describe a discordance between the in vivo and in vitro findings, but do not allow to make any conclusion on whether the decrease in bone mass is partly caused by increased bone resorption.  
+
+Figure 3. The histomorphometric data should be confirmed in more mice, as 3 mice per group for histomorphometric analysis is often not sufficient (Figure 3F- H). In addition, the bone formation data are puzzling, as osteoblast surface and osteoid surface are normal, but MS/BS and DB/BS are decreased. The authors interpret these data as a 'lower number of osteoblasts' but this statement does not fit with the normal osteoblast surface that is observed. Since dynamic bone formation parameters primarily measure the incorporation of minerals, these data might suggest that the formation of bone matrix by the osteoblasts is normal, but that the mineralisation of this bone matrix is impaired (and likely some osteoblasts are not mineralizing the matrix, whereas others show normal mineralization capacity as MAR is normal). Gene expression analysis might provide some more insight. The gene expression analysis (Figure 3J) is now restricted to genes that typify mature osteoblasts, but the expression of genes involved in mineralization is not analysed. In addition, the
+
+<--- Page Split --->
+
+variation of the gene expression data reported in Figure 3J and M, is rather high and this quantification should be validated with qRT- PCR data and using more mice.  
+
+Figure 4. Panel D shows the relative expression of selected genes; are these the most differentially expressed genes between the different genotypes? To appreciate the importance of these differences, it will be important to provide also the unbiased ranked overview/list of pathways which differ the most between genotypes, based on genes involved. In addition, since mutant SSC maintain their potential to differentiate normally to chondrocytes and adipocytes, are SOX9 and Pparg expression normal in mutant SSC? Furthermore, it remains hard to understand that a decrease in OPC number (Figure 2B, Figure 4I) does not affect osteoblast or osteoid surface. How do the authors reconcile these data?  
+
+Figure 5 and 3. The data suggest that osteogenic differentiation starting from mutant SSC is reduced (Figure 5E- G), but once mutant SSC become OPC they can differentiate normally (Figure 3L). It should be good to confirm this observation, by performing the same assays on OPC as shown for SSC (Figure 5: differentiation with Alp quantification and gene expression analysis). In addition, it is rather particular that after 21 days of osteogenic differentiation, most of the cells are still SSC (Figure S1), and intermediate cells account only for \(15\%\) of the population, whereas the \(0\%\) of ALP+ cells, reported in Figure 5E, seems much higher. Same comment for the low \(0\%\) of mature cells compared to reported homogeneous and abundant alizarin red staining (Figure 5F).  
+
+Figure 5 in vivo data. The authors state that especially the cortical bone is rescued in mutant mice, but not the trabecular phenotype, based on lumbar spine BMD data. To validate this statement, \(\mu \mathrm{CT}\) analysis of cortex of long bones should be analysed with and without AMD3100 treatment. In addition, these data also suggest that CXCR4 desensitisation in osteogenic lineage cells is likely not responsible for the trabecular bone phenotype, and that other cell types/mechanisms might be involved. This site- specificity should be reflected in the title and in the abstract.  
+
+## Minor comments  
+
+Perilipin staining should be quantified as the observation that CXCR4 specifically reduces the osteogenic, but not the adipogenic differentiation is interesting, but should be validated by quantitative data.  
+
+Figure 1D: it is not clear whether the total number of mice used is 7- 14, coming from 3 experiments, or that in each of the 3 experiments there were 7- 14 mice, thus 21- 42 mice in total. Similar comment to all experiments using mice.  
+
+Figure 2J: the \(0\%\) of apoptotic OPC is around \(30\%\) , which is rather high, and should be commented on.
+
+<--- Page Split --->
+
+## Reviewer #2 (Remarks to the Author):  
+
+WHIM syndrome (WS) is a rare immunodeficiency caused by gain- of- function CXCR4 mutations. The authors have demonstrated for the first time a substantial decrease in bone mineral density in \(25\%\) of WS patients and osteoporosis in a WS mouse model. Interestingly, wild- type mice transplanted with bone marrow hematopoietic cells from mice with a WS- linked CXCR4 mutation (Cxcr4+/1013 or Cxcr4 1013/1013) had reduced trabecular bone content compared with wild- type chimeras. On the other hand, transplantation of wild- type bone marrow cells did not rescue the reduced trabecular bone content in the mutant chimeras. Osteogenic differentiation of cultured bone marrow skeletal stem cells (SSCs) from the mutants was impaired in vitro. The CXCR4 antagonist AMD3100 normalized in vitro osteogenic potential of SSCs and reversed an in vivo decrease in Sca- 1- PDGFRα- cells in the mutants. These results are interesting and important; however the major concern remains at this time. There is the possibility that osteopenia in mice and patients, which carry the WS- linked CXCR4 mutation, is the result of only enhanced osteoclast function but not reduced osteogenic differentiation of SSCs.  
+
+1. As the authors described, it has been shown previously that deletion of CXCR4 in mesenchymal cells, including SSCs, resulted in osteopenia (Tzeng et al., J. Bone Miner. Res. 2018; Zhu et al., J. Biol/ Chem. 2011). These results argue against the authors' conclusion that gain-of-function CXCR4 mutations in SSCs resulted in osteopenia. Thus, I would recommend the authors to generate and analyze the mice, in which mesenchymal cells, including SSCs, but not hematopoietic cells carry WS-linked CXCR4 mutations.  
+
+2. The authors show the reduced trabecular bone content of mice with a WS-linked CXCR4 mutation transplanted with bone marrow hematopoietic cells from wild-type mice was not rescued 3 and 16 weeks after transplantation. However, wild-type hematopoietic cells might be able to rescue the reduced trabecular bone content of the mutants earlier in development.  
+
+3. The authors describe Sca-1+PDGFRα+ cells as SSCs (Page 8, line 142); however, the major population of bone marrow SSCs is defined as Sca-1-PDGFRα+PDGFRb+LePR+CD31-cells (Omatsu et al., Immunity 2010; Zhou et al., Cell Stem Cell 2014; Seike et al., Genes Dev 2018).  
+
+4. The evidence that Sca-1-PDGFRα-cells are committed osteoblasts (OPCs) in the bone marrow would not be convincing (Page 8, line 143).
+
+<--- Page Split --->
+
+## Reviewer #4 (Remarks to the Author):  
+
+The manuscript by Anginot and colleagues provides novel insights into the role of CXCR4- mediated signaling in skeletal stromal/stem cell osteogenic specification. The authors describe a series of experiments characterizing the anatomic, developmental and functional properties of the skeletal and osteogenic compartment in a knock- in mouse model of the human genetic disorder WHIM syndrome. The significance of the deficits in skeletal remodeling and stem cell differentiation identified in the mouse model in human bone biology are validated in cohort of WHIM syndrome patients carrying gain- of- function mutations in CXCR4. These findings represent a novel contribution elucidating an important new role for CXCR4 in bone biology.  
+
+The authors characterized the effects of increased CXCR4 signaling in vivo through standard histomorphometric of bone anatomy and flow cytometric analyses of various progenitor cell populations in the mouse model. The data in Figure 1 are well presented and convincing in regard to the gene- dose dependent skeletal effects as well as the specificity of the changes to cortical and trabecular bone. Figure 2 is overly dense and contains information that could be moved to the supplement without impacting the major findings of the work. In particular, the experiments demonstrating the functional effects of the mutant CXCR4 receptor recapitulate characteristics of CXCR4 C- terminal truncations that have been well studied in other contexts. It would suffice to state that the mutant receptor localization, internalization and intracellular signaling were similar to what has been seen in other contexts and move panels 2E- J to the supplement. The bone marrow reconstitution experiments shown in the remainder of the figure demonstrate clearly the contribution of cell- extrinsic as well as cell- intrinsic factors to the observed skeletal changes. The Similarly, the effects on bone resorption and formation shown in Figure 3 panels C- E can be moved to supplement to better focus on the transcriptional effects shown in the subsequent panels.  
+
+The data in Figures 4 and 5 provide compelling data regarding the impact of aberrant CXCR4 signaling on osteogenic specification at the level of transcriptional effects and cell cycle progression. The PCA data shown in Figure 4C is not well explained as the 48 genes used for expression profiling are not described in the text nor the supplement, which lists a smaller number of genes. The data in the subsequent panels are more informative. I would consider removing panel 4C or moving it to the supplement with a better description of the analysis. The experiments shown in Figure 5 document the selective reduction in osteogenic differentiation capacity of stromal stem cells carrying one or two mutant CXCR4 receptors in a dose dependent fashion and the reversal of this phenotype with treatment of the receptor inhibitor AMD3100. The relevance of these data in mice to human bone biology are supported with the data shown in Figure 6 which revealed a selective osteogenic differentiation defect in bone marrow cells derived from WHIM syndrome patients.  
+
+With regards to the conclusion that a skeletal phenotype is present in a subset of WHIM syndrome patients, given that treatment of neutropenia with G- CSF is associated with osteopenia as side effect of therapy, it would be useful to know the total number of patients treated with G- CSF in the cohort to address the concern that the enrichment in osteopenic patients is restricted to those patients that have been so treated as well as their ages and genders given the impact of these variables on risk for osteopenia in general.
+
+<--- Page Split --->
+
+Apart from these concerns, the quality of the data presented is good and the conclusions supported by the evidence. The manuscript is well written and the references appropriate, though it was notable that the initial description of the cause of WHIM syndrome as gain- of- function truncation mutations in CXCR4 was not cited, this should be added.
+
+<--- Page Split --->
+
+## Point-to-point response to the reviewers' comments  
+
+## Reviewer #1 comments:  
+
+Anginot et al. report on the importance of CXCR4 desensitization in skeletal stem cells (SSCs) in order to allow SSCs to proliferate adequately and differentiate into the osteogenic lineage, whereas chondrogenic and adipogenic differentiation seem not to be affected by gain of function mutation of CXCR4. These novel data certainly increase our understanding on CXCR4 signalling in osteogenic lineage cells. In addition, the authors combined numerous well- designed in vivo and in vitro experiments to elucidate the cellular mechanisms. However, several inconsistencies between the findings are present, especially concerning the effect of CXCR4 desensitization on SSC properties and their osteogenic differentiation potential.  
+
+## Major concerns:  
+
+1. "Figure 2B. The decrease in stroma cell number in mutant mice ( \(60 \times 10^{3}\) versus \(100 \times 10^{3}\) in WT mice) cannot be explained by the combined decrease in SSC ( \(3 \times 10^{3}\) versus 5 \(\times 10^{3}\) ) and OPC ( \(7 \times 10^{3}\) versus \(13 \times 10^{3}\) ). The question is therefore which other bone cell types are decreased in mutant mice as these other cell types might also contribute to the observed decrease in bone mass. Are endothelial cells decreased (H-type and L-type) in mutant mice as they express CXCR4 and might provide a vascular niche for the SSC?"  
+
+We are grateful to the reviewer for this constructive comment and agree that some populations are likely missing in our flow- cytometric analyses. In particular, we did not consider the CD51- Sca1- cell population which is non- hematopoietic (CD45- ) and non- vascular (CD31- ) but appeared to be decreased in an allele- dose dependent manner in mutant mice. Thus, this stromal population might contribute to the overall decrease in stroma cell number in mutant mice. Because we do not know anything about this population, we propose to remove the stroma quantification panel to better focus on SSCs and OPCs (new Figures 2A and 2B). Whether endothelial cell (EC) numbers are affected is an interesting question raised by the reviewer. Different types of bone marrow (BM) ECs have been phenotypically identified in long bones (see for instance Kusumbe Nature 2014; Balzano Cell Rep 2019). The bone fraction is reported to be enriched for arteriolar ECs (Sca1+CD31+Emcn- ), few L- type sinusoidal ECs and CD31hiEmcnhi H- type ECs, a small fraction of the ECs at the end of the CD31+Emcn- arteriolar network. Based on Sca1 and CD31 expression, we observed by flow cytometry a decrease in ECs in the bone fraction of mutant mice (see below Figure 1 for reviewers). Although these preliminary findings are very interesting, we feel that they deserve to be strengthened by adding notably the Endomucin marker to visualize by immunofluorescence the impact of the Cxcr4 mutation on H- type and L- type EC architecture and numbers. This would constitute the subject of another study that will be dedicated to vascular modifications in WS mice. However, the well- established regulatory role of the vascular system on the mesenchymal one has been discussed in the revised version of the manuscript (page 22, lines 474 and 482). In particular, whether vascular cells participate in the defective osteolineage specification of SSCs in Cxcr4<sup>1013</sup>- bearing mice deserves further investigations.  
+
+![PLACEHOLDER_6_0]
+
+<center>Figure 1: Reduced endothelial cells in the bone fraction of mutant mice. Absolute numbers of the indicated stroma cell subsets from bone fractions were determined by flow cytometry in WT, \(+ / 1013\) and 1013/1013 mice. Data (means + SEM) are from three independent experiments with 6 mice in total per group. \*, P < 0.05; and \*\*, P < 0.005 compared with WT cells. §§, P < 0.005 compared with \(+ / 1013\) cells. (as determined using the two-tailed Student's t test). </center>
+
+<--- Page Split --->
+
+2. "Figure 2N and 2S. Parameters of TBV and cortical bone should be quantified, preferable by \(\mu \mathrm{CT}\) (or quantitative histological analysis). At this moment, only 1 image per condition is shown and this is an Opn staining, which is not considered to be the appropriate approach for quantitative bone measurements. This quantification of bone parameters is especially necessary to verify the bone loss that occurs when recipient mice are WT (Figure 2S), as the bone loss that is induced by transplantation of mutant donor cells in WT recipient mice is hard to be explained only by a reduced number of SSC and OPC, as is now suggested."  
+
+We are grateful to the reviewer for this helpful suggestion and as requested, we have quantified trabecular and cortical bone parameters by \(\mu \mathrm{CT}\) (new Figures 20 and 2P and new supplemental Figure 1G). By this way, we confirmed the bone loss in WT recipient upon transplantation of mutant BM, thereby indicating cell-extrinsic (hematopoietic) Cxcr4-mediated regulation of the skeletal landscape. The text has been modified accordingly (page 11, line 230; page 9, line 183). One can speculate that myeloid cells including OCLs as well as lymphoid cells may actively participate in promoting bone remodeling in BM chimeric WT recipient mice. Indeed, the laboratory of Pr. A. Bozec among others recently reported that prolonged HIF- 1α signaling in B cells leads to enhanced RANKL production and OCL formation in the BM (Meng et al., Bone Research 2022). Likewise, BM T cells are known to produce RANKL and to regulate OCL compartment within the BM (see for review for instance Corrado et al., IJMS 2020; Mori et al., Clin Dev Immunol 2013; Zhang et al., Front Endocrinol 2020). Whether the transplantation of mutant BM recreates a pro- osteoclastogenic environment through a remodeling of the lymphoid compartment deserves further investigations. This point has now been discussed in the revised version of the manuscript (page 21, line 459).  
+
+3. "Figure 3. The authors suggest that the in vivo observed increase in osteoclasts in mutant mice is linked to an altered BM environment. To strengthen this statement, coculture experiments of osteogenic cells and osteoclast-precursors, in different combinations of WT vs mutant cells (treated with PTH, Pg or 1,25-vitamin D) should be performed. At this moment, the data only describe a discordance between the in vivo and in vitro findings, but do not allow to make any conclusion on whether the decrease in bone mass is partly caused by increased bone resorption."  
+
+We sincerely thank the reviewer for this very relevant and helpful comment. We fully agree with the point that making a link between osteogenic cells and osteoclast precursors is of importance. As recommended by the reviewer, we addressed it using a co- culture system between in vitro expanded osteogenic cells carrying or not the Cxcr4 mutation and WT OCL precursors, ie., BM CD11b+ myeloid cells. As shown in the new Figure 3L, mutant osteogenic cells promoted exacerbated OCL differentiation compared to WT cells. Soluble factors seem to be not sufficient to explain this bias as the supernatants of stimulated expanded osteogenic cells (WT or mutant) did not induce OCL differentiation. Additionally, transcriptomic analyses of stimulated osteogenic cells carrying or not the Cxcr4 mutation did not reveal any major changes in expression levels of master genes regulating osteoclastogenesis such as the RANKL/OPG balance or the M-Csf cytokine (see new Figure 3M). These findings suggest a juxtacrine function of osteogenic cells toward OCL differentiation that likely relies on direct interactions between both cell types and involves the Cxcl12/Cxcr4 axis. As adding the osteogenic component carrying the Cxcr4 mutation is sufficient to promote in vitro enhancement of OCL differentiation, we propose that the overall decrease in bone mass in mutant mice involves remodeling of osteogenic and osteoclastogenic components leading to decreased bone formation and increased bone resorption. Although the use of a conditional mutant mouse model would be the ideal way to confirm these findings, such a model is not currently available to our knowledge. In such a process, the osteogenic lineage would act as the driver and the OCL one as a passenger. The underlying molecular mechanism(s)
+
+<--- Page Split --->
+
+of this cross- talk remains to be elucidated, but seems to require direct contact between both cell types. The text has been modified accordingly (page 11, line 230; page 21, line 451).  
+
+4. "Figure 3. The histomorphometric data should be confirmed in more mice, as 3 mice per group for histomorphometric analysis is often not sufficient (Figure 3F-H). In addition, the bone formation data are puzzling, as osteoblast surface and osteoid surface are normal, but MS/BS and DB/BS are decreased. The authors interpret these data as a 'lower number of osteoblasts' but this statement does not fit with the normal osteoblast surface that is observed. Since dynamic bone formation parameters primarily measure the incorporation of minerals, these data might suggest that the formation of bone matrix by the osteoblasts is normal, but that the mineralisation of this bone matrix is impaired (and likely some osteoblasts are not mineralizing the matrix, whereas others show normal mineralization capacity as MAR is normal). Gene expression analysis might provide some more insight. The gene expression analysis (Figure 3J) is now restricted to genes that typify mature osteoblasts, but the expression of genes involved in mineralization is not analysed. In addition, the variation of the gene expression data reported in Figure 3J and M, is rather high and this quantification should be validated with qRT-PCR data and using more mice." As requested by the reviewer, histomorphometric and osteoclast data have been implemented by adding two to three mice per group. These results that are now displayed in Figure 3A-3E confirmed the previous ones, ie. increased OCL surface and number and decreased total and double labelled bone surfaces in mutant mice compared to WT ones. Mineral apposition rate was similar in WT and Cxcr4<sup>1013</sup>-bearing mice, while bone formation rate is lower in mutant mice. These data prompt us to suggest a decrease in bone formation related to a lower number of OBLs with maintained activity of each individual OBL. In line with preserved intrinsic bone formation capacities of active osteoblastic lineage cells in mutant mice, our RNA-seq analyses of bulks sorted from the bone fraction highlighted in mutant OPCs a gene signature with preserved mineralized matrix potential that has been confirmed by qPCR analyses (see new Figures 3F-H and S1K-M). In agreement, sorted OPCs from mutant mice were as efficient as WT ones in vitro at producing differentiated OBLs and mineralized nodules after 14- or 21- days culture in osteogenic medium as determined by Alkaline phosphatase and Alizarin Red staining respectively (see new Figures 3I and S1N). This was confirmed by qPCR analyses with no changes in expression of genes encoding osteogenic regulators in mutant cultures (see new Figure S1O). These findings are in line with efficient terminal osteogenic differentiation and preserved bone formation and mineralization capacities in Cxcr4<sup>1013</sup>-bearing mice. The text has been modified accordingly (page 11, lines 208 & 219).  
+
+5. "Figure 4. Panel D shows the relative expression of selected genes; are these the most differentially expressed genes between the different genotypes? To appreciate the importance of these differences, it will be important to provide also the unbiased ranked overview/list of pathways which differ the most between genotypes, based on genes involved. In addition, since mutant SSC maintain their potential to differentiate normally to chondrocytes and adipocytes, are SOX9 and Pparg expression normal in mutant SSC? Furthermore, it remains hard to understand that a decrease in OPC number (Figure 2B, Figure 4I) does not affect osteoblast or osteoid surface. How do the authors reconcile these data?"  
+
+We sincerely thank the reviewer for bringing to our attention that unbiased transcriptomic analyses of WT and mutant SSC are needed. As requested, we investigated the impact of the gain- of- Cxcr4- function on the molecular identity of SSCs by performing RNA- seq analyses of sorted bulk cells from WT and mutant bone fractions. Biological processes related to cell cycle and osteogenic differentiation were significantly modulated in 1013/1013 SSCs compared to WT SSCs as determined by GSEA (Gene set enrichment analysis) (see new Figure 4C). The gene signature related to cell cycle progression and regulation was reduced in 1013/1013 SSCs compared to WT
+
+<--- Page Split --->
+
+ones (see new Figures S2A and S2B). Likewise, genes related to osteogenic differentiation appeared to be decreased in mutant SSCs (see new Figures 4D and 4E). In contrast, key genes involved in both adipogenesis and chondrogenesis were not differentially expressed in mutant SSCs (see new Figure S2C). These results were confirmed by microfluidic- based multiplex gene expression analyses (see Figures 4F and 4G and Figure S2D), thus suggesting that proper Cxcr4 signaling is required for regulating osteogenic specification of SSCs at the transcriptional level. The text has been modified accordingly (page 13, line 264). Regarding the last point about our flow- cytometric and histomorphometric results, we agree that decreased OPC number cannot fully be explained by the unremarkable osteoid and osteoblast number. We therefore measured the labelled surfaces and MAR and also calculated the bone formation rate, which are more accurate indices of dynamic bone formation. Indeed, labelled surfaces and bone formation rate are decreased, which is in favor of reduced OBL differentiation, while the MAR remained identical, thus suggesting a maintained capacity of osteoblast to produce matrix once differentiated.  
+
+6. "Figure 5 and 3. The data suggest that osteogenic differentiation starting from mutant SSC is reduced (Figure 5E-G), but once mutant SSC become OPC they can differentiate normally (Figure 3L). It should be good to confirm this observation, by performing the same assays on OPC as shown for SSC (Figure 5: differentiation with Alp quantification and gene expression analysis). In addition, it is rather particular that after 21 days of osteogenic differentiation, most of the cells are still SSC (Figure S1), and intermediate cells account only for 15% of the population, whereas the % of ALP+ cells, reported in Figure 5E, seems much higher. Same comment for the low % of mature cells compared to reported homogeneous and abundant alizarin red staining (Figure 5F)."  
+
+We are grateful to the reviewer for this constructive suggestion and as requested we performed Alp quantification and gene expression analyses as already explained in response to the point#4 above. Our novel data showed that sorted OPCs from mutant mice were as efficient as WT ones in vitro at generating bone- making OBLs after 14- days culture in osteogenic medium as determined by Alp staining (see new Figure S1N). This was further confirmed by qPCR analyses with no changes in expression of genes encoding osteogenic regulators in mutant cultures (see new Figure S1O). These findings are in line with efficient terminal osteogenic differentiation and preserved bone formation and mineralization capacities in Cxcr4<sup>1013</sup>- bearing mice. The text has been modified accordingly (page 11, line 219). We fully agree with the reviewer that the yield of immature and mature osteogenic cells recovered by flow cytometry was not as high as expected in light of Alp and Alizarin red staining, and this was likely due to the difficulty we experimented to collect and separate homogenously the cells from the mineralized matrix at the end of the culture. Although real- time quantitative PCR analyses of Sca- 1 and PDGFRα markers corroborated the flow cytometric results (see Figure S2E), these flow cytometric results are rather dispensable for the paper and therefore we propose to remove them to clarify the message. We thank the reviewer for having pointed this inconsistency.  
+
+7. "Figure 5 in vivo data. The authors state that especially the cortical bone is rescued in mutant mice, but not the trabecular phenotype, based on lumbar spine BMD data. To validate this statement, μCT analysis of cortex of long bones should be analysed with and without AMD3100 treatment. In addition, these data also suggest that CXCR4 desensitisation in osteogenic lineage cells is likely not responsible for the trabecular bone phenotype, and that other cell types/mechanisms might be involved. This site-specificity should be reflected in the title and in the abstract."  
+
+We thank the reviewer for this relevant comment. Our original version of the manuscript stated a suggestion for a correcting effect of Cxcr4- dependent signaling dampening on the cortical, rather than trabecular, bone based on BMD values of lumbar spine in mutant mice. Because μCT analyses were not carried out for this experiment, we sought to measure the cortical thickness in paraffin- embedded sections stained with Toluidine Blue. The two cortices were measured using
+
+<--- Page Split --->
+
+histomorphometry software and expressed as mean of both cortices for each sample. As shown in the new Figure 5L, AMD3100 treatment for 3 weeks did not ameliorate the cortical network in mutant mice, thus suggesting that either the treatment procedure should be further optimized in terms of duration and kinetics or, as anticipated by the reviewer, that other cell types/mechanisms might be involved at this stage such as OCLs or perivascular SSCs as recently reported by Jeffery and coll. (Cell Stem Cell, 2022). This warrants further investigations. This point has been mentioned in the revised version of the manuscript (page 16, line 345).  
+
+## Minor concerns:  
+
+1. "Perilipin staining should be quantified as the observation that CXCR4 specifically reduces the osteogenic, but not the adipogenic differentiation is interesting, but should be validated by quantitative data."  
+
+We thank the reviewer for bringing to our attention that quantification data would be helpful to strengthen the significance of our findings. As requested, Figure 1H mentioned by the reviewer has been edited with quantification data and shows no change in adipocyte content in the BM of mutant mice, as compared to WT mice (see new Figure 1J). Congruent with immunostaining on bone sections, RNA-seq analyses performed during the reviewing period show that mutant SSCs displayed a gene signature consistent with preserved adipogenic potential (see new Figures S2C and S2D). These cells also differentiated into adipocytes similarly to WT SSCs when cultured in vitro in adipogenic medium (Figure S2G). These results suggest that proper Cxcr4 signaling is required for regulating the osteogenic specification of SSCs specifically. The text has been modified accordingly (page 7, line 130; page 13, line 264, page 15 line 316).  
+
+2. "Figure 1D: it is not clear whether the total number of mice used is 7-14, coming from 3 experiments, or that in each of the 3 experiments there were 7-14 mice, thus 21-42 mice in total. Similar comment to all experiments using mice."  
+
+We thank the reviewer for bringing to our attention that the total number of mice used in each experiment was not clear and we apologize for that. In fact, each number mentioned represents the total number of mice used, in 3 independent experiments or more. The legends have been modified accordingly.  
+
+3. "Figure 2J: the % of apoptotic OPC is around 30%, which is rather high, and should be commented on."  
+
+We thank the reviewer for this relevant comment. Although the reason why the apoptosis rate is high among OPCs is unclear, we obtained similar results using cleaved caspase 3 staining instead of Annexin V staining. One can speculate that experimental procedures make these cells more fragile and prone to undergo apoptosis. In both assays, we were unable to observe differences between WT and mutant OPCs, thus strongly suggesting that increased apoptosis of OPCs does not contribute to bone loss in mutant mice. As requested by the Reviewer#4, this panel has been moved to the supplemental Figure 1 (see Figure S1F).  
+
+## Reviewer #2 comments:  
+
+WHIM syndrome (WS) is a rare immunodeficiency caused by gain- of- function CXCR4 mutations. The authors have demonstrated for the first time a substantial decrease in bone mineral density in \(25\%\) of WS patients and osteoporosis in a WS mouse model. Interestingly, wild- type mice transplanted with bone marrow hematopoietic cells from mice with a WS- linked CXCR4 mutation (Cxcr4+/1013 or Cxcr4 1013/1013) had reduced trabecular bone content compared with wild- type chimeras. On the other hand,
+
+<--- Page Split --->
+
+transplantation of wild- type bone marrow cells did not rescue the reduced trabecular bone content in the mutant chimeras. Osteogenic differentiation of cultured bone marrow skeletal stem cells (SSCs) from the mutants was impaired in vitro. The CXCR4 antagonist AMD3100 normalized in vitro osteogenic potential of SSCs and reversed an in vivo decrease in Sca- 1- PDGFRa- cells in the mutants. These results are interesting and important; however the major concern remains at this time. There is the possibility that osteopenia in mice and patients, which carry the WS- linked CXCR4 mutation, is the result of only enhanced osteoclast function but not reduced osteogenic differentiation of SSCs.  
+
+## Major concerns:  
+
+1. "As the authors described, it has been shown previously that deletion of CXCR4 in mesenchymal cells, including SSCs, resulted in osteopenia (Tzeng et al., J. Bone Miner. Res. 2018; Zhu et al., J. Biol/ Chem. 2011). These results argue against the authors' conclusion that gain-of-function CXCR4 mutations in SSCs resulted in osteopenia. Thus, I would recommend the authors to generate and analyze the mice, in which mesenchymal cells, including SSCs, but not hematopoietic cells carry WS-linked CXCR4 mutations."  
+
+We are grateful to the reviewer for this relevant and constructive comment. Indeed, truncating mutations in CXCR4 which cause the WHIM syndrome (WS) in humans lead in vitro to a typical gain- of- function response to CXCL12 as exemplified by enhanced chemotaxis. However, in several cellular contexts (e.g., HSC lymphoid differentiation, B cell development...), we observed that loss of CXCR4 and gain of function of CXCR4 translated into similar phenotypes. This likely relates to the intensity and the strength of CXCR4 signaling that should be tightly regulated to permit the occurrence of physiological functions. Our findings unveil that mutant SSCs from the bone fraction are impaired in their capacities to generate OBLs as illustrated notably in vitro thus implying a cell- autonomous effect of the Cxcr4 mutation in the bone phenotype. In line with this, J. Pereira's laboratory recently showed using a second mouse model of the WS, carrying the gain- of- function CXCR4 R334X mutation, that lymphopoiesis is reduced because of a dysregulated transcriptome of mesenchymal stem cell isolated from the flushed marrow fraction and characterized by a switch from an adipogenic to an osteolineage- prone program with limited lymphopoietic activity (Zehentmeier et al., Science Immunology 2022). These results agree with ours and suggest that both hematopoietic and stromal cells are affected by the Cxcr4 gain of function mutation. The text has been modified accordingly (page 5, line 90; page 19, line 403).  
+
+Our reciprocal BM reconstitution experiments support this assumption since transplantation of WT BM into lethally irradiated mutant recipients was not sufficient to rescue the skeletal landscape phenotype, and conversely, transplantation of mutant BM induced bone dysregulation in WT recipient (see Figures 3E- P and S1G). Although we are aware of the fact that BM chimera do not constitute perfect models, we do believe they are informative notably when hematopoietic cells that are engrafted do not carry WS- linked CXCR4 mutations. Moreover, we think that our ubiquitous mouse model is relevant since it closely phenocopies the immune- hematological phenotype of the human pathology in which both hematopoietic and stromal cells harbor the Cxcr4 mutation. To confirm that, a conditional mouse model would have been ideal and not beyond the scope but we are not aware that such a model exists and it was not feasible de novo in the frame of a reviewing period. Rather, as suggested by the Reviewer#1, we set- up a co- culture system between in vitro expanded osteogenic cells carrying or not the Cxcr4 mutation and WT OCL precursors, ie., BM CD11b+ myeloid cells. As shown in the new Figure 3L, mutant osteogenic cells promoted exacerbated OCL differentiation compared to WT cells. Soluble factors do not seem sufficient as the supernatants of such stimulated expanded osteogenic cells (WT or mutant) did not induce OCL differentiation. Additionally, transcriptomic analyses of stimulated osteogenic cells carrying or not the Cxcr4 mutation did not reveal any major changes in expression levels of master genes regulating osteoclastogenesis such as the RANKL/OPG
+
+<--- Page Split --->
+
+balance (see new Figure 3M). These findings suggest a juxtacrine function of osteogenic cells toward OCL differentiation that likely relies on direct interactions between both cell types and involves the Cxcl12/Cxcr4 axis. As adding the osteogenic component carrying the Cxcr4 mutation is sufficient to promote in vitro enhancement of OCL differentiation, we propose that the overall decrease in bone mass in mutant mice involves remodeling of osteogenic and osteoclastogenic components leading to decreased bone formation and increased bone resorption. In such a process, the osteogenic lineage would act as the driver and the OCL one as a passenger. The underlying molecular mechanism(s) of this cross- talk remains to be elucidated but seems to require direct contact between both cell types. The entire manuscript as well as the title have been modified accordingly (page 11, line 230; page 21, lines 451 & 459) and a graphical abstract has been designed consequently.  
+
+2. "The authors show the reduced trabecular bone content of mice with a WS-linked CXCR4 mutation transplanted with bone marrow hematopoietic cells from wild-type mice was not rescued 3 and 16 weeks after transplantation. However, wild-type hematopoietic cells might be able to rescue the reduced trabecular bone content of the mutants earlier in development."  
+
+We thank the reviewer for this relevant comment. However, we have to stress that currently we do not have the ethical authorization to transplant BM into mice younger than seven/eight weeks but we are aware that it would be interesting to do it. This has been clearly mentioned in the revised version of the manuscript (page 9, line 175).  
+
+3. "The authors describe Sca-1+PDGFRa+ cells as SSCs (Page 8, line 142); however, the major population of bone marrow SSCs is defined as Sca-1-PDGFra+PDGFRb+LePr+CD31-cells (Omatsu et al., Immunity 2010; Zhou et al., Cell Stem Cell 2014; Seike et al., Genes Dev 2018)."  
+
+We are grateful to the referee for pointing out that the phenotype of SSCs we used could be a matter of debate and should be better justified. To the best of our knowledge, there is currently no consensual denomination for the different BM mesenchymal subpopulations and we agree with the reviewer that we should have been more precise on this point. As shown in the paper of Zhou et al. (Cell Stem Cell, 2014), the highest CFU- F clonogenic potential is observed in the Sca1+PDGFRa+ subset and not in the Sca1- PDGFRa+ population. This has been confirmed and extended to SSCs in the periosteum (Jeffery et al., Cell Stem Cell, 2022). Furthermore, 16wks after transplantation of GFP+ Sca1+PDGFRa+ into WT mice (Morikawa et al., JEM 2009), it was shown that among the GFP+ cells recovered, a few were Sca1+PDGFRa+ and most of them were Sca1- PDGFRa+, indicating that Sca1+PDGFRa+ cells are at the top of the hierarchy. This is why we chose to consider the Sca1+PDGFRa+ cells in the bone fraction as skeletal stem cells as compared to Sca1- PDGFRa+ that are more engaged in differentiation, as osteoblast progenitor cells. This point has been mentioned in the revised version of the manuscript (page 8, line 146).  
+
+4. "The evidence that Sca-1-PDGFra- cells are committed osteoblasts (OPCs) in the bone marrow would not be convincing (Page 8, line 143)."  
+
+We apologize for the lack of clarity with this sentence. As explained in the point 3, we consider the CD51+Sca1- population as more differentiated than its Sca1+ counterpart and the sentence has been modified accordingly (page 8, line 146). There was also a typo and we should have referred to the CD51+Sca1- population as PDGFRa+/- as it includes both PDGFRa positive and negative subsets. In line with this, we already consider early OPCs with multipotent adipo/osteogenic potential in the flushed stromal marrow fraction as Sca-1- negative and PDGFRa- positive (see new Figure 4M). Our previous results showed that the Sca1- PDGFRa- population highly express committed osteoblast markers such as Bglap, Col1a1 and Pth1r1 (see Balzano et al., Cell Reports 2019).
+
+<--- Page Split --->
+
+## Reviewer #4 comments:  
+
+The manuscript by Anginot and colleagues provides novel insights into the role of CXCR4- mediated signaling in skeletal stromal/stem cell osteogenic specification. The authors describe a series of experiments characterizing the anatomic, developmental and functional properties of the skeletal and osteogenic compartment in a knock- in mouse model of the human genetic disorder WHIM syndrome. The significance of the deficits in skeletal remodeling and stem cell differentiation identified in the mouse model in human bone biology are validated in cohort of WHIM syndrome patients carrying gain- of- function mutations in CXCR4. These findings represent a novel contribution elucidating an important new role for CXCR4 in bone biology.  
+
+## Major concerns:  
+
+1. "The authors characterized the effects of increased CXCR4 signaling in vivo through standard histomorphometric of bone anatomy and flow cytometric analyses of various progenitor cell populations in the mouse model. The data in Figure 1 are well presented and convincing in regard to the gene-dose dependent skeletal effects as well as the specificity of the changes to cortical and trabecular bone. Figure 2 is overly dense and contains information that could be moved to the supplement without impacting the major findings of the work. In particular, the experiments demonstrating the functional effects of the mutant CXCR4 receptor recapitulate characteristics of CXCR4 C-terminal truncations that have been well studied in other contexts. It would suffice to state that the mutant receptor localization, internalization and intracellular signaling were similar to what has been seen in other contexts and move panels 2E-J to the supplement. The bone marrow reconstitution experiments shown in the remainder of the figure demonstrate clearly the contribution of cell-extrinsic as well as cell-intrinsic factors to the observed skeletal changes. Similarly, the effects on bone resorption and formation shown in Figure 3 panels C-E can be moved to supplement to better focus on the transcriptional effects shown in the subsequent panels."  
+
+We are grateful to the reviewer for these constructive suggestions and as requested, the panels 2E- J and 3C- E have been moved to the new Supplemental Figure 1 (see panels S1A- S1F and S1H- S1J).  
+
+2. "The data in Figures 4 and 5 provide compelling data regarding the impact of aberrant CXCR4 signaling on osteogenic specification at the level of transcriptional effects and cell cycle progression. The PCA data shown in Figure 4C is not well explained as the 48 genes used for expression profiling are not described in the text nor the supplement, which lists a smaller number of genes. The data in the subsequent panels are more informative. I would consider removing panel 4C or moving it to the supplement with a better description of the analysis. The experiments shown in Figure 5 document the selective reduction in osteogenic differentiation capacity of stromal stem cells carrying one or two mutant CXCR4 receptors in a dose dependent fashion and the reversal of this phenotype with treatment of the receptor inhibitor AMD3100. The relevance of these data in mice to human bone biology are supported with the data shown in Figure 6 which revealed a selective osteogenic differentiation defect in bone marrow cells derived from WHIM syndrome patients."  
+
+We thank the reviewer for bringing to our attention that the PCA data shown in Figure 4C was not clear and we apologize for that. This panel has now been removed. As suggested by Reviewer#1, we decided to investigate the impact of the gain- of- Cxcr4- function on the molecular identity of SSCs by RNA- seq analyses of sorted bulk cells from WT and mutant bone fractions. Biological processes related to cell cycle and osteogenic differentiation were significantly modulated in
+
+<--- Page Split --->
+
+1013/1013 SSCs as determined by GSEA (Gene set enrichment analysis) (see new Figure 4C). The SSC signature in 1013/1013 mice was reduced for genes related to cell cycle progression and regulation (see new Figures S2A and S2B). Likewise, genes related to osteogenic differentiation appeared to be decreased in mutant SSCs (see new Figures 4D and 4E). In contrast, key genes involved in both adipogenesis and chondrogenesis were not differentially expressed in mutant SSCs (see new Figure S2C). These results were confirmed by microfluidic-based multiplex gene expression analyses (see Figures 4F and 4G and Figure S2D), thus suggesting that proper Cxcr4 signaling is required for regulating osteogenic specification of SSCs at the transcriptional level. The text has been modified accordingly (page 13, line 264).  
+
+3. "With regards to the conclusion that a skeletal phenotype is present in a subset of WHIM syndrome patients, given that treatment of neutropenia with G-CSF is associated with osteopenia as side effect of therapy, it would be useful to know the total number of patients treated with G-CSF in the cohort to address the concern that the enrichment in osteopenic patients is restricted to those patients that have been so treated as well as their ages and genders given the impact of these variables on risk for osteopenia in general."  
+
+We thank the reviewer for this very relevant comment. Nineteen WS patients had a baseline bone density scan as part of a drug treatment trial (NCT02231879) comparing 1 year of twice daily filgrastim (Neupogen) versus plerixafor (Mozobil) in a randomized, blinded crossover design. There were 13 women and 6 men with an average age of 30.5 years (range 10- 56). Patients had been on filgrastim for an average of 5.7 years prior to enrolling in the trial (range 0- 27). Six of the 19 had not used filgrastim regularly prior to trial enrollment. These findings suggest that the enrichment in osteopenic WS patients is not merely due to treatment regimen, age or gender parameters. This point is now mentioned in the revised version of the manuscript (page 22, line 487; page 23, line 507).  
+
+4. "Apart from these concerns, the quality of the data presented is good and the conclusions supported by the evidence. The manuscript is well written and the references appropriate, though it was notable that the initial description of the cause of WHIM syndrome as gain-of-function truncation mutations in CXCR4 was not cited, this should be added."  
+
+We thank the reviewer for this relevant comment and apologize for this oversight. The initial description of inherited CXCR4 mutations in the WS has been reported by Hernandez and collaborators in 2003 (Nature Genetics, PMID: 12692554). The appropriate reference (n°50 in the list of references) has been added accordingly (page 5, line 98).
+
+<--- Page Split --->
+
+## REVIEWER COMMENTS  
+
+## Reviewer #1 (Remarks to the Author):  
+
+The authors answered adequately to the comments and questions by performing additional experiments and adapting the text. The claims are now well supported by their findings, making it an interesting study providing further insight in the skeletal effects of CXCR4 mutations.  
+
+Minor comments:  
+
+Page 11, line 208: the following sentence is difficult to interpret: Cxcr41013- bearing mice exhibited unremarkable bone formation. Not clear what is meant by 'unremarkable'.  
+
+Page 11, line 225: It is mentioned that OBL differentiation is reduced in mutant mice, whereas the previous lines describe normal osteogenic differentiation when cultures are started with OPCs. To avoid misunderstanding, some other wording should be used to describe that the transition of SSCs to OPCs is impaired or that there is reduced osteogenic lineage commitment.  
+
+## Reviewer #2 (Remarks to the Author):  
+
+The authors have given a satisfactory response to some of this reviewer's concerns, improving the manuscript. However, their answers to several issues remain incomplete, and therefore their conclusions are still not convincing.  
+
+1. The new data that transplantation of Cxcr4 1013/1013 mutant bone marrow cells markedly reduced trabecular bone content (BV/TV and Tb.Nb) of wild-type recipient mice (Fig. 2P) are interesting and important. The magnitude of the decrease seems to be much larger compared with Cxcr4 1013/1013 mutant mice, suggesting that microenvironments with gain-of-function Cxcr4 1013/1013 mutations increased and rescued trabecular bone content. This is consistent with previous findings that deletion of CXCR4 in mesenchymal cells reduced trabecular bone content (Tzeng et al., J. Bone Miner. Res. 2018; Zhu et al., J. Biol. Chem. 2011).  
+
+2. Again I would recommend the authors to generate and analyze the mice, in which mesenchymal cells, including SSCs, but not hematopoietic cells carry WS-linked CXCR4 mutations. However, the authors mentioned that it was not feasible in the frame of a reviewing period. Then, the authors should at least show trabecular bone content (BV/TV and Tb.Nb) of Cxcr4 1013/1013 mutant mice transplanted with bone marrow cells from wild-type mice and compare the results with those of wild-type mice transplanted with mutant bone marrow cells.
+
+<--- Page Split --->
+
+## Reviewer #4 (Remarks to the Author):  
+
+The authors have addressed all of the issues raised by me adequately. I am satisfied with the responses to the other reviewers as well and have no additional concerns.
+
+<--- Page Split --->
+
+## Point-to-point response to the reviewers' comments  
+
+## Reviewer #1 comments:  
+
+The authors answered adequately to the comments and questions by performing additional experiments and adapting the text. The claims are now well supported by their findings, making it an interesting study providing further insight in the skeletal effects of CXCR4 mutations.  
+
+## Minor comments:  
+
+1. Page 11, line 208: the following sentence is difficult to interpret: Cxcr41013-bearing mice exhibited unremarkable bone formation. Not clear what is meant by 'unremarkable'.  
+
+We thank the reviewer for bringing to our attention that the use of the term "unremarkable" was not clear and probably not appropriate. The sentence has been modified as follow: "Cxcr41013- bearing mice exhibited similar bone formation as revealed by osteoid surface (OS/BS) and osteoblast surface (Obl.S/BS) compared to WT mice (Fig. 3C)". The text has been modified accordingly (page 11, line 208).  
+
+2. Page 11, line 225: It is mentioned that OBL differentiation is reduced in mutant mice, whereas the previous lines describe normal osteogenic differentiation when cultures are started with OPCs. To avoid misunderstanding, some other wording should be used to describe that the transition of SSCs to OPCs is impaired or that there is reduced osteogenic lineage commitment.  
+
+We thank the reviewer for this very relevant comment. The sentence has been changed as follow: "These findings suggest reduced osteogenic lineage commitment in Cxcr41013-bearing mice". The text has been modified accordingly (page 11, line 224).  
+
+## Reviewer #2 comments:  
+
+The authors have given a satisfactory response to some of this reviewer's concerns, improving the manuscript. However, their answers to several issues remain incomplete, and therefore their conclusions are still not convincing.  
+
+1. The new data that transplantation of Cxcr4 1013/1013 mutant bone marrow cells markedly reduced trabecular bone content (BV/TV and Tb.Nb) of wild-type recipient mice (Fig. 2P) are interesting and important. The magnitude of the decrease seems to be much larger compared with Cxcr4 1013/1013 mutant mice, suggesting that microenvironments with gain-of-function Cxcr4 1013/1013 mutations increased and rescued trabecular bone content. This is consistent with previous findings that deletion of CXCR4 in mesenchymal cells reduced trabecular bone content (Tzeng et al., J. Bone Miner. Res. 2018; Zhu et al., J. Biol. Chem. 2011).  
+
+We thank the reviewer for these valuable remarks and suggestions. Although it is difficult to compare chimeric and steady state mice, especially considering the whole- body irradiation and the 4- month reconstitution period, it appears indeed that WT mice reconstituted with mutant BM display stronger trabecular bone defects than younger mutant mice at steady state. It is indeed possible that, as put by the reviewer, "microenvironments with gain- of- function Cxcr4 1013/1013 mutations increased and rescued trabecular bone content". Besides mesenchymal and osteolineage cells, the BM ecosystem contains other possible effector cells including hematopoietic and mature immune cells, but also some radiosistant endothelial cells, and other stromal cells such as adipocytes that all express the CXCR4 receptor, which emerge during bone development and reach homeostasis at the adult stage. One can assume that the gain of CXCR4 function might modulate, positively or negatively, one, or several, BM landscape component(s) that could balance the trabecular bone defect in 1013/1013 mice. This is indeed consistent with
+
+<--- Page Split --->
+
+previous works reporting a cell- autonomous Cxcl12- Cxcr4 signaling on the MSPC osteogenesis but not adipogenesis (Tzeng et al., J. Bone Miner. Res. 2018; Zhu et al., J. Biol. Chem. 2011). Future studies are necessary to identify such cells by tissue- specific CXCR4 targeting, as suggested by the reviewer. This point has been now discussed (page 21, line 469).  
+
+The magnitude of the bone loss in WT recipient upon transplantation of mutant BM is surprising but is indicative of cell- extrinsic hematopoietic- driven Cxcr4- mediated regulation of the skeletal landscape. This may be due to the presence of effector mature leukocytes in the BM graft as we suggested previously in response to the point 2 raised by Reviewer#1. Indeed, one can speculate that myeloid cells including OCL progenitors as well as lymphoid cells may actively participate in promoting bone remodeling in BM chimeric WT recipient mice. Indeed, the laboratory of Pr. A. Bozec among others recently reported that prolonged HIF- 1α signaling in B cells leads to enhanced RANKL production and OCL formation in the BM (Meng et al., Bone Research 2022). Likewise, BM T cells are known to produce RANKL and to regulate OCL compartment within the BM (see for review for instance Corrado et al., IJMS 2020; Mori et al., Clin Dev Immunol 2013; Zhang et al., Front Endocrinol 2020). Whether the transplantation of mutant BM recreates a pro- osteoclastogenic environment through a remodeling of the lymphoid compartment deserves further investigations.  
+
+2. Again I would recommend the authors to generate and analyze the mice, in which mesenchymal cells, including SSCs, but not hematopoietic cells carry WS-linked CXCR4 mutations. However, the authors mentioned that it was not feasible in the frame of a reviewing period. Then, the authors should at least show trabecular bone content (BV/TV and Tb.Nb) of Cxcr4 1013/1013 mutant mice transplanted with bone marrow cells from wild-type mice and compare the results with those of wild-type mice transplanted with mutant bone marrow cells.  
+
+We are grateful to the reviewer for this helpful suggestion and as requested, we have quantified trabecular bone parameters by \(\mu \mathrm{CT}\) (see new Figures 2H and 2I). These new analyses extend the flow- cytometric and histological ones and indicate a persistent bone loss in mutant recipient upon transplantation of WT BM, thereby supporting the hypothesis of a cell- intrinsic CXCR4- mediated regulation of the skeletal landscape. However, the extent of the bone loss appears to be less marked compared with WT recipients transplanted with mutant BM cells. As discussed above, this could rely on the modulatory effect due to the gain of CXCR4 function on one, or several, stromal component(s) that could compensate the trabecular bone defect particularly in 1013/1013 mice. As stated in the manuscript, these findings suggest that impaired CXCR4 desensitization in both skeletal and hematopoietic cells have combinatorial effects on bone landscape dysregulation in adult Cxcr4<sup>1013</sup>- bearing mice.  
+
+## Reviewer #4 comments:  
+
+The authors have addressed all of the issues raised by me adequately. I am satisfied with the responses to the other reviewers as well and have no additional concerns. We thank the reviewer for the previous suggestions and are happy that all concerns were addressed.
+
+<--- Page Split --->
+
+## REVIEWERS' COMMENTS  
+
+## Reviewer #2 (Remarks to the Author):  
+
+The authors have given a satisfactory response to this reviewer's concerns, improving the manuscript.  
+
+Minor pointsFigure 21: How about p- values in Tb.Nb? They appear to be significantly less than 0.005 as seen in BV/TV.
+
+<--- Page Split --->
+
+## Point-to-point response to the reviewers' comments  
+
+Reviewer #2 comments: The authors have given a satisfactory response to this reviewer's concerns, improving the manuscript.  
+
+Minor points Figure 2l: How about p-values in Tb.Nb? They appear to be significantly less than 0.005 as seen in BV/TV.  
+
+We thank the reviewer for his/her previous constructive comments that have helped us improving greatly the quality of our manuscript. We are glad to read that all concerns were now addressed. Regarding the p- values of the data shown in Figure 2l, they have been determined using the twotailed Student's t test and are as follow:  
+
+For the BV/TV parameter: WT BM- chimeric \(\mathsf{CD45.2 + }\) WT vs WT BM- chimeric \(\mathsf{CD45.2 + }\) +/1013 mice: \(\mathsf{P} = 0.033\) WT BM- chimeric \(\mathsf{CD45.2 + }\) WT vs WT BM- chimeric \(\mathsf{CD45.2 + }\) 1013/1013 mice: \(\mathsf{P} = 0.0244\) For the Tb.Nb parameter: WT BM- chimeric \(\mathsf{CD45.2 + }\) WT vs WT BM- chimeric \(\mathsf{CD45.2 + }\) +/1013 mice: \(\mathsf{P} = 0.0702\) WT BM- chimeric \(\mathsf{CD45.2 + }\) WT vs WT BM- chimeric \(\mathsf{CD45.2 + }\) 1013/1013 mice: \(\mathsf{P} = 0.0710\)  
+
+The exact p- values have been now provided in the legend of Figure 2.
+
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+<|ref|>title<|/ref|><|det|>[[60, 40, 507, 90]]<|/det|>
+# nature portfolio  
+
+<|ref|>text<|/ref|><|det|>[[67, 111, 360, 140]]<|/det|>
+Peer Review File  
+
+<|ref|>text<|/ref|><|det|>[[83, 154, 920, 181]]<|/det|>
+WHIM Syndrome- linked CXCR4 mutations drive osteoporosis  
+
+<|ref|>image<|/ref|><|det|>[[56, 732, 240, 784]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[250, 732, 912, 784]]<|/det|>
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[118, 86, 348, 104]]<|/det|>
+## REVIEWER COMMENTS  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 150, 410, 166]]<|/det|>
+## Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[117, 185, 880, 322]]<|/det|>
+Anginot et al. report on the importance of CXCR4 desensitization in skeletal stem cells (SSCs) in order to allow SSCs to proliferate adequately and differentiate into the osteogenic lineage, whereas chondrogenic and adipogenic differentiation seem not to be affected by gain of function mutation of CXCR4. These novel data certainly increase our understanding on CXCR4 signalling in osteogenic lineage cells. In addition, the authors combined numerous well- designed in vivo and in vitro experiments to elucidate the cellular mechanisms. However, several inconsistencies between the findings are present, especially concerning the effect of CXCR4 desensitization on SSC properties and their osteogenic differentiation potential.  
+
+<|ref|>text<|/ref|><|det|>[[117, 339, 875, 441]]<|/det|>
+Figure 2B. The decrease in stroma cell number in mutant mice ( \(60 \times 10^{3}\) versus \(100 \times 10^{3}\) in WT mice) cannot be explained by the combined decrease in SSC ( \(3 \times 10^{3}\) versus \(5 \times 10^{3}\) ) and OPC ( \(7 \times 10^{3}\) versus \(13 \times 10^{3}\) ). The question is therefore which other bone cell types are decreased in mutant mice as these other cell types might also contribute to the observed decrease in bone mass. Are endothelial cells decreased (H- type and L- type) in mutant mice as they express CXCR4 and might provide a vascular niche for the SSC?  
+
+<|ref|>text<|/ref|><|det|>[[117, 459, 878, 579]]<|/det|>
+Figure 2N and 2S. Parameters of TBV and cortical bone should be quantified, preferable by \(\mu \mathrm{CT}\) (or quantitative histological analysis). At this moment, only 1 image per condition is shown and this is an Opn staining, which is not considered to be the appropriate approach for quantitative bone measurements. This quantification of bone parameters is especially necessary to verify the bone loss that occurs when recipient mice are WT (Figure 2S), as the bone loss that is induced by transplantation of mutant donor cells in WT recipient mice is hard to be explained only by a reduced number of SSC and OPC, as is now suggested.  
+
+<|ref|>text<|/ref|><|det|>[[117, 597, 872, 700]]<|/det|>
+Figure 3. The authors suggest that the in vivo observed increase in osteoclasts in mutant mice is linked to an altered BM environment. To strengthen this statement, coculture experiments of osteogenic cells and osteoclast- precursors, in different combinations of WT vs mutant cells (treated with PTH, \(\mathrm{Pg}\) or 1,25- vitamin D) should be performed. At this moment, the data only describe a discordance between the in vivo and in vitro findings, but do not allow to make any conclusion on whether the decrease in bone mass is partly caused by increased bone resorption.  
+
+<|ref|>text<|/ref|><|det|>[[117, 718, 880, 906]]<|/det|>
+Figure 3. The histomorphometric data should be confirmed in more mice, as 3 mice per group for histomorphometric analysis is often not sufficient (Figure 3F- H). In addition, the bone formation data are puzzling, as osteoblast surface and osteoid surface are normal, but MS/BS and DB/BS are decreased. The authors interpret these data as a 'lower number of osteoblasts' but this statement does not fit with the normal osteoblast surface that is observed. Since dynamic bone formation parameters primarily measure the incorporation of minerals, these data might suggest that the formation of bone matrix by the osteoblasts is normal, but that the mineralisation of this bone matrix is impaired (and likely some osteoblasts are not mineralizing the matrix, whereas others show normal mineralization capacity as MAR is normal). Gene expression analysis might provide some more insight. The gene expression analysis (Figure 3J) is now restricted to genes that typify mature osteoblasts, but the expression of genes involved in mineralization is not analysed. In addition, the
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 84, 784, 118]]<|/det|>
+variation of the gene expression data reported in Figure 3J and M, is rather high and this quantification should be validated with qRT- PCR data and using more mice.  
+
+<|ref|>text<|/ref|><|det|>[[118, 135, 880, 273]]<|/det|>
+Figure 4. Panel D shows the relative expression of selected genes; are these the most differentially expressed genes between the different genotypes? To appreciate the importance of these differences, it will be important to provide also the unbiased ranked overview/list of pathways which differ the most between genotypes, based on genes involved. In addition, since mutant SSC maintain their potential to differentiate normally to chondrocytes and adipocytes, are SOX9 and Pparg expression normal in mutant SSC? Furthermore, it remains hard to understand that a decrease in OPC number (Figure 2B, Figure 4I) does not affect osteoblast or osteoid surface. How do the authors reconcile these data?  
+
+<|ref|>text<|/ref|><|det|>[[118, 290, 880, 428]]<|/det|>
+Figure 5 and 3. The data suggest that osteogenic differentiation starting from mutant SSC is reduced (Figure 5E- G), but once mutant SSC become OPC they can differentiate normally (Figure 3L). It should be good to confirm this observation, by performing the same assays on OPC as shown for SSC (Figure 5: differentiation with Alp quantification and gene expression analysis). In addition, it is rather particular that after 21 days of osteogenic differentiation, most of the cells are still SSC (Figure S1), and intermediate cells account only for \(15\%\) of the population, whereas the \(0\%\) of ALP+ cells, reported in Figure 5E, seems much higher. Same comment for the low \(0\%\) of mature cells compared to reported homogeneous and abundant alizarin red staining (Figure 5F).  
+
+<|ref|>text<|/ref|><|det|>[[118, 445, 875, 547]]<|/det|>
+Figure 5 in vivo data. The authors state that especially the cortical bone is rescued in mutant mice, but not the trabecular phenotype, based on lumbar spine BMD data. To validate this statement, \(\mu \mathrm{CT}\) analysis of cortex of long bones should be analysed with and without AMD3100 treatment. In addition, these data also suggest that CXCR4 desensitisation in osteogenic lineage cells is likely not responsible for the trabecular bone phenotype, and that other cell types/mechanisms might be involved. This site- specificity should be reflected in the title and in the abstract.  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 566, 251, 580]]<|/det|>
+## Minor comments  
+
+<|ref|>text<|/ref|><|det|>[[119, 582, 815, 633]]<|/det|>
+Perilipin staining should be quantified as the observation that CXCR4 specifically reduces the osteogenic, but not the adipogenic differentiation is interesting, but should be validated by quantitative data.  
+
+<|ref|>text<|/ref|><|det|>[[118, 652, 880, 702]]<|/det|>
+Figure 1D: it is not clear whether the total number of mice used is 7- 14, coming from 3 experiments, or that in each of the 3 experiments there were 7- 14 mice, thus 21- 42 mice in total. Similar comment to all experiments using mice.  
+
+<|ref|>text<|/ref|><|det|>[[115, 704, 875, 720]]<|/det|>
+Figure 2J: the \(0\%\) of apoptotic OPC is around \(30\%\) , which is rather high, and should be commented on.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[119, 85, 411, 100]]<|/det|>
+## Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[117, 118, 875, 342]]<|/det|>
+WHIM syndrome (WS) is a rare immunodeficiency caused by gain- of- function CXCR4 mutations. The authors have demonstrated for the first time a substantial decrease in bone mineral density in \(25\%\) of WS patients and osteoporosis in a WS mouse model. Interestingly, wild- type mice transplanted with bone marrow hematopoietic cells from mice with a WS- linked CXCR4 mutation (Cxcr4+/1013 or Cxcr4 1013/1013) had reduced trabecular bone content compared with wild- type chimeras. On the other hand, transplantation of wild- type bone marrow cells did not rescue the reduced trabecular bone content in the mutant chimeras. Osteogenic differentiation of cultured bone marrow skeletal stem cells (SSCs) from the mutants was impaired in vitro. The CXCR4 antagonist AMD3100 normalized in vitro osteogenic potential of SSCs and reversed an in vivo decrease in Sca- 1- PDGFRα- cells in the mutants. These results are interesting and important; however the major concern remains at this time. There is the possibility that osteopenia in mice and patients, which carry the WS- linked CXCR4 mutation, is the result of only enhanced osteoclast function but not reduced osteogenic differentiation of SSCs.  
+
+<|ref|>text<|/ref|><|det|>[[118, 359, 874, 461]]<|/det|>
+1. As the authors described, it has been shown previously that deletion of CXCR4 in mesenchymal cells, including SSCs, resulted in osteopenia (Tzeng et al., J. Bone Miner. Res. 2018; Zhu et al., J. Biol/ Chem. 2011). These results argue against the authors' conclusion that gain-of-function CXCR4 mutations in SSCs resulted in osteopenia. Thus, I would recommend the authors to generate and analyze the mice, in which mesenchymal cells, including SSCs, but not hematopoietic cells carry WS-linked CXCR4 mutations.  
+
+<|ref|>text<|/ref|><|det|>[[118, 479, 872, 547]]<|/det|>
+2. The authors show the reduced trabecular bone content of mice with a WS-linked CXCR4 mutation transplanted with bone marrow hematopoietic cells from wild-type mice was not rescued 3 and 16 weeks after transplantation. However, wild-type hematopoietic cells might be able to rescue the reduced trabecular bone content of the mutants earlier in development.  
+
+<|ref|>text<|/ref|><|det|>[[118, 564, 872, 615]]<|/det|>
+3. The authors describe Sca-1+PDGFRα+ cells as SSCs (Page 8, line 142); however, the major population of bone marrow SSCs is defined as Sca-1-PDGFRα+PDGFRb+LePR+CD31-cells (Omatsu et al., Immunity 2010; Zhou et al., Cell Stem Cell 2014; Seike et al., Genes Dev 2018).  
+
+<|ref|>text<|/ref|><|det|>[[118, 633, 840, 667]]<|/det|>
+4. The evidence that Sca-1-PDGFRα-cells are committed osteoblasts (OPCs) in the bone marrow would not be convincing (Page 8, line 143).
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[119, 85, 410, 100]]<|/det|>
+## Reviewer #4 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 135, 874, 273]]<|/det|>
+The manuscript by Anginot and colleagues provides novel insights into the role of CXCR4- mediated signaling in skeletal stromal/stem cell osteogenic specification. The authors describe a series of experiments characterizing the anatomic, developmental and functional properties of the skeletal and osteogenic compartment in a knock- in mouse model of the human genetic disorder WHIM syndrome. The significance of the deficits in skeletal remodeling and stem cell differentiation identified in the mouse model in human bone biology are validated in cohort of WHIM syndrome patients carrying gain- of- function mutations in CXCR4. These findings represent a novel contribution elucidating an important new role for CXCR4 in bone biology.  
+
+<|ref|>text<|/ref|><|det|>[[117, 290, 876, 530]]<|/det|>
+The authors characterized the effects of increased CXCR4 signaling in vivo through standard histomorphometric of bone anatomy and flow cytometric analyses of various progenitor cell populations in the mouse model. The data in Figure 1 are well presented and convincing in regard to the gene- dose dependent skeletal effects as well as the specificity of the changes to cortical and trabecular bone. Figure 2 is overly dense and contains information that could be moved to the supplement without impacting the major findings of the work. In particular, the experiments demonstrating the functional effects of the mutant CXCR4 receptor recapitulate characteristics of CXCR4 C- terminal truncations that have been well studied in other contexts. It would suffice to state that the mutant receptor localization, internalization and intracellular signaling were similar to what has been seen in other contexts and move panels 2E- J to the supplement. The bone marrow reconstitution experiments shown in the remainder of the figure demonstrate clearly the contribution of cell- extrinsic as well as cell- intrinsic factors to the observed skeletal changes. The Similarly, the effects on bone resorption and formation shown in Figure 3 panels C- E can be moved to supplement to better focus on the transcriptional effects shown in the subsequent panels.  
+
+<|ref|>text<|/ref|><|det|>[[117, 547, 878, 737]]<|/det|>
+The data in Figures 4 and 5 provide compelling data regarding the impact of aberrant CXCR4 signaling on osteogenic specification at the level of transcriptional effects and cell cycle progression. The PCA data shown in Figure 4C is not well explained as the 48 genes used for expression profiling are not described in the text nor the supplement, which lists a smaller number of genes. The data in the subsequent panels are more informative. I would consider removing panel 4C or moving it to the supplement with a better description of the analysis. The experiments shown in Figure 5 document the selective reduction in osteogenic differentiation capacity of stromal stem cells carrying one or two mutant CXCR4 receptors in a dose dependent fashion and the reversal of this phenotype with treatment of the receptor inhibitor AMD3100. The relevance of these data in mice to human bone biology are supported with the data shown in Figure 6 which revealed a selective osteogenic differentiation defect in bone marrow cells derived from WHIM syndrome patients.  
+
+<|ref|>text<|/ref|><|det|>[[118, 789, 880, 892]]<|/det|>
+With regards to the conclusion that a skeletal phenotype is present in a subset of WHIM syndrome patients, given that treatment of neutropenia with G- CSF is associated with osteopenia as side effect of therapy, it would be useful to know the total number of patients treated with G- CSF in the cohort to address the concern that the enrichment in osteopenic patients is restricted to those patients that have been so treated as well as their ages and genders given the impact of these variables on risk for osteopenia in general.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 84, 872, 152]]<|/det|>
+Apart from these concerns, the quality of the data presented is good and the conclusions supported by the evidence. The manuscript is well written and the references appropriate, though it was notable that the initial description of the cause of WHIM syndrome as gain- of- function truncation mutations in CXCR4 was not cited, this should be added.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[116, 63, 562, 80]]<|/det|>
+## Point-to-point response to the reviewers' comments  
+
+<|ref|>sub_title<|/ref|><|det|>[[117, 91, 322, 108]]<|/det|>
+## Reviewer #1 comments:  
+
+<|ref|>text<|/ref|><|det|>[[115, 117, 883, 264]]<|/det|>
+Anginot et al. report on the importance of CXCR4 desensitization in skeletal stem cells (SSCs) in order to allow SSCs to proliferate adequately and differentiate into the osteogenic lineage, whereas chondrogenic and adipogenic differentiation seem not to be affected by gain of function mutation of CXCR4. These novel data certainly increase our understanding on CXCR4 signalling in osteogenic lineage cells. In addition, the authors combined numerous well- designed in vivo and in vitro experiments to elucidate the cellular mechanisms. However, several inconsistencies between the findings are present, especially concerning the effect of CXCR4 desensitization on SSC properties and their osteogenic differentiation potential.  
+
+<|ref|>sub_title<|/ref|><|det|>[[116, 279, 256, 295]]<|/det|>
+## Major concerns:  
+
+<|ref|>text<|/ref|><|det|>[[115, 301, 883, 396]]<|/det|>
+1. "Figure 2B. The decrease in stroma cell number in mutant mice ( \(60 \times 10^{3}\) versus \(100 \times 10^{3}\) in WT mice) cannot be explained by the combined decrease in SSC ( \(3 \times 10^{3}\) versus 5 \(\times 10^{3}\) ) and OPC ( \(7 \times 10^{3}\) versus \(13 \times 10^{3}\) ). The question is therefore which other bone cell types are decreased in mutant mice as these other cell types might also contribute to the observed decrease in bone mass. Are endothelial cells decreased (H-type and L-type) in mutant mice as they express CXCR4 and might provide a vascular niche for the SSC?"  
+
+<|ref|>text<|/ref|><|det|>[[115, 396, 882, 733]]<|/det|>
+We are grateful to the reviewer for this constructive comment and agree that some populations are likely missing in our flow- cytometric analyses. In particular, we did not consider the CD51- Sca1- cell population which is non- hematopoietic (CD45- ) and non- vascular (CD31- ) but appeared to be decreased in an allele- dose dependent manner in mutant mice. Thus, this stromal population might contribute to the overall decrease in stroma cell number in mutant mice. Because we do not know anything about this population, we propose to remove the stroma quantification panel to better focus on SSCs and OPCs (new Figures 2A and 2B). Whether endothelial cell (EC) numbers are affected is an interesting question raised by the reviewer. Different types of bone marrow (BM) ECs have been phenotypically identified in long bones (see for instance Kusumbe Nature 2014; Balzano Cell Rep 2019). The bone fraction is reported to be enriched for arteriolar ECs (Sca1+CD31+Emcn- ), few L- type sinusoidal ECs and CD31hiEmcnhi H- type ECs, a small fraction of the ECs at the end of the CD31+Emcn- arteriolar network. Based on Sca1 and CD31 expression, we observed by flow cytometry a decrease in ECs in the bone fraction of mutant mice (see below Figure 1 for reviewers). Although these preliminary findings are very interesting, we feel that they deserve to be strengthened by adding notably the Endomucin marker to visualize by immunofluorescence the impact of the Cxcr4 mutation on H- type and L- type EC architecture and numbers. This would constitute the subject of another study that will be dedicated to vascular modifications in WS mice. However, the well- established regulatory role of the vascular system on the mesenchymal one has been discussed in the revised version of the manuscript (page 22, lines 474 and 482). In particular, whether vascular cells participate in the defective osteolineage specification of SSCs in Cxcr4<sup>1013</sup>- bearing mice deserves further investigations.  
+
+<|ref|>image<|/ref|><|det|>[[123, 754, 416, 888]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[488, 757, 858, 886]]<|/det|>
+<center>Figure 1: Reduced endothelial cells in the bone fraction of mutant mice. Absolute numbers of the indicated stroma cell subsets from bone fractions were determined by flow cytometry in WT, \(+ / 1013\) and 1013/1013 mice. Data (means + SEM) are from three independent experiments with 6 mice in total per group. \*, P < 0.05; and \*\*, P < 0.005 compared with WT cells. §§, P < 0.005 compared with \(+ / 1013\) cells. (as determined using the two-tailed Student's t test). </center>
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 78, 883, 208]]<|/det|>
+2. "Figure 2N and 2S. Parameters of TBV and cortical bone should be quantified, preferable by \(\mu \mathrm{CT}\) (or quantitative histological analysis). At this moment, only 1 image per condition is shown and this is an Opn staining, which is not considered to be the appropriate approach for quantitative bone measurements. This quantification of bone parameters is especially necessary to verify the bone loss that occurs when recipient mice are WT (Figure 2S), as the bone loss that is induced by transplantation of mutant donor cells in WT recipient mice is hard to be explained only by a reduced number of SSC and OPC, as is now suggested."  
+
+<|ref|>text<|/ref|><|det|>[[115, 207, 883, 450]]<|/det|>
+We are grateful to the reviewer for this helpful suggestion and as requested, we have quantified trabecular and cortical bone parameters by \(\mu \mathrm{CT}\) (new Figures 20 and 2P and new supplemental Figure 1G). By this way, we confirmed the bone loss in WT recipient upon transplantation of mutant BM, thereby indicating cell-extrinsic (hematopoietic) Cxcr4-mediated regulation of the skeletal landscape. The text has been modified accordingly (page 11, line 230; page 9, line 183). One can speculate that myeloid cells including OCLs as well as lymphoid cells may actively participate in promoting bone remodeling in BM chimeric WT recipient mice. Indeed, the laboratory of Pr. A. Bozec among others recently reported that prolonged HIF- 1α signaling in B cells leads to enhanced RANKL production and OCL formation in the BM (Meng et al., Bone Research 2022). Likewise, BM T cells are known to produce RANKL and to regulate OCL compartment within the BM (see for review for instance Corrado et al., IJMS 2020; Mori et al., Clin Dev Immunol 2013; Zhang et al., Front Endocrinol 2020). Whether the transplantation of mutant BM recreates a pro- osteoclastogenic environment through a remodeling of the lymphoid compartment deserves further investigations. This point has now been discussed in the revised version of the manuscript (page 21, line 459).  
+
+<|ref|>text<|/ref|><|det|>[[115, 462, 883, 576]]<|/det|>
+3. "Figure 3. The authors suggest that the in vivo observed increase in osteoclasts in mutant mice is linked to an altered BM environment. To strengthen this statement, coculture experiments of osteogenic cells and osteoclast-precursors, in different combinations of WT vs mutant cells (treated with PTH, Pg or 1,25-vitamin D) should be performed. At this moment, the data only describe a discordance between the in vivo and in vitro findings, but do not allow to make any conclusion on whether the decrease in bone mass is partly caused by increased bone resorption."  
+
+<|ref|>text<|/ref|><|det|>[[115, 575, 883, 878]]<|/det|>
+We sincerely thank the reviewer for this very relevant and helpful comment. We fully agree with the point that making a link between osteogenic cells and osteoclast precursors is of importance. As recommended by the reviewer, we addressed it using a co- culture system between in vitro expanded osteogenic cells carrying or not the Cxcr4 mutation and WT OCL precursors, ie., BM CD11b+ myeloid cells. As shown in the new Figure 3L, mutant osteogenic cells promoted exacerbated OCL differentiation compared to WT cells. Soluble factors seem to be not sufficient to explain this bias as the supernatants of stimulated expanded osteogenic cells (WT or mutant) did not induce OCL differentiation. Additionally, transcriptomic analyses of stimulated osteogenic cells carrying or not the Cxcr4 mutation did not reveal any major changes in expression levels of master genes regulating osteoclastogenesis such as the RANKL/OPG balance or the M-Csf cytokine (see new Figure 3M). These findings suggest a juxtacrine function of osteogenic cells toward OCL differentiation that likely relies on direct interactions between both cell types and involves the Cxcl12/Cxcr4 axis. As adding the osteogenic component carrying the Cxcr4 mutation is sufficient to promote in vitro enhancement of OCL differentiation, we propose that the overall decrease in bone mass in mutant mice involves remodeling of osteogenic and osteoclastogenic components leading to decreased bone formation and increased bone resorption. Although the use of a conditional mutant mouse model would be the ideal way to confirm these findings, such a model is not currently available to our knowledge. In such a process, the osteogenic lineage would act as the driver and the OCL one as a passenger. The underlying molecular mechanism(s)
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 62, 883, 97]]<|/det|>
+of this cross- talk remains to be elucidated, but seems to require direct contact between both cell types. The text has been modified accordingly (page 11, line 230; page 21, line 451).  
+
+<|ref|>text<|/ref|><|det|>[[114, 108, 883, 625]]<|/det|>
+4. "Figure 3. The histomorphometric data should be confirmed in more mice, as 3 mice per group for histomorphometric analysis is often not sufficient (Figure 3F-H). In addition, the bone formation data are puzzling, as osteoblast surface and osteoid surface are normal, but MS/BS and DB/BS are decreased. The authors interpret these data as a 'lower number of osteoblasts' but this statement does not fit with the normal osteoblast surface that is observed. Since dynamic bone formation parameters primarily measure the incorporation of minerals, these data might suggest that the formation of bone matrix by the osteoblasts is normal, but that the mineralisation of this bone matrix is impaired (and likely some osteoblasts are not mineralizing the matrix, whereas others show normal mineralization capacity as MAR is normal). Gene expression analysis might provide some more insight. The gene expression analysis (Figure 3J) is now restricted to genes that typify mature osteoblasts, but the expression of genes involved in mineralization is not analysed. In addition, the variation of the gene expression data reported in Figure 3J and M, is rather high and this quantification should be validated with qRT-PCR data and using more mice." As requested by the reviewer, histomorphometric and osteoclast data have been implemented by adding two to three mice per group. These results that are now displayed in Figure 3A-3E confirmed the previous ones, ie. increased OCL surface and number and decreased total and double labelled bone surfaces in mutant mice compared to WT ones. Mineral apposition rate was similar in WT and Cxcr4<sup>1013</sup>-bearing mice, while bone formation rate is lower in mutant mice. These data prompt us to suggest a decrease in bone formation related to a lower number of OBLs with maintained activity of each individual OBL. In line with preserved intrinsic bone formation capacities of active osteoblastic lineage cells in mutant mice, our RNA-seq analyses of bulks sorted from the bone fraction highlighted in mutant OPCs a gene signature with preserved mineralized matrix potential that has been confirmed by qPCR analyses (see new Figures 3F-H and S1K-M). In agreement, sorted OPCs from mutant mice were as efficient as WT ones in vitro at producing differentiated OBLs and mineralized nodules after 14- or 21- days culture in osteogenic medium as determined by Alkaline phosphatase and Alizarin Red staining respectively (see new Figures 3I and S1N). This was confirmed by qPCR analyses with no changes in expression of genes encoding osteogenic regulators in mutant cultures (see new Figure S1O). These findings are in line with efficient terminal osteogenic differentiation and preserved bone formation and mineralization capacities in Cxcr4<sup>1013</sup>-bearing mice. The text has been modified accordingly (page 11, lines 208 & 219).  
+
+<|ref|>text<|/ref|><|det|>[[115, 637, 883, 783]]<|/det|>
+5. "Figure 4. Panel D shows the relative expression of selected genes; are these the most differentially expressed genes between the different genotypes? To appreciate the importance of these differences, it will be important to provide also the unbiased ranked overview/list of pathways which differ the most between genotypes, based on genes involved. In addition, since mutant SSC maintain their potential to differentiate normally to chondrocytes and adipocytes, are SOX9 and Pparg expression normal in mutant SSC? Furthermore, it remains hard to understand that a decrease in OPC number (Figure 2B, Figure 4I) does not affect osteoblast or osteoid surface. How do the authors reconcile these data?"  
+
+<|ref|>text<|/ref|><|det|>[[115, 782, 883, 895]]<|/det|>
+We sincerely thank the reviewer for bringing to our attention that unbiased transcriptomic analyses of WT and mutant SSC are needed. As requested, we investigated the impact of the gain- of- Cxcr4- function on the molecular identity of SSCs by performing RNA- seq analyses of sorted bulk cells from WT and mutant bone fractions. Biological processes related to cell cycle and osteogenic differentiation were significantly modulated in 1013/1013 SSCs compared to WT SSCs as determined by GSEA (Gene set enrichment analysis) (see new Figure 4C). The gene signature related to cell cycle progression and regulation was reduced in 1013/1013 SSCs compared to WT
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 62, 883, 272]]<|/det|>
+ones (see new Figures S2A and S2B). Likewise, genes related to osteogenic differentiation appeared to be decreased in mutant SSCs (see new Figures 4D and 4E). In contrast, key genes involved in both adipogenesis and chondrogenesis were not differentially expressed in mutant SSCs (see new Figure S2C). These results were confirmed by microfluidic- based multiplex gene expression analyses (see Figures 4F and 4G and Figure S2D), thus suggesting that proper Cxcr4 signaling is required for regulating osteogenic specification of SSCs at the transcriptional level. The text has been modified accordingly (page 13, line 264). Regarding the last point about our flow- cytometric and histomorphometric results, we agree that decreased OPC number cannot fully be explained by the unremarkable osteoid and osteoblast number. We therefore measured the labelled surfaces and MAR and also calculated the bone formation rate, which are more accurate indices of dynamic bone formation. Indeed, labelled surfaces and bone formation rate are decreased, which is in favor of reduced OBL differentiation, while the MAR remained identical, thus suggesting a maintained capacity of osteoblast to produce matrix once differentiated.  
+
+<|ref|>text<|/ref|><|det|>[[115, 286, 884, 431]]<|/det|>
+6. "Figure 5 and 3. The data suggest that osteogenic differentiation starting from mutant SSC is reduced (Figure 5E-G), but once mutant SSC become OPC they can differentiate normally (Figure 3L). It should be good to confirm this observation, by performing the same assays on OPC as shown for SSC (Figure 5: differentiation with Alp quantification and gene expression analysis). In addition, it is rather particular that after 21 days of osteogenic differentiation, most of the cells are still SSC (Figure S1), and intermediate cells account only for 15% of the population, whereas the % of ALP+ cells, reported in Figure 5E, seems much higher. Same comment for the low % of mature cells compared to reported homogeneous and abundant alizarin red staining (Figure 5F)."  
+
+<|ref|>text<|/ref|><|det|>[[115, 431, 883, 687]]<|/det|>
+We are grateful to the reviewer for this constructive suggestion and as requested we performed Alp quantification and gene expression analyses as already explained in response to the point#4 above. Our novel data showed that sorted OPCs from mutant mice were as efficient as WT ones in vitro at generating bone- making OBLs after 14- days culture in osteogenic medium as determined by Alp staining (see new Figure S1N). This was further confirmed by qPCR analyses with no changes in expression of genes encoding osteogenic regulators in mutant cultures (see new Figure S1O). These findings are in line with efficient terminal osteogenic differentiation and preserved bone formation and mineralization capacities in Cxcr4<sup>1013</sup>- bearing mice. The text has been modified accordingly (page 11, line 219). We fully agree with the reviewer that the yield of immature and mature osteogenic cells recovered by flow cytometry was not as high as expected in light of Alp and Alizarin red staining, and this was likely due to the difficulty we experimented to collect and separate homogenously the cells from the mineralized matrix at the end of the culture. Although real- time quantitative PCR analyses of Sca- 1 and PDGFRα markers corroborated the flow cytometric results (see Figure S2E), these flow cytometric results are rather dispensable for the paper and therefore we propose to remove them to clarify the message. We thank the reviewer for having pointed this inconsistency.  
+
+<|ref|>text<|/ref|><|det|>[[115, 702, 883, 816]]<|/det|>
+7. "Figure 5 in vivo data. The authors state that especially the cortical bone is rescued in mutant mice, but not the trabecular phenotype, based on lumbar spine BMD data. To validate this statement, μCT analysis of cortex of long bones should be analysed with and without AMD3100 treatment. In addition, these data also suggest that CXCR4 desensitisation in osteogenic lineage cells is likely not responsible for the trabecular bone phenotype, and that other cell types/mechanisms might be involved. This site-specificity should be reflected in the title and in the abstract."  
+
+<|ref|>text<|/ref|><|det|>[[115, 816, 883, 896]]<|/det|>
+We thank the reviewer for this relevant comment. Our original version of the manuscript stated a suggestion for a correcting effect of Cxcr4- dependent signaling dampening on the cortical, rather than trabecular, bone based on BMD values of lumbar spine in mutant mice. Because μCT analyses were not carried out for this experiment, we sought to measure the cortical thickness in paraffin- embedded sections stained with Toluidine Blue. The two cortices were measured using
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 62, 883, 177]]<|/det|>
+histomorphometry software and expressed as mean of both cortices for each sample. As shown in the new Figure 5L, AMD3100 treatment for 3 weeks did not ameliorate the cortical network in mutant mice, thus suggesting that either the treatment procedure should be further optimized in terms of duration and kinetics or, as anticipated by the reviewer, that other cell types/mechanisms might be involved at this stage such as OCLs or perivascular SSCs as recently reported by Jeffery and coll. (Cell Stem Cell, 2022). This warrants further investigations. This point has been mentioned in the revised version of the manuscript (page 16, line 345).  
+
+<|ref|>sub_title<|/ref|><|det|>[[116, 204, 257, 220]]<|/det|>
+## Minor concerns:  
+
+<|ref|>text<|/ref|><|det|>[[116, 225, 884, 277]]<|/det|>
+1. "Perilipin staining should be quantified as the observation that CXCR4 specifically reduces the osteogenic, but not the adipogenic differentiation is interesting, but should be validated by quantitative data."  
+
+<|ref|>text<|/ref|><|det|>[[115, 278, 883, 450]]<|/det|>
+We thank the reviewer for bringing to our attention that quantification data would be helpful to strengthen the significance of our findings. As requested, Figure 1H mentioned by the reviewer has been edited with quantification data and shows no change in adipocyte content in the BM of mutant mice, as compared to WT mice (see new Figure 1J). Congruent with immunostaining on bone sections, RNA-seq analyses performed during the reviewing period show that mutant SSCs displayed a gene signature consistent with preserved adipogenic potential (see new Figures S2C and S2D). These cells also differentiated into adipocytes similarly to WT SSCs when cultured in vitro in adipogenic medium (Figure S2G). These results suggest that proper Cxcr4 signaling is required for regulating the osteogenic specification of SSCs specifically. The text has been modified accordingly (page 7, line 130; page 13, line 264, page 15 line 316).  
+
+<|ref|>text<|/ref|><|det|>[[115, 467, 883, 518]]<|/det|>
+2. "Figure 1D: it is not clear whether the total number of mice used is 7-14, coming from 3 experiments, or that in each of the 3 experiments there were 7-14 mice, thus 21-42 mice in total. Similar comment to all experiments using mice."  
+
+<|ref|>text<|/ref|><|det|>[[115, 519, 883, 587]]<|/det|>
+We thank the reviewer for bringing to our attention that the total number of mice used in each experiment was not clear and we apologize for that. In fact, each number mentioned represents the total number of mice used, in 3 independent experiments or more. The legends have been modified accordingly.  
+
+<|ref|>text<|/ref|><|det|>[[115, 604, 883, 638]]<|/det|>
+3. "Figure 2J: the % of apoptotic OPC is around 30%, which is rather high, and should be commented on."  
+
+<|ref|>text<|/ref|><|det|>[[115, 639, 883, 760]]<|/det|>
+We thank the reviewer for this relevant comment. Although the reason why the apoptosis rate is high among OPCs is unclear, we obtained similar results using cleaved caspase 3 staining instead of Annexin V staining. One can speculate that experimental procedures make these cells more fragile and prone to undergo apoptosis. In both assays, we were unable to observe differences between WT and mutant OPCs, thus strongly suggesting that increased apoptosis of OPCs does not contribute to bone loss in mutant mice. As requested by the Reviewer#4, this panel has been moved to the supplemental Figure 1 (see Figure S1F).  
+
+<|ref|>sub_title<|/ref|><|det|>[[116, 778, 322, 794]]<|/det|>
+## Reviewer #2 comments:  
+
+<|ref|>text<|/ref|><|det|>[[115, 808, 883, 905]]<|/det|>
+WHIM syndrome (WS) is a rare immunodeficiency caused by gain- of- function CXCR4 mutations. The authors have demonstrated for the first time a substantial decrease in bone mineral density in \(25\%\) of WS patients and osteoporosis in a WS mouse model. Interestingly, wild- type mice transplanted with bone marrow hematopoietic cells from mice with a WS- linked CXCR4 mutation (Cxcr4+/1013 or Cxcr4 1013/1013) had reduced trabecular bone content compared with wild- type chimeras. On the other hand,
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 62, 884, 208]]<|/det|>
+transplantation of wild- type bone marrow cells did not rescue the reduced trabecular bone content in the mutant chimeras. Osteogenic differentiation of cultured bone marrow skeletal stem cells (SSCs) from the mutants was impaired in vitro. The CXCR4 antagonist AMD3100 normalized in vitro osteogenic potential of SSCs and reversed an in vivo decrease in Sca- 1- PDGFRa- cells in the mutants. These results are interesting and important; however the major concern remains at this time. There is the possibility that osteopenia in mice and patients, which carry the WS- linked CXCR4 mutation, is the result of only enhanced osteoclast function but not reduced osteogenic differentiation of SSCs.  
+
+<|ref|>sub_title<|/ref|><|det|>[[116, 236, 256, 252]]<|/det|>
+## Major concerns:  
+
+<|ref|>text<|/ref|><|det|>[[116, 257, 884, 355]]<|/det|>
+1. "As the authors described, it has been shown previously that deletion of CXCR4 in mesenchymal cells, including SSCs, resulted in osteopenia (Tzeng et al., J. Bone Miner. Res. 2018; Zhu et al., J. Biol/ Chem. 2011). These results argue against the authors' conclusion that gain-of-function CXCR4 mutations in SSCs resulted in osteopenia. Thus, I would recommend the authors to generate and analyze the mice, in which mesenchymal cells, including SSCs, but not hematopoietic cells carry WS-linked CXCR4 mutations."  
+
+<|ref|>text<|/ref|><|det|>[[115, 360, 883, 617]]<|/det|>
+We are grateful to the reviewer for this relevant and constructive comment. Indeed, truncating mutations in CXCR4 which cause the WHIM syndrome (WS) in humans lead in vitro to a typical gain- of- function response to CXCL12 as exemplified by enhanced chemotaxis. However, in several cellular contexts (e.g., HSC lymphoid differentiation, B cell development...), we observed that loss of CXCR4 and gain of function of CXCR4 translated into similar phenotypes. This likely relates to the intensity and the strength of CXCR4 signaling that should be tightly regulated to permit the occurrence of physiological functions. Our findings unveil that mutant SSCs from the bone fraction are impaired in their capacities to generate OBLs as illustrated notably in vitro thus implying a cell- autonomous effect of the Cxcr4 mutation in the bone phenotype. In line with this, J. Pereira's laboratory recently showed using a second mouse model of the WS, carrying the gain- of- function CXCR4 R334X mutation, that lymphopoiesis is reduced because of a dysregulated transcriptome of mesenchymal stem cell isolated from the flushed marrow fraction and characterized by a switch from an adipogenic to an osteolineage- prone program with limited lymphopoietic activity (Zehentmeier et al., Science Immunology 2022). These results agree with ours and suggest that both hematopoietic and stromal cells are affected by the Cxcr4 gain of function mutation. The text has been modified accordingly (page 5, line 90; page 19, line 403).  
+
+<|ref|>text<|/ref|><|det|>[[115, 617, 883, 903]]<|/det|>
+Our reciprocal BM reconstitution experiments support this assumption since transplantation of WT BM into lethally irradiated mutant recipients was not sufficient to rescue the skeletal landscape phenotype, and conversely, transplantation of mutant BM induced bone dysregulation in WT recipient (see Figures 3E- P and S1G). Although we are aware of the fact that BM chimera do not constitute perfect models, we do believe they are informative notably when hematopoietic cells that are engrafted do not carry WS- linked CXCR4 mutations. Moreover, we think that our ubiquitous mouse model is relevant since it closely phenocopies the immune- hematological phenotype of the human pathology in which both hematopoietic and stromal cells harbor the Cxcr4 mutation. To confirm that, a conditional mouse model would have been ideal and not beyond the scope but we are not aware that such a model exists and it was not feasible de novo in the frame of a reviewing period. Rather, as suggested by the Reviewer#1, we set- up a co- culture system between in vitro expanded osteogenic cells carrying or not the Cxcr4 mutation and WT OCL precursors, ie., BM CD11b+ myeloid cells. As shown in the new Figure 3L, mutant osteogenic cells promoted exacerbated OCL differentiation compared to WT cells. Soluble factors do not seem sufficient as the supernatants of such stimulated expanded osteogenic cells (WT or mutant) did not induce OCL differentiation. Additionally, transcriptomic analyses of stimulated osteogenic cells carrying or not the Cxcr4 mutation did not reveal any major changes in expression levels of master genes regulating osteoclastogenesis such as the RANKL/OPG
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 62, 883, 240]]<|/det|>
+balance (see new Figure 3M). These findings suggest a juxtacrine function of osteogenic cells toward OCL differentiation that likely relies on direct interactions between both cell types and involves the Cxcl12/Cxcr4 axis. As adding the osteogenic component carrying the Cxcr4 mutation is sufficient to promote in vitro enhancement of OCL differentiation, we propose that the overall decrease in bone mass in mutant mice involves remodeling of osteogenic and osteoclastogenic components leading to decreased bone formation and increased bone resorption. In such a process, the osteogenic lineage would act as the driver and the OCL one as a passenger. The underlying molecular mechanism(s) of this cross- talk remains to be elucidated but seems to require direct contact between both cell types. The entire manuscript as well as the title have been modified accordingly (page 11, line 230; page 21, lines 451 & 459) and a graphical abstract has been designed consequently.  
+
+<|ref|>text<|/ref|><|det|>[[115, 254, 883, 335]]<|/det|>
+2. "The authors show the reduced trabecular bone content of mice with a WS-linked CXCR4 mutation transplanted with bone marrow hematopoietic cells from wild-type mice was not rescued 3 and 16 weeks after transplantation. However, wild-type hematopoietic cells might be able to rescue the reduced trabecular bone content of the mutants earlier in development."  
+
+<|ref|>text<|/ref|><|det|>[[115, 335, 883, 400]]<|/det|>
+We thank the reviewer for this relevant comment. However, we have to stress that currently we do not have the ethical authorization to transplant BM into mice younger than seven/eight weeks but we are aware that it would be interesting to do it. This has been clearly mentioned in the revised version of the manuscript (page 9, line 175).  
+
+<|ref|>text<|/ref|><|det|>[[115, 414, 883, 479]]<|/det|>
+3. "The authors describe Sca-1+PDGFRa+ cells as SSCs (Page 8, line 142); however, the major population of bone marrow SSCs is defined as Sca-1-PDGFra+PDGFRb+LePr+CD31-cells (Omatsu et al., Immunity 2010; Zhou et al., Cell Stem Cell 2014; Seike et al., Genes Dev 2018)."  
+
+<|ref|>text<|/ref|><|det|>[[115, 479, 883, 687]]<|/det|>
+We are grateful to the referee for pointing out that the phenotype of SSCs we used could be a matter of debate and should be better justified. To the best of our knowledge, there is currently no consensual denomination for the different BM mesenchymal subpopulations and we agree with the reviewer that we should have been more precise on this point. As shown in the paper of Zhou et al. (Cell Stem Cell, 2014), the highest CFU- F clonogenic potential is observed in the Sca1+PDGFRa+ subset and not in the Sca1- PDGFRa+ population. This has been confirmed and extended to SSCs in the periosteum (Jeffery et al., Cell Stem Cell, 2022). Furthermore, 16wks after transplantation of GFP+ Sca1+PDGFRa+ into WT mice (Morikawa et al., JEM 2009), it was shown that among the GFP+ cells recovered, a few were Sca1+PDGFRa+ and most of them were Sca1- PDGFRa+, indicating that Sca1+PDGFRa+ cells are at the top of the hierarchy. This is why we chose to consider the Sca1+PDGFRa+ cells in the bone fraction as skeletal stem cells as compared to Sca1- PDGFRa+ that are more engaged in differentiation, as osteoblast progenitor cells. This point has been mentioned in the revised version of the manuscript (page 8, line 146).  
+
+<|ref|>text<|/ref|><|det|>[[115, 702, 881, 735]]<|/det|>
+4. "The evidence that Sca-1-PDGFra- cells are committed osteoblasts (OPCs) in the bone marrow would not be convincing (Page 8, line 143)."  
+
+<|ref|>text<|/ref|><|det|>[[115, 735, 883, 877]]<|/det|>
+We apologize for the lack of clarity with this sentence. As explained in the point 3, we consider the CD51+Sca1- population as more differentiated than its Sca1+ counterpart and the sentence has been modified accordingly (page 8, line 146). There was also a typo and we should have referred to the CD51+Sca1- population as PDGFRa+/- as it includes both PDGFRa positive and negative subsets. In line with this, we already consider early OPCs with multipotent adipo/osteogenic potential in the flushed stromal marrow fraction as Sca-1- negative and PDGFRa- positive (see new Figure 4M). Our previous results showed that the Sca1- PDGFRa- population highly express committed osteoblast markers such as Bglap, Col1a1 and Pth1r1 (see Balzano et al., Cell Reports 2019).
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[117, 64, 321, 80]]<|/det|>
+## Reviewer #4 comments:  
+
+<|ref|>text<|/ref|><|det|>[[116, 92, 883, 239]]<|/det|>
+The manuscript by Anginot and colleagues provides novel insights into the role of CXCR4- mediated signaling in skeletal stromal/stem cell osteogenic specification. The authors describe a series of experiments characterizing the anatomic, developmental and functional properties of the skeletal and osteogenic compartment in a knock- in mouse model of the human genetic disorder WHIM syndrome. The significance of the deficits in skeletal remodeling and stem cell differentiation identified in the mouse model in human bone biology are validated in cohort of WHIM syndrome patients carrying gain- of- function mutations in CXCR4. These findings represent a novel contribution elucidating an important new role for CXCR4 in bone biology.  
+
+<|ref|>sub_title<|/ref|><|det|>[[116, 266, 256, 283]]<|/det|>
+## Major concerns:  
+
+<|ref|>text<|/ref|><|det|>[[115, 287, 884, 544]]<|/det|>
+1. "The authors characterized the effects of increased CXCR4 signaling in vivo through standard histomorphometric of bone anatomy and flow cytometric analyses of various progenitor cell populations in the mouse model. The data in Figure 1 are well presented and convincing in regard to the gene-dose dependent skeletal effects as well as the specificity of the changes to cortical and trabecular bone. Figure 2 is overly dense and contains information that could be moved to the supplement without impacting the major findings of the work. In particular, the experiments demonstrating the functional effects of the mutant CXCR4 receptor recapitulate characteristics of CXCR4 C-terminal truncations that have been well studied in other contexts. It would suffice to state that the mutant receptor localization, internalization and intracellular signaling were similar to what has been seen in other contexts and move panels 2E-J to the supplement. The bone marrow reconstitution experiments shown in the remainder of the figure demonstrate clearly the contribution of cell-extrinsic as well as cell-intrinsic factors to the observed skeletal changes. Similarly, the effects on bone resorption and formation shown in Figure 3 panels C-E can be moved to supplement to better focus on the transcriptional effects shown in the subsequent panels."  
+
+<|ref|>text<|/ref|><|det|>[[116, 544, 883, 592]]<|/det|>
+We are grateful to the reviewer for these constructive suggestions and as requested, the panels 2E- J and 3C- E have been moved to the new Supplemental Figure 1 (see panels S1A- S1F and S1H- S1J).  
+
+<|ref|>text<|/ref|><|det|>[[115, 606, 884, 815]]<|/det|>
+2. "The data in Figures 4 and 5 provide compelling data regarding the impact of aberrant CXCR4 signaling on osteogenic specification at the level of transcriptional effects and cell cycle progression. The PCA data shown in Figure 4C is not well explained as the 48 genes used for expression profiling are not described in the text nor the supplement, which lists a smaller number of genes. The data in the subsequent panels are more informative. I would consider removing panel 4C or moving it to the supplement with a better description of the analysis. The experiments shown in Figure 5 document the selective reduction in osteogenic differentiation capacity of stromal stem cells carrying one or two mutant CXCR4 receptors in a dose dependent fashion and the reversal of this phenotype with treatment of the receptor inhibitor AMD3100. The relevance of these data in mice to human bone biology are supported with the data shown in Figure 6 which revealed a selective osteogenic differentiation defect in bone marrow cells derived from WHIM syndrome patients."  
+
+<|ref|>text<|/ref|><|det|>[[116, 815, 883, 895]]<|/det|>
+We thank the reviewer for bringing to our attention that the PCA data shown in Figure 4C was not clear and we apologize for that. This panel has now been removed. As suggested by Reviewer#1, we decided to investigate the impact of the gain- of- Cxcr4- function on the molecular identity of SSCs by RNA- seq analyses of sorted bulk cells from WT and mutant bone fractions. Biological processes related to cell cycle and osteogenic differentiation were significantly modulated in
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 62, 883, 208]]<|/det|>
+1013/1013 SSCs as determined by GSEA (Gene set enrichment analysis) (see new Figure 4C). The SSC signature in 1013/1013 mice was reduced for genes related to cell cycle progression and regulation (see new Figures S2A and S2B). Likewise, genes related to osteogenic differentiation appeared to be decreased in mutant SSCs (see new Figures 4D and 4E). In contrast, key genes involved in both adipogenesis and chondrogenesis were not differentially expressed in mutant SSCs (see new Figure S2C). These results were confirmed by microfluidic-based multiplex gene expression analyses (see Figures 4F and 4G and Figure S2D), thus suggesting that proper Cxcr4 signaling is required for regulating osteogenic specification of SSCs at the transcriptional level. The text has been modified accordingly (page 13, line 264).  
+
+<|ref|>text<|/ref|><|det|>[[115, 222, 884, 333]]<|/det|>
+3. "With regards to the conclusion that a skeletal phenotype is present in a subset of WHIM syndrome patients, given that treatment of neutropenia with G-CSF is associated with osteopenia as side effect of therapy, it would be useful to know the total number of patients treated with G-CSF in the cohort to address the concern that the enrichment in osteopenic patients is restricted to those patients that have been so treated as well as their ages and genders given the impact of these variables on risk for osteopenia in general."  
+
+<|ref|>text<|/ref|><|det|>[[115, 321, 883, 464]]<|/det|>
+We thank the reviewer for this very relevant comment. Nineteen WS patients had a baseline bone density scan as part of a drug treatment trial (NCT02231879) comparing 1 year of twice daily filgrastim (Neupogen) versus plerixafor (Mozobil) in a randomized, blinded crossover design. There were 13 women and 6 men with an average age of 30.5 years (range 10- 56). Patients had been on filgrastim for an average of 5.7 years prior to enrolling in the trial (range 0- 27). Six of the 19 had not used filgrastim regularly prior to trial enrollment. These findings suggest that the enrichment in osteopenic WS patients is not merely due to treatment regimen, age or gender parameters. This point is now mentioned in the revised version of the manuscript (page 22, line 487; page 23, line 507).  
+
+<|ref|>text<|/ref|><|det|>[[115, 478, 883, 560]]<|/det|>
+4. "Apart from these concerns, the quality of the data presented is good and the conclusions supported by the evidence. The manuscript is well written and the references appropriate, though it was notable that the initial description of the cause of WHIM syndrome as gain-of-function truncation mutations in CXCR4 was not cited, this should be added."  
+
+<|ref|>text<|/ref|><|det|>[[115, 565, 882, 630]]<|/det|>
+We thank the reviewer for this relevant comment and apologize for this oversight. The initial description of inherited CXCR4 mutations in the WS has been reported by Hernandez and collaborators in 2003 (Nature Genetics, PMID: 12692554). The appropriate reference (n°50 in the list of references) has been added accordingly (page 5, line 98).
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[119, 86, 348, 105]]<|/det|>
+## REVIEWER COMMENTS  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 150, 411, 166]]<|/det|>
+## Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[119, 184, 878, 235]]<|/det|>
+The authors answered adequately to the comments and questions by performing additional experiments and adapting the text. The claims are now well supported by their findings, making it an interesting study providing further insight in the skeletal effects of CXCR4 mutations.  
+
+<|ref|>text<|/ref|><|det|>[[119, 254, 255, 268]]<|/det|>
+Minor comments:  
+
+<|ref|>text<|/ref|><|det|>[[119, 270, 864, 303]]<|/det|>
+Page 11, line 208: the following sentence is difficult to interpret: Cxcr41013- bearing mice exhibited unremarkable bone formation. Not clear what is meant by 'unremarkable'.  
+
+<|ref|>text<|/ref|><|det|>[[118, 320, 868, 389]]<|/det|>
+Page 11, line 225: It is mentioned that OBL differentiation is reduced in mutant mice, whereas the previous lines describe normal osteogenic differentiation when cultures are started with OPCs. To avoid misunderstanding, some other wording should be used to describe that the transition of SSCs to OPCs is impaired or that there is reduced osteogenic lineage commitment.  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 468, 411, 484]]<|/det|>
+## Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[119, 503, 858, 554]]<|/det|>
+The authors have given a satisfactory response to some of this reviewer's concerns, improving the manuscript. However, their answers to several issues remain incomplete, and therefore their conclusions are still not convincing.  
+
+<|ref|>text<|/ref|><|det|>[[118, 571, 866, 692]]<|/det|>
+1. The new data that transplantation of Cxcr4 1013/1013 mutant bone marrow cells markedly reduced trabecular bone content (BV/TV and Tb.Nb) of wild-type recipient mice (Fig. 2P) are interesting and important. The magnitude of the decrease seems to be much larger compared with Cxcr4 1013/1013 mutant mice, suggesting that microenvironments with gain-of-function Cxcr4 1013/1013 mutations increased and rescued trabecular bone content. This is consistent with previous findings that deletion of CXCR4 in mesenchymal cells reduced trabecular bone content (Tzeng et al., J. Bone Miner. Res. 2018; Zhu et al., J. Biol. Chem. 2011).  
+
+<|ref|>text<|/ref|><|det|>[[118, 709, 863, 812]]<|/det|>
+2. Again I would recommend the authors to generate and analyze the mice, in which mesenchymal cells, including SSCs, but not hematopoietic cells carry WS-linked CXCR4 mutations. However, the authors mentioned that it was not feasible in the frame of a reviewing period. Then, the authors should at least show trabecular bone content (BV/TV and Tb.Nb) of Cxcr4 1013/1013 mutant mice transplanted with bone marrow cells from wild-type mice and compare the results with those of wild-type mice transplanted with mutant bone marrow cells.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[119, 85, 411, 100]]<|/det|>
+## Reviewer #4 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 119, 816, 152]]<|/det|>
+The authors have addressed all of the issues raised by me adequately. I am satisfied with the responses to the other reviewers as well and have no additional concerns.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[116, 64, 562, 81]]<|/det|>
+## Point-to-point response to the reviewers' comments  
+
+<|ref|>sub_title<|/ref|><|det|>[[117, 92, 322, 108]]<|/det|>
+## Reviewer #1 comments:  
+
+<|ref|>text<|/ref|><|det|>[[116, 118, 884, 183]]<|/det|>
+The authors answered adequately to the comments and questions by performing additional experiments and adapting the text. The claims are now well supported by their findings, making it an interesting study providing further insight in the skeletal effects of CXCR4 mutations.  
+
+<|ref|>sub_title<|/ref|><|det|>[[116, 199, 267, 214]]<|/det|>
+## Minor comments:  
+
+<|ref|>text<|/ref|><|det|>[[116, 215, 884, 247]]<|/det|>
+1. Page 11, line 208: the following sentence is difficult to interpret: Cxcr41013-bearing mice exhibited unremarkable bone formation. Not clear what is meant by 'unremarkable'.  
+
+<|ref|>text<|/ref|><|det|>[[116, 247, 883, 333]]<|/det|>
+We thank the reviewer for bringing to our attention that the use of the term "unremarkable" was not clear and probably not appropriate. The sentence has been modified as follow: "Cxcr41013- bearing mice exhibited similar bone formation as revealed by osteoid surface (OS/BS) and osteoblast surface (Obl.S/BS) compared to WT mice (Fig. 3C)". The text has been modified accordingly (page 11, line 208).  
+
+<|ref|>text<|/ref|><|det|>[[115, 348, 883, 428]]<|/det|>
+2. Page 11, line 225: It is mentioned that OBL differentiation is reduced in mutant mice, whereas the previous lines describe normal osteogenic differentiation when cultures are started with OPCs. To avoid misunderstanding, some other wording should be used to describe that the transition of SSCs to OPCs is impaired or that there is reduced osteogenic lineage commitment.  
+
+<|ref|>text<|/ref|><|det|>[[116, 428, 883, 477]]<|/det|>
+We thank the reviewer for this very relevant comment. The sentence has been changed as follow: "These findings suggest reduced osteogenic lineage commitment in Cxcr41013-bearing mice". The text has been modified accordingly (page 11, line 224).  
+
+<|ref|>sub_title<|/ref|><|det|>[[116, 494, 322, 509]]<|/det|>
+## Reviewer #2 comments:  
+
+<|ref|>text<|/ref|><|det|>[[116, 510, 883, 559]]<|/det|>
+The authors have given a satisfactory response to some of this reviewer's concerns, improving the manuscript. However, their answers to several issues remain incomplete, and therefore their conclusions are still not convincing.  
+
+<|ref|>text<|/ref|><|det|>[[115, 580, 883, 708]]<|/det|>
+1. The new data that transplantation of Cxcr4 1013/1013 mutant bone marrow cells markedly reduced trabecular bone content (BV/TV and Tb.Nb) of wild-type recipient mice (Fig. 2P) are interesting and important. The magnitude of the decrease seems to be much larger compared with Cxcr4 1013/1013 mutant mice, suggesting that microenvironments with gain-of-function Cxcr4 1013/1013 mutations increased and rescued trabecular bone content. This is consistent with previous findings that deletion of CXCR4 in mesenchymal cells reduced trabecular bone content (Tzeng et al., J. Bone Miner. Res. 2018; Zhu et al., J. Biol. Chem. 2011).  
+
+<|ref|>text<|/ref|><|det|>[[115, 714, 883, 905]]<|/det|>
+We thank the reviewer for these valuable remarks and suggestions. Although it is difficult to compare chimeric and steady state mice, especially considering the whole- body irradiation and the 4- month reconstitution period, it appears indeed that WT mice reconstituted with mutant BM display stronger trabecular bone defects than younger mutant mice at steady state. It is indeed possible that, as put by the reviewer, "microenvironments with gain- of- function Cxcr4 1013/1013 mutations increased and rescued trabecular bone content". Besides mesenchymal and osteolineage cells, the BM ecosystem contains other possible effector cells including hematopoietic and mature immune cells, but also some radiosistant endothelial cells, and other stromal cells such as adipocytes that all express the CXCR4 receptor, which emerge during bone development and reach homeostasis at the adult stage. One can assume that the gain of CXCR4 function might modulate, positively or negatively, one, or several, BM landscape component(s) that could balance the trabecular bone defect in 1013/1013 mice. This is indeed consistent with
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 62, 883, 128]]<|/det|>
+previous works reporting a cell- autonomous Cxcl12- Cxcr4 signaling on the MSPC osteogenesis but not adipogenesis (Tzeng et al., J. Bone Miner. Res. 2018; Zhu et al., J. Biol. Chem. 2011). Future studies are necessary to identify such cells by tissue- specific CXCR4 targeting, as suggested by the reviewer. This point has been now discussed (page 21, line 469).  
+
+<|ref|>text<|/ref|><|det|>[[115, 127, 883, 336]]<|/det|>
+The magnitude of the bone loss in WT recipient upon transplantation of mutant BM is surprising but is indicative of cell- extrinsic hematopoietic- driven Cxcr4- mediated regulation of the skeletal landscape. This may be due to the presence of effector mature leukocytes in the BM graft as we suggested previously in response to the point 2 raised by Reviewer#1. Indeed, one can speculate that myeloid cells including OCL progenitors as well as lymphoid cells may actively participate in promoting bone remodeling in BM chimeric WT recipient mice. Indeed, the laboratory of Pr. A. Bozec among others recently reported that prolonged HIF- 1α signaling in B cells leads to enhanced RANKL production and OCL formation in the BM (Meng et al., Bone Research 2022). Likewise, BM T cells are known to produce RANKL and to regulate OCL compartment within the BM (see for review for instance Corrado et al., IJMS 2020; Mori et al., Clin Dev Immunol 2013; Zhang et al., Front Endocrinol 2020). Whether the transplantation of mutant BM recreates a pro- osteoclastogenic environment through a remodeling of the lymphoid compartment deserves further investigations.  
+
+<|ref|>text<|/ref|><|det|>[[115, 351, 883, 463]]<|/det|>
+2. Again I would recommend the authors to generate and analyze the mice, in which mesenchymal cells, including SSCs, but not hematopoietic cells carry WS-linked CXCR4 mutations. However, the authors mentioned that it was not feasible in the frame of a reviewing period. Then, the authors should at least show trabecular bone content (BV/TV and Tb.Nb) of Cxcr4 1013/1013 mutant mice transplanted with bone marrow cells from wild-type mice and compare the results with those of wild-type mice transplanted with mutant bone marrow cells.  
+
+<|ref|>text<|/ref|><|det|>[[115, 462, 883, 640]]<|/det|>
+We are grateful to the reviewer for this helpful suggestion and as requested, we have quantified trabecular bone parameters by \(\mu \mathrm{CT}\) (see new Figures 2H and 2I). These new analyses extend the flow- cytometric and histological ones and indicate a persistent bone loss in mutant recipient upon transplantation of WT BM, thereby supporting the hypothesis of a cell- intrinsic CXCR4- mediated regulation of the skeletal landscape. However, the extent of the bone loss appears to be less marked compared with WT recipients transplanted with mutant BM cells. As discussed above, this could rely on the modulatory effect due to the gain of CXCR4 function on one, or several, stromal component(s) that could compensate the trabecular bone defect particularly in 1013/1013 mice. As stated in the manuscript, these findings suggest that impaired CXCR4 desensitization in both skeletal and hematopoietic cells have combinatorial effects on bone landscape dysregulation in adult Cxcr4<sup>1013</sup>- bearing mice.  
+
+<|ref|>sub_title<|/ref|><|det|>[[117, 661, 321, 678]]<|/det|>
+## Reviewer #4 comments:  
+
+<|ref|>text<|/ref|><|det|>[[115, 690, 884, 755]]<|/det|>
+The authors have addressed all of the issues raised by me adequately. I am satisfied with the responses to the other reviewers as well and have no additional concerns. We thank the reviewer for the previous suggestions and are happy that all concerns were addressed.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[119, 85, 377, 101]]<|/det|>
+## REVIEWERS' COMMENTS  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 138, 449, 153]]<|/det|>
+## Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[119, 167, 808, 196]]<|/det|>
+The authors have given a satisfactory response to this reviewer's concerns, improving the manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[118, 210, 875, 251]]<|/det|>
+Minor pointsFigure 21: How about p- values in Tb.Nb? They appear to be significantly less than 0.005 as seen in BV/TV.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[115, 114, 562, 131]]<|/det|>
+## Point-to-point response to the reviewers' comments  
+
+<|ref|>text<|/ref|><|det|>[[115, 142, 884, 192]]<|/det|>
+Reviewer #2 comments: The authors have given a satisfactory response to this reviewer's concerns, improving the manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[115, 207, 884, 255]]<|/det|>
+Minor points Figure 2l: How about p-values in Tb.Nb? They appear to be significantly less than 0.005 as seen in BV/TV.  
+
+<|ref|>text<|/ref|><|det|>[[115, 270, 883, 335]]<|/det|>
+We thank the reviewer for his/her previous constructive comments that have helped us improving greatly the quality of our manuscript. We are glad to read that all concerns were now addressed. Regarding the p- values of the data shown in Figure 2l, they have been determined using the twotailed Student's t test and are as follow:  
+
+<|ref|>text<|/ref|><|det|>[[115, 342, 830, 450]]<|/det|>
+For the BV/TV parameter: WT BM- chimeric \(\mathsf{CD45.2 + }\) WT vs WT BM- chimeric \(\mathsf{CD45.2 + }\) +/1013 mice: \(\mathsf{P} = 0.033\) WT BM- chimeric \(\mathsf{CD45.2 + }\) WT vs WT BM- chimeric \(\mathsf{CD45.2 + }\) 1013/1013 mice: \(\mathsf{P} = 0.0244\) For the Tb.Nb parameter: WT BM- chimeric \(\mathsf{CD45.2 + }\) WT vs WT BM- chimeric \(\mathsf{CD45.2 + }\) +/1013 mice: \(\mathsf{P} = 0.0702\) WT BM- chimeric \(\mathsf{CD45.2 + }\) WT vs WT BM- chimeric \(\mathsf{CD45.2 + }\) 1013/1013 mice: \(\mathsf{P} = 0.0710\)  
+
+<|ref|>text<|/ref|><|det|>[[115, 465, 673, 483]]<|/det|>
+The exact p- values have been now provided in the legend of Figure 2.
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__0120cbbdf5abcce247bf35686d0d3fbc3c94f93c709d874b56ecf9271a6516aa/images_list.json

@@ -0,0 +1,520 @@
+[
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_0.jpg",
+    "caption": "Fig. R1 | Absorption of PtNP-shell. a, Absorption curves of PtNP-shell with different concentrations (10, 25, 50 and \\(75\\mu \\mathrm{g}\\cdot \\mathrm{mL}^{-1}\\) ). b, Mass extinction coefficient of PtNP-shell at \\(1064\\mathrm{nm}\\) . Normalized absorbance intensity at \\(\\lambda = 1064\\mathrm{nm}\\) divided by the characteristic length of the cell (A/L) at different concentrations (10, 25, 50 and \\(75\\mu \\mathrm{g}\\cdot \\mathrm{mL}^{-1}\\) ).",
+    "footnote": [],
+    "bbox": [],
+    "page_idx": 0
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_1.jpg",
+    "caption": "Fig. R2 | Maximal HR changes of beagle treatment with PtNP-shell or control a, before and b, after NIR-II exposure, \\(n = 6\\) .",
+    "footnote": [],
+    "bbox": [
+      [
+        195,
+        650,
+        803,
+        821
+      ]
+    ],
+    "page_idx": 7
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_2.jpg",
+    "caption": "Fig. R3 | Ganglion biocompatibility of targeted injections of PtNP or PBS after NIR-II irradiation and after 30 days of follow-up. a, Representative images of H&E and TUNEL staining of NG from different treatment groups immediately after NIR-II irradiation or after 30 days of follow-up. b, Representative images of H&E and TUNEL staining of LSG from different treatment groups immediately after NIR-II irradiation or after 30 days of follow-up.",
+    "footnote": [],
+    "bbox": [
+      [
+        144,
+        360,
+        850,
+        731
+      ]
+    ],
+    "page_idx": 12
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_3.jpg",
+    "caption": "Fig. R4 | Long term biosafety of PtNP-shell microinjection. Long-term in vivo biosafety was assessed by local injection of PtNP-shell into the ganglion of Beagle or by injection of equal doses of PtNP-shell into the tail vein of Sprague-Dawley rats. a, Representative H&E staining of major organs of beagles following different treatments. Blood biochemical analyses including b, ALT, c, AST, d, Urea, e, Crea, f, LDH1, g, TNF- \\(\\alpha\\) , and h, IL-6 were performed on Beagles in different treatment groups. i, Representative H&E staining of major organs of rats following different treatments. Blood biochemical analyses including j, ALT, k, AST, l, Urea, m, Crea, n, LDH1, o, TNF- \\(\\alpha\\) and p, IL-6 were performed on rats in different treatment groups.",
+    "footnote": [],
+    "bbox": [
+      [
+        140,
+        58,
+        856,
+        505
+      ]
+    ],
+    "page_idx": 13
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_4.jpg",
+    "caption": "Fig. R5 | Quantitative analysis of VF threshold in different groups. Data are shown as the mean \\(\\pm\\) S.E.M. \\*P < 0.05, \\*\\*P < 0.01, \\*\\*\\*P < 0.001.",
+    "footnote": [],
+    "bbox": [
+      [
+        396,
+        199,
+        598,
+        370
+      ]
+    ],
+    "page_idx": 14
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_5.jpg",
+    "caption": "Fig. R6 | Statistical analysis of recorded ventricular arrhythmia events post myocardial ischemia. Quantitative analysis the number of a, VTs and b, the duration of sVT of beagles with MI. Data are shown as the mean \\(\\pm\\) S.E.M. ns means that the difference is not statistically significant.",
+    "footnote": [],
+    "bbox": [
+      [
+        312,
+        565,
+        682,
+        738
+      ]
+    ],
+    "page_idx": 16
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_6.jpg",
+    "caption": "Fig. R7 | Effect of NIR-II laser irradiation with varying durations on the viability of HT-22 cells treated with PtNP-shell (50 µg·mL⁻¹) (Power densities: a, 0.75 W·cm⁻² and b, 1 W·cm⁻²). The error bar indicates S.E.M.",
+    "footnote": [],
+    "bbox": [
+      [
+        219,
+        150,
+        777,
+        320
+      ]
+    ],
+    "page_idx": 16
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_7.jpg",
+    "caption": "Fig. R8 | Blood biochemical analyses including a, TNF-α, and b, IL-6 were performed on Beagles in different treatment groups. Blood biochemical analyses including c, TNF-α and d, IL-6 were performed on rats in different treatment groups.",
+    "footnote": [],
+    "bbox": [
+      [
+        315,
+        58,
+        683,
+        396
+      ]
+    ],
+    "page_idx": 17
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_8.jpg",
+    "caption": "Fig. R9 | Characterization of the smaller PtNP-shell (100 nm). a, TEM image and b, element mapping of the smaller PtNP-shell (100 nm).",
+    "footnote": [],
+    "bbox": [
+      [
+        225,
+        185,
+        771,
+        343
+      ]
+    ],
+    "page_idx": 18
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_9.jpg",
+    "caption": "Fig. R10 | Hydrodynamic size size of PtNP-shell after a, 1, b, 4, c, 7, and d, 14 days of standing (Inset: digital photograph).",
+    "footnote": [],
+    "bbox": [
+      [
+        255,
+        563,
+        740,
+        840
+      ]
+    ],
+    "page_idx": 20
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_10.jpg",
+    "caption": "Fig. R11 | UV-vis-NIR absorption spectrum of GaNPs, Ga@Pt NPs and PtNP-shell (75 \\(\\mu \\mathrm{g}\\cdot \\mathrm{mL}^{-1}\\) ).",
+    "footnote": [],
+    "bbox": [
+      [
+        352,
+        149,
+        644,
+        318
+      ]
+    ],
+    "page_idx": 20
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_11.jpg",
+    "caption": "Fig. R12 | Measurement of PtNP-shell blackness. a, Visual appearance of GaNPs, Ga@Pt NPs and PtNP-shell at different concentrations. b, Position of each color in the RGB cube, obtained by extracting the relative components of red, green and blue from Fig. R8a.",
+    "footnote": [],
+    "bbox": [
+      [
+        230,
+        376,
+        768,
+        560
+      ]
+    ],
+    "page_idx": 23
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_12.jpg",
+    "caption": "Fig. R13 | The synthesis steps of the PtNP-shell and the concept of mediating precise photothermal effects for cardioprotection. a, The synthesis steps of PtNP-shell and schematic diagram of photothermal effect. b, Schematic diagram of multifunctional autonomic modulation mediated by photothermal effect of PtNP-shell for precise cardioprotection against myocardial I/R injury and MI-induced VAs.",
+    "footnote": [],
+    "bbox": [
+      [
+        147,
+        58,
+        844,
+        416
+      ]
+    ],
+    "page_idx": 23
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_13.jpg",
+    "caption": "Fig. R14 | UV-vis-NIR absorption spectrum of GaNPs, Ga@Pt NPs and PtNP-shell (75 \\(\\mu \\mathrm{g}\\cdot \\mathrm{mL}^{-1}\\) ).",
+    "footnote": [],
+    "bbox": [
+      [
+        355,
+        56,
+        642,
+        222
+      ]
+    ],
+    "page_idx": 24
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_14.jpg",
+    "caption": "Fig. R15 | Temperature elevation curves of PBS, GaNPs, Ga@Pt NPs and PtNP-shell (50 \\(\\mu \\mathrm{g}\\cdot \\mathrm{mL}^{-1}\\) ) under NIR-II laser irradiation (1 \\(\\mathrm{W}\\cdot \\mathrm{cm}^{-2}\\) ).",
+    "footnote": [],
+    "bbox": [
+      [
+        350,
+        501,
+        647,
+        667
+      ]
+    ],
+    "page_idx": 25
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_15.jpg",
+    "caption": "Fig. R16 | The XPS survey spectrum of a, GaNPs, b, Ga@Pt NPs and c, PtNP-shell.",
+    "footnote": [],
+    "bbox": [
+      [
+        146,
+        150,
+        852,
+        291
+      ]
+    ],
+    "page_idx": 25
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_16.jpg",
+    "caption": "Fig. R17 | High-resolution XPS spectra and fitting results of a, GaNPs, b, Ga@Pt NPs and c, PtNP-shell.",
+    "footnote": [],
+    "bbox": [
+      [
+        146,
+        350,
+        850,
+        735
+      ]
+    ],
+    "page_idx": 26
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_17.jpg",
+    "caption": "Fig. R18 | Hydrodynamic size of PtNP-shell after a, 1, b, 4, c, 7, and d, 14 days of standing (Inset: digital photograph).",
+    "footnote": [],
+    "bbox": [
+      [
+        210,
+        521,
+        785,
+        850
+      ]
+    ],
+    "page_idx": 26
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_18.jpg",
+    "caption": "Fig. R19 | The impact of PEG on the photothermal properties of PtNP-shell. a, Temperature elevation curves of SH-PEG modified and unmodified PtNP-shell. The hydrodynamic size of PtNP-shell b, before and c, after SH-PEG modification (after 600 s of 1064 nm laser irradiation). d, TEM image of PtNP-shell before SH-PEG modification (after 600 s of 1064 nm laser irradiation).",
+    "footnote": [],
+    "bbox": [
+      [
+        234,
+        405,
+        755,
+        710
+      ]
+    ],
+    "page_idx": 27
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_19.jpg",
+    "caption": "Fig. R20 | PtNP-shell co-cultured with neurons. a and b, Cross-sectional TEM and c, SEM of the neurons incubated with PtNP-shell particles for \\(24\\mathrm{h}\\) .",
+    "footnote": [],
+    "bbox": [
+      [
+        260,
+        60,
+        736,
+        597
+      ]
+    ],
+    "page_idx": 28
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_20.jpg",
+    "caption": "Fig. R21 | a, Cell viability of HT-22 treated with different concentrations of PtNP-shell for \\(24\\mathrm{h}\\) . Effect of NIR-II laser irradiation with varying durations on the viability of HT-22 cells treated with PtNP-shell (50 \\(\\mu \\mathrm{g}\\cdot \\mathrm{mL}^{-1}\\) ) (Power densities: b, \\(0.75\\mathrm{W}\\cdot \\mathrm{cm}^{-2}\\) and c, \\(1\\mathrm{W}\\cdot \\mathrm{cm}^{-2}\\) ). The error bar indicates S.E.M.",
+    "footnote": [],
+    "bbox": [
+      [
+        147,
+        59,
+        850,
+        202
+      ]
+    ],
+    "page_idx": 30
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_21.jpg",
+    "caption": "Fig. R22 | Ganglion biocompatibility of targeted injections of PtNP or PBS after NIR-II irradiation and after 30 days of follow-up. a, Representative images of H&E and TUNEL staining of NG from different treatment groups immediately after NIR-II irradiation or after 30 days of follow-up. b, Representative images of H&E and TUNEL staining of LSG from different treatment groups immediately after NIR-II irradiation or after 30 days of follow-up.",
+    "footnote": [],
+    "bbox": [
+      [
+        144,
+        300,
+        850,
+        668
+      ]
+    ],
+    "page_idx": 31
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_22.jpg",
+    "caption": "Fig. R23 | Long term biosafety of PtNP-shell microinjection. Long-term in vivo biosafety was assessed by local injection of PtNP-shell into the ganglion of Beagle or by injection of equal doses of PtNP-shell into the tail vein of Sprague-Dawley rats. a, Representative H&E staining of major organs of beagles following different treatments. Blood biochemical analyses including b, ALT, c, AST, d, Urea, e, Crea, f, LDH1, g, TNF- \\(\\alpha\\) , and h, IL-6 were performed on Beagles in different treatment groups. i, Representative H&E staining of major organs of rats following different treatments. Blood biochemical analyses including j, ALT, k, AST, l, Urea, m, Crea, n, LDH1, o, TNF- \\(\\alpha\\) and p, IL-6 were performed on rats in different treatment groups.",
+    "footnote": [],
+    "bbox": [
+      [
+        144,
+        60,
+        850,
+        500
+      ]
+    ],
+    "page_idx": 31
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_23.jpg",
+    "caption": "Fig. R24 | Effect of NIR-II laser irradiation with varying durations on the viability of HT-22 cells treated with PtNP-shell (50 \\(\\mu \\mathrm{g}\\cdot \\mathrm{mL}^{-1}\\) ) (Power densities: i, \\(0.75\\mathrm{W}\\cdot \\mathrm{cm}^{-2}\\) and j, \\(1\\mathrm{W}\\cdot \\mathrm{cm}^{-2}\\) ). The error bar indicates S.E.M.",
+    "footnote": [],
+    "bbox": [
+      [
+        210,
+        58,
+        788,
+        234
+      ]
+    ],
+    "page_idx": 32
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_24.jpg",
+    "caption": "Fig. R25 | Effect of NIR-II laser irradiation with varying durations on the viability of HT-22 cells treated with PtNP-shell (50 \\(\\mu \\mathrm{g}\\cdot \\mathrm{mL}^{-1}\\) ) (Power densities: i, \\(0.75\\mathrm{W}\\cdot \\mathrm{cm}^{-2}\\) and j, \\(1\\mathrm{W}\\cdot \\mathrm{cm}^{-2}\\) ). The error bar indicates S.E.M.",
+    "footnote": [],
+    "bbox": [
+      [
+        218,
+        515,
+        777,
+        686
+      ]
+    ],
+    "page_idx": 33
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_25.jpg",
+    "caption": "Fig. R26 | Direct effect of NIR irradiation of NG. a, Local temperature curve of NG under NIR-II irradiation. b, Neural function of NG before and after NIR-II irradiation.",
+    "footnote": [],
+    "bbox": [
+      [
+        181,
+        352,
+        816,
+        523
+      ]
+    ],
+    "page_idx": 33
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_26.jpg",
+    "caption": "Fig. R27 | Direct effect of NIR irradiation of LSG. a, Local temperature curve of LSG under NIR-II irradiation. b, Neural function of LSG before and after NIR-II irradiation.",
+    "footnote": [],
+    "bbox": [
+      [
+        181,
+        602,
+        816,
+        775
+      ]
+    ],
+    "page_idx": 34
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_27.jpg",
+    "caption": "**Fig. R28 | Ganglion biocompatibility of targeted injections of PtNP or PBS after NIR-II irradiation and after 30 days of follow-up. a,** Representative images of H&E and TUNEL staining of NG from different treatment groups immediately after NIR-II irradiation or after 30 days of follow-up. **b,** Representative images of H&E and TUNEL staining of LSG from different treatment groups immediately after NIR-II irradiation or after 30 days of follow-up.",
+    "footnote": [],
+    "bbox": [
+      [
+        147,
+        201,
+        850,
+        570
+      ]
+    ],
+    "page_idx": 34
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_28.jpg",
+    "caption": "**Fig. R29 | Effect of PtNP-shell photothermal stimulation of NG.** Maximal HR changes of beagles treatment with PtNP-shell or control from 1 to 3 hours after NIR irradiation, \\(n = 6\\). Data are shown as the mean \\(\\pm\\) S.E.M. *\\(P < 0.05\\), **\\(P < 0.01\\), ns means that the difference is not statistically significant.",
+    "footnote": [],
+    "bbox": [
+      [
+        101,
+        707,
+        897,
+        848
+      ]
+    ],
+    "page_idx": 35
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_29.jpg",
+    "caption": "Fig. R30 | Effect of PtNP-shell photothermal inhibition of LSG. Maximal SBP changes of beagles treatment with PtNP-shell or control from 1 to 3 hours after NIR irradiation, \\(\\mathrm{n} = 6\\) . Data are shown as the mean \\(\\pm\\) S.E.M. \\(^\\ast \\mathrm{P}< 0.05\\) , \\(^{**}P< 0.01\\) , ns means that the difference is not statistically significant.",
+    "footnote": [],
+    "bbox": [
+      [
+        100,
+        58,
+        896,
+        199
+      ]
+    ],
+    "page_idx": 35
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_30.jpg",
+    "caption": "Fig. R31 | Long term biosafety of PtNP-shell microinjection. Long-term in vivo biosafety was assessed by local injection of PtNP-shell into the ganglion of Beagle or by injection of equal doses of PtNP-shell into the tail vein of Sprague-Dawley rats. a, Representative H&E staining of major organs of beagles following different treatments. Blood biochemical analyses including b, ALT, c, AST, d, Urea, e, Crea, f, LDH1, g, TNF- \\(\\alpha\\) , and h, IL-6 were performed on Beagles in different treatment groups. i, Representative H&E staining of major organs of rats following different treatments. Blood biochemical analyses including j, ALT, k, AST, l, Urea, m, Crea, n, LDH1, o, TNF- \\(\\alpha\\) and p, IL-6 were performed on rats in different treatment groups.",
+    "footnote": [],
+    "bbox": [
+      [
+        144,
+        295,
+        851,
+        740
+      ]
+    ],
+    "page_idx": 36
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_31.jpg",
+    "caption": "Fig. R1 | Flowchart of regulating NG to protect against myocardial I/R injury and associated VAs.",
+    "footnote": [],
+    "bbox": [
+      [
+        241,
+        781,
+        756,
+        895
+      ]
+    ],
+    "page_idx": 36
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_32.jpg",
+    "caption": "Fig. R2 | Take home figure working model for b-adrenergic activation induced cardiac inflammatory cascade which finally results in cardiac remodeling (left) and therapeutic strategy (right) (Eur. Heart J. 2018, 39, 60).",
+    "footnote": [],
+    "bbox": [
+      [
+        205,
+        66,
+        792,
+        383
+      ]
+    ],
+    "page_idx": 41
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_33.jpg",
+    "caption": "Fig. R3 | Myocardial area at risk and infarct size. a, Area at risk expressed as \\(\\%\\) of left ventricle and b, infarct size (with representative images of infarcted myocardium) expressed as \\(\\%\\) of the area at risk following \\(30\\mathrm{min}\\) ischaemia and 2-h reperfusion in control animals (Control IR; \\(\\mathrm{n} = 14\\) ), after vagal nerve stimulation (VNS + IR; \\(\\mathrm{n} = 13\\) ), \\(\\alpha 7\\mathrm{nAChR}\\) blockade and VNS (MLA + VNS + IR; \\(\\mathrm{n} = 7\\) ), the arginase inhibitor nor-NOHA and IR (nor-NOHA + IR, \\(\\mathrm{n} = 5\\) ), nor-NOHA+VNS+IR ( \\(\\mathrm{n} = 6\\) ) and MLA alone ( \\(\\mathrm{n} = 5\\) ). Data are shown as mean \\(\\pm\\) SEM. Significant differences between groups are shown; \\(*P< 0.05\\) and \\(\\ddagger P< 0.001\\) (Acta Physiol. 2017, 221, 174).",
+    "footnote": [],
+    "bbox": [
+      [
+        179,
+        470,
+        812,
+        778
+      ]
+    ],
+    "page_idx": 44
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_34.jpg",
+    "caption": "Fig. R4 | The XPS survey spectra of a, GaNPs, b, Ga@Pt NPs and c, PtNP-shell.",
+    "footnote": [],
+    "bbox": [
+      [
+        130,
+        633,
+        866,
+        784
+      ]
+    ],
+    "page_idx": 44
+  }
+]

peer_reviews/supplementary_0_Peer Review File__0120cbbdf5abcce247bf35686d0d3fbc3c94f93c709d874b56ecf9271a6516aa/supplementary_0_Peer Review File__0120cbbdf5abcce247bf35686d0d3fbc3c94f93c709d874b56ecf9271a6516aa.mmd

@@ -0,0 +1,685 @@
+
+# nature portfolio  
+
+Peer Review File  
+
+Pt nanoshell with ultra- high NIR- II photothermal conversion efficiency mediates multimodal neuromodulation against ventricular arrhythmias  
+
+![](images/Figure_unknown_0.jpg)
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+Reviewers' Comments:  
+
+Reviewer #1:  
+
+Remarks to the Author:  
+
+In this manuscript, the authors synthesized Pt nanoshells for cardiac protection through photothermal therapy. The photothermal conversion efficiency of the Pt nanoshells was studied. In the in vitro and in vivo studies, the effect of photothermal therapy on TRPV1 and TREK1 channels were studied. I have the following comments.  
+
+1. The effect of photothermal therapy in the NIR-I window on ventricular arrhythmias was reported by the authors previously. In this study, the authors used the laser irradiation in the NIR-II instead. In fact, the irradiation was directly performed at the NG and LSG with microinjection of the nanoparticles. There is no need to use PTT in the NIR-II. Therefore, this work lacks novelty. 
+2. In the supporting information, the authors suggested that the photothermal conversion efficiency of Pt nanoshells was higher than other nanoparticles in previous reports. It is not accurate. 
+3. The Pt nanoshells don't have a unique strong absorption peak in the NIR-II range. Therefore, they are not good candidates for PTT in the NIR-II window. 
+4. In the in vitro cell study, the PTT study was performed in a 35 mm confocal dish. However, the irradiation diameter of the laser was very small. Most the cells in the dish cannot received the laser irradiation. It should be another concern.  
+
+Reviewer #2:  
+
+Remarks to the Author:  
+
+Wang, Zhou and Liu present a paper describing a \(\sim 200 \text{nm}\) platinum- based, PEG- modified nanoparticle (NP) with high absorbance of near infra- red \((1,000 \text{nm} +)\) light. The synthesised NP has high photothermal efficiency, which the authors used to stimulate heat- sensitive TRPV1 and TREK1 ion channels. First, they demonstrate this using HT- 22 hippocampus neuronal cells, they then applied it to a canine myocardial infarction model, stimulating channels in the nodose ganglion (NG) and left stellate ganglion (LSG). The aim is to stimulate the parasympathetic nervous system and inhibit the sympathetic nervous system. The authors then show changes in ventricular electrophysiology and reduced biomarkers of cardiac injury, plus some basic biocompatibility data.  
+
+Overall, there are many positive aspects of the work. I find that the paper is very well presented, with nicely laid out, attractive and well designed figures with clear illustrations. The methods section, at the end of the main document, is also very detailed. However, I find that the main manuscript is quite difficult to follow in some places. There are a lot of abbreviations - many are used without first defining them (though they can be found in the methods section). There is also very little introduction or background information given, meaning that the rationale of the study is not very clearly spelled out. No hypothesis or research aims are stated.  
+
+I think it is also somewhat unusual that the introduction section starts to describe results (line 54 onwards) and even refers to figures (even if they are just schematic diagrams.)  
+
+Similarly, the results section offers very little narrative or explanation of the results, which makes it difficult to follow the rationale of each experiment. For example, Vacht and c- fos staining are introduced with no explanation of what those are, why they are being stained, or what those results actually mean.  
+
+The work appears to be original. The authors have previously used the same canine I/R models and stimulation of the NG and LSG by other means, but I do not find this NP formulation described previously. Statistical tests, sample sizes and P values are mostly clearly annotated and described, and seem appropriate for the types of data being analysed. Sample sizes also seem reasonable, though it not always clear which data points are replicates, independent biological samples, or
+
+<--- Page Split --->
+
+actual experimental repeats.  
+
+I do not find any major flaws with the NP synthesis or characterisation aspects, though this is not my specialty. However, I do have a few questions about the canine I/R model:  
+
+The text mentions that "The NG was subsequently exposed to NIR- II laser irradiation for a duration of 5 minutes prior to occlusion of the left anterior descending (LAD) coronary artery for reperfusion therapy." This text, and the diagram in Figures 4B, 5B and 6B, explain that the treatment was done before the I/R was induced. I am curious about why the authors choose a pre- treatment experimental design, rather than initiating the treatment after reperfusion. Obviously investigating only pre- treatment greatly lowers the clinical/therapeutic relevance of the research.  
+
+Related to this, why do the authors think there is a reduction of serum troponin and myoglobin? (It is also not clear at what time point these samples were taken. Does this reflect acute cardio protection?) In my opinion, if the authors want to say that the system is cardioprotective (line 292) or protects against cardiac damage (line 350), additional metrics (echocardiography, infarct volume etc) would be required to support this claim.  
+
+Figure 4f and 4g are a little difficult to understand. The Y axis is different between the two graphs, but it seems that the effect on max HR is quite mild? I think it would make sense to put the baseline and laser irradiation groups on the same graph and statistically compare those too.  
+
+I am curious about the overall delivery efficiency, and biodistribution of the NPs and whether direct injection into the NG/LSG is clinically applicable.  
+
+I also note that the in vivo comparisons are simply nanoparticles vs. PBS. This can clearly demonstrate that the NPs have some activity; but how do they compare to other methods of stimulating the NG/LSG, or drugs which stimulate the sympathetic/parasympathetic nervous system? Without these sorts of comparison, it is difficult to put the significance of authors' findings into context. If the authors can demonstrate that their approach is better than other approaches, this could be a lot more convincing.  
+
+In terms of overall interest, I think is a good technical demonstration of clever system; but what is the real- world application? Could the authors envisage a way in which this technology can actually be applied to MI patients?  
+
+The abstract mentions that the NPs conferred protection against ventricular arrhythmias following MI. However, supplementary figure 25 seems to show that there was no difference in overall VA events.  
+
+Minor points:  
+
+Figure 3i is not very easy to read or understand.  
+
+Line 373, I think that more than a few blood tests and some organ histologically is required to make such a strong claim of "unequivocally demonstrate" long- term safety.  
+
+Supplementary figure 6, 13. I think these would be more readable as tables rather than bar charts.  
+
+Supplementary figure 7; this is quite a broad range of nanoparticle sizes. What makes up the smaller (100 nm) particles? Is there aggregation to produce larger particles?  
+
+Reviewer #3:  
+
+Remarks to the Author:  
+
+The manuscript describes photothermal neuromodulation via Pt nano- shell nanoparticles. Ga nanoparticles are used as a template for electrocoupling substitution- based synthesis of the Pt nano- shell. Using KOH wet etching, Ga core is etched and a Pt nano- shell structure is obtained. The rough surface topography of the Pt nano- shell structure allows the particles to exhibit high
+
+<--- Page Split --->
+
+optical absorbance. It is claimed that these particles have one of the highest photothermal energy conversion efficiencies. The photothermal conversion of optical irradiation is then utilized for stimulation of target cells and tissues via temperature activated ion channels- TRPV1 and TREK1. The potential application of such photothermal modulation technique in regulating cardiac pulsing is demonstrated with regards to protecting against acute ventricular arrhythmias. However, the manuscript does not include proper controls to demonstrate that in- vivo photothermal modulation is achieved exclusively through the Pt nanoparticles. In addition, there are certain claims and results that need to be better corroborated to reach the scientific requirements of the journal. Therefore, I cannot recommend that this manuscript be accepted at Nature Communications in its current form.  
+
+1. Abstract: It is claimed that "the autonomic nervous system plays a pivotal role in the pathophysiology of cardiovascular diseases." This sentence is misleading since the dysregulation of the autonomic nervous system can contribute to cardiovascular diseases. However, it is not the primary contributor to the diseases, autonomous nervous system in fact regulates normal functioning of the cardiovascular system. (see: Purves D, Augustine GJ, Fitzpatrick D, LaMantia AS, McNamara JO, Williams SM. Autonomic regulation of cardiovascular function. Neuroscience. 2001:491-3 AND Gordan R, Gwathmey JK, Xie LH. Autonomic and endocrine control of cardiovascular function. World journal of cardiology. 2015 Apr 4;7(4):204.) 
+2. The authors claim that bi-directional reversible autonomic modulation is achieved via NIR-II photothermal modulation using Pt nano-shell nanoparticles. The manuscript presents unidirectional modulation where the target tissues are stimulated. Bi-directionality isn't demonstrated since in terms of neural interfaces, bi-directionality refers to the ability to record neural activity as well as stimulate (see: Song KI, Seo H, Seong D, Kim S, Yu KJ, Kim YC, Kim J, Kwon SJ, Han HS, Youn I, Lee H. Adaptive self-healing electronic epineurium for chronic bidirectional neural interfaces. Nature communications. 2020 Aug 21;11(1):4195. AND Hughes C, Herrera A, Gaunt R, Collinger J. Bidirectional brain-computer interfaces. Handbook of clinical neurology. 2020 Jan 1;168:163-81.). 
+3. It is unclear why the NIR-II range was utilized in this work. This is important for selecting the right materials, models, and experiments. (see: nature.com/articles/s44222-023-00022-y AND nature.com/articles/s41551-022-00862-w). 
+4. Instead of using terminologies like "nearly perfect blackbody absorption", the actual optical properties and metrics should be presented. 
+5. Figure 1 presents how the nanoparticles will interact with the biological systems, however it does not show how light pulses/irradiation will be delivered to the target tissues/sites. This should be discussed in the figure and the manuscript since it is important for clinical translation. 
+6. Adequate controls should be provided to better compare the physical properties of PtNP-shells. That is, please provide the optical absorbance of GaNPs, Pt coated GaNPs for Figure 2.d; similar controls should be provided for Figure 2.e (including the thermal transients of such the solvent under irradiation. 
+7. For the XPS characterization, a survey scan of presentative sample should be presented along with the detailed XPS characterization of oxygen (O1s) and potassium (K2p). The elemental composition of the PtNP-shells, Pt coated GaNPs, and GaNPs should be compared as well. This will better elucidate the composition of effectiveness of the synthesis protocols. 
+8. The stability of the Pt-nanoshell suspensions should be evaluated as a function of time. Do the nanoparticle aggregate over time? Will this be a concern when the Pt-nanoshells are injected into biological systems. 
+9. How does the addition of mPEG-SH5000 effect the photothermal properties of the nanoparticles? 
+10. Critical information from the methods section is missing- for example, details regarding the cell culture protocol and photothermal stimulation (such as power and pulse duration of optical irradiation are missing). How long was the ECG data recorded for? What were the exact stimulation conditions for all in-vivo experiments? 
+11. For the in-vitro experiments, are the Pt nanoparticles engulfed by the target cells or are they localized in the vicinity of the cell membrane?
+
+<--- Page Split --->
+
+12. Both in-vivo and in-vitro photothermal stimulation experiments require the cells' microenvironment to reach temperatures greater than \(42^{\circ}C\) . Does repeated photothermal stimulation using such high temperatures adversely affect cellular health by disrupting the cell membrane or trigger heat shock response?  
+13. The claim that Pt-NP shell does not induce significant damage to neurons under controlled NIR-II laser irradiation is incorrect since there is \(\sim 10\%\) loss in cellular viability.  
+14. It will be recommended that the data presentation in Figure 3.1 be changed since the details of the data are difficult to comprehend through a 3-D plot.  
+15. For the in-vivo photothermal stimulation experiments, can similar affects be achieved without the presence of the Pt-nanoshell particles? Figure 4.d presents high temperature gradients for the surrounding tissue as well. Stimulation using infra-red radiation has been demonstrated previously, see: doi.org/10.1364/OL.30.000504 and doi.org/10.1117/1.2121772.  
+16. The biosafety of Pt-nanoshell particles was evaluated after a rapid excision of the LSG and NG tissues. Can the authors comments on the long-term biosafety of the nanoparticles in passive (without photothermal stimulation) and active (with photothermal stimulation) states?  
+17. Page 2, line 21: Please include examples and appropriate references for "conventional international procedures for MI."  
+18. Page 4, line 16: Please change the word "encapsulated on" since Pt is not encapsulated on the surface of GaNPs. Pt is deposited onto of GaNP core then it encapsulates GaNP core.
+
+<--- Page Split --->
+
+## Reply to the referees  
+
+## To Referee #1  
+
+First of all, we really appreciate your constructive comments. We have made a point- by- point response to your comments and carefully revised the manuscript as you suggested. For your reference, please find our revisions marked in red color.  
+
+Comment 1: In this manuscript, the authors synthesized Pt nanoshells for cardiac protection through photothermal therapy. The photothermal conversion efficiency of the Pt nanoshells was studied. In the in vitro and in vivo studies, the effect of photothermal therapy on TRPV1 and TREK1 channels were studied. I have the following comments. The effect of photothermal therapy in the NIR- I window on ventricular arrhythmias was reported by the authors previously. In this study, the authors used the laser irradiation in the NIR- II instead. In fact, the irradiation was directly performed at the NG and LSG with microinjection of the nanoparticles. There is no need to use PTT in the NIR- II. Therefore, this work lacks novelty.  
+
+Author reply: Thank you for your comments. The focus of our research is to utilize PtNP- shell with near- perfect blackbody absorption and high photothermal conversion efficiency, enabling safer and more precise bidirectional deep neural modulation of the nodose ganglion (NG) and the left stellate ganglion (LSG). Moreover, compared to the first near- infrared (NIR- I, 650–900 nm) and visible window, the photons in the second near- infrared window (NIR- II, 900–1700 nm) exhibit reduced tissue scattering and absorption, thereby increasing the maximum allowable exposure (MPE) of biological tissues. This means photons within the NIR- II window exhibit significantly enhanced tissue penetration depths (up to 5–20 mm) (Nat. Nanotech. 2009, 4, 710; Nat. Med. 2012, 18, 1841; Nat. Biomed. Eng. 2017, 1, 0010). We developed PtNP- shell and validated its photothermal neuromodulation efficacy in the NIR- II window both in vivo and in vitro. Given its wavelength independence, further investigations may facilitate the selection of a more suitable laser for achieving deeper tissue penetration while adhering to the MPE range. The exceptional potential of PtNP- shell makes it highly promising for precise neural regulation in deeper tissue. Moreover, NG and LSG are pivotal nodes of the vagal loop and sympathetic loop, respectively. In contrast to previous studies, our approach not only achieves neural activity inhibition but also enables nerve activation for bidirectional reversible modulation. Furthermore, we substantiate the therapeutic efficacy of this strategy in diverse models of cardiac injury.  
+
+Comment 2: In the supporting information, the authors suggested that the photothermal conversion efficiency of Pt nanoshells was higher than other nanoparticles in previous reports. It is not accurate.
+
+<--- Page Split --->
+
+**Author reply:** Thank you for the comment. The PtNP-shell exhibits an exceptionally high photothermal conversion efficiency, surpassing the values reported in Supplementary Table 2 (Table R1). To ensure utmost rigor, we have revised the statement to “among the highest”.
+
+Table R1 | Comparison of photothermal conversion efficiency.
+
+<table><tr><td></td><td>Photothermal conversion efficiency(%)</td></tr><tr><td>This work</td><td>73.70</td></tr><tr><td>PEDOT:ICG@PEG-GTA1</td><td>71.10</td></tr><tr><td>MINDS2</td><td>71.00</td></tr><tr><td>PTG NPs3</td><td>67.60</td></tr><tr><td>RBC@Cu2-xSeNPs4</td><td>67.20</td></tr><tr><td>AuDAg2S5</td><td>67.10</td></tr><tr><td>MAPSULES6</td><td>67.00</td></tr><tr><td>Fe3O4@PPy@GOD NCs7</td><td>66.40</td></tr><tr><td>NPPBTPBF-BT8</td><td>66.40</td></tr><tr><td>AS10649</td><td>65.92</td></tr><tr><td>Gold Nanorasperrry10</td><td>65.00</td></tr><tr><td>P-Pc-HSA11</td><td>64.70</td></tr><tr><td>Ultrathin polypyrrole nanosheets12</td><td>64.60</td></tr><tr><td>\(\mathrm {H}_{x}\mathrm {MoO}_{3}^{13}\)</td><td>60.90</td></tr><tr><td>NiP PHNPs14</td><td>56.80</td></tr><tr><td>FP NRs15</td><td>56.60</td></tr><tr><td>COF16</td><td>55.20</td></tr><tr><td>2MPT-\(-CB^{17}\)</td><td>54.60</td></tr><tr><td>SPN-PT18</td><td>53.00</td></tr><tr><td>Pdots19</td><td>53.00</td></tr><tr><td>Pt Spirals20</td><td>52.50</td></tr><tr><td>TBDOPV-DT21</td><td>50.50</td></tr><tr><td>\(\mathrm {Ti}_{2}\mathrm {O}_{3}\)@HA NPs22</td><td>50.20</td></tr><tr><td>TBDOPV-DT NP23</td><td>50.00</td></tr><tr><td>DPP-IID-FA NPs24</td><td>49.50</td></tr><tr><td>SPN-DT18</td><td>49.00</td></tr><tr><td>NP25</td><td>49.00</td></tr><tr><td>FTQ nanoparticles26</td><td>49.00</td></tr><tr><td>CNPs27</td><td>49.00</td></tr><tr><td>\(\mathrm {H-SiO}_{x}\) NPs28</td><td>48.60</td></tr><tr><td>BETA NPs29</td><td>47.60</td></tr><tr><td>CN-NPs30</td><td>47.60</td></tr><tr><td>Pt-NDs31</td><td>46.90</td></tr><tr><td>\(\mathrm {MoO}_{3-x}\)nanobelts32</td><td>46.90</td></tr></table>
+
+<--- Page Split --->
+
+
+<table><tr><td>P3 NPs33</td><td>46.00</td></tr><tr><td>Ni3S34</td><td>46.00</td></tr><tr><td>PtAg nanosheets35</td><td>45.70</td></tr><tr><td>Nb2C NSs36</td><td>45.65</td></tr><tr><td>V2C-TAT@Ex-RGD37</td><td>45.10</td></tr><tr><td>PEG-TONW NRs38</td><td>43.60</td></tr><tr><td>SPNI-II39</td><td>43.40</td></tr><tr><td>1T-MoS240</td><td>43.30</td></tr><tr><td>Bi@C NPs41</td><td>43.20</td></tr><tr><td>Au NPL@TiO242</td><td>42.10</td></tr><tr><td>CT NPs43</td><td>42.00</td></tr><tr><td>PPy-PEG NPs44</td><td>41.97</td></tr><tr><td>AuPt@CuS NSs45</td><td>41.56</td></tr><tr><td>Bi15S27I3 nanorods46</td><td>41.50</td></tr><tr><td>Cu3BiS3 NR47</td><td>40.70</td></tr><tr><td>MPAE-NPS48</td><td>40.07</td></tr></table>  
+
+Comment 3: The Pt nanoshells don't have a unique strong absorption peak in the NIR- II range. Therefore, they are not good candidates for PTT in the NIR- II window.  
+
+Author reply: The PtNP shell exhibits strong absorption across a broad range of wavelengths, including the NIR- II window. We posit that the PTNP- shell holds promise as an excellent candidate for photothermal therapy (PTT) within the NIR- II window. Given its wavelength independence, further investigations may facilitate selection of a more suitable laser for achieving deeper tissue penetration while adhering to the MPE range, thereby enabling precise and noninvasive neural modulation.  
+
+![](images/Figure_unknown_1.jpg)
+
+<center>Fig. R1 | Absorption of PtNP-shell. a, Absorption curves of PtNP-shell with different concentrations (10, 25, 50 and \(75\mu \mathrm{g}\cdot \mathrm{mL}^{-1}\) ). b, Mass extinction coefficient of PtNP-shell at \(1064\mathrm{nm}\) . Normalized absorbance intensity at \(\lambda = 1064\mathrm{nm}\) divided by the characteristic length of the cell (A/L) at different concentrations (10, 25, 50 and \(75\mu \mathrm{g}\cdot \mathrm{mL}^{-1}\) ). </center>
+
+<--- Page Split --->
+
+Comment 4: In the in vitro cell study, the PTT study was performed in a 35 mm confocal dish. However, the irradiation diameter of the laser was very small. Most the cells in the dish cannot received the laser irradiation. It should be another concern.  
+
+Author reply: Thank you for the comment. With reference to previous photothermal research on cells (Nat. Biomed. Eng. 2022, 6, 754; Nano Converg. 2022, 9, 13), we conducted a cell calcium imaging experiment wherein the NIR- II laser was utilized to activate neuron cells within the confocal dish covered by the laser beam. We employed confocal microscopy to capture images of these neuron cells covered by laser beam and make quantitative analysis of calcium imaging. Consequently, the dimensions of the confocal dish did not influence the outcomes of the cellular calcium imaging experiment. A more comprehensive explanation is provided in the “Method” section:  
+
+“XYT images in the region of 1064 nm illumination were acquired and collected under a 20x objective lens.”
+
+<--- Page Split --->
+
+## To Referee #2  
+
+We appreciate your positive evaluation on our manuscript. We have made a point- by- point response to your comments and carefully revised the manuscript as you suggested. For your reference, please find our revisions marked in red color.  
+
+Comment 1: Wang, Zhou and Liu present a paper describing a \(\sim 200 \mathrm{nm}\) platinum- based, PEGmodified nanoparticle (NP) with high absorbance of near infra- red (1,000 \(\mathrm{nm + }\) ) light. The synthesised NP has high photothermal efficiency, which the authors used to stimulate heat- sensitive TRPV1 and TREK1 ion channels. First, they demonstrate this using HT- 22 hippocampus neuronal cells, they then applied it to a canine myocardial infarction model, stimulating channels in the nodose ganglion (NG) and left stellate ganglion (LSG). The aim is to stimulate the parasympathetic nervous system and inhibit the sympathetic nervous system. The authors then show changes in ventricular electrophysiology and reduced biomarkers of cardiac injury, plus some basic biocompatibility data. Overall, there are many positive aspects of the work. I find that the paper is very well presented, with nicely laid out, attractive and well designed figures with clear illustrations. The methods section, at the end of the main document, is also very detailed. However, I find that the main manuscript is quite difficult to follow in some places. There are a lot of abbreviations - many are used without first defining them (though they can be found in the methods section). There is also very little introduction or background information given, meaning that the rationale of the study is not very clearly spelled out. No hypothesis or research aims are stated.  
+
+Author reply: Thank you for your recognition of our work. The advices you put forward are very constructive and help a lot for us to improve our manuscript.  
+
+We have provided reasonable descriptions of specialized acronyms that appear for the first time to increase readability. In addition, we have followed up on your comments to optimize the "introduction". The background and rationale for the development and application of this strategy are described in more detail, and our research hypotheses and objectives are added.  
+
+"However, left cardiac sympathetic denervation (LCSD), stellate ganglion block (SGB), and renal denervation (RDN) are associated with certain adverse effects, including Horner's syndrome (Circ.- Arrhythmia Electrophysiol. 2015, 8, 1007), inadvertent bleeding, and inconsistent ablation outcomes (N. Engl. J. Med. 2014, 370, 1393). Both conventional low- level vagus nerve stimulation (LL- VNS) and optogenetic neuromodulation necessitate the implantation of in vivo electrical stimulation (JACC- Clin. Electrophysiol. 2017, 3, 929) or light source devices (J. Am. Coll. Cardiol. 2017, 70, 2778). Furthermore, optogenetic neuromodulation requires viral transfection of photosensitive proteins (J. Am. Coll. Cardiol. 2017, 70, 2778), thereby limiting the clinical advancement of these therapeutic approaches."
+
+<--- Page Split --->
+
+"The light in the second near-infrared window (NIR- II, 900- 1700 nm) has reduced tissue scattering and absorption and increased maximum permissible exposure (MPE) for biological tissues compared to the light in the first near- infrared (NIR- I, 650- 900 nm) and visible window (Acc. Chem. Res. 2018, 51, 1840). Consequently, this enables non- invasive and non- implantable neuromodulation using NIR- II photothermal."  
+
+"Therefore, the objective of this study is to develop a Pt nanoparticle shell (PtNP- shell) with near- blackbody properties and ultra- high PCE in the NIR- II window (Fig. 1a) and investigate its potential in protecting against MI and myocardial reperfusion injury accompanying intervention through rapid, efficient, and precise multifunctional autonomic neuromodulation (Fig. 1b)."  
+
+Comment 2: I think it is also somewhat unusual that the introduction section starts to describe results (line 54 onwards) and even refers to figures (even if they are just schematic diagrams).  
+
+Author reply: Following your suggestion, we have revised the introduction by modifying the section describing results into one that outlines research assumptions and objectives, while enhancing the background information and fundamental principles underlying the development and application of this strategy.  
+
+"Therefore, the objective of this study is to develop a Pt nanoparticle shell (PtNP- shell) with near- blackbody properties and ultra- high PCE in the NIR- II window (Fig. 1a) and investigate its potential in protecting against MI and myocardial reperfusion injury accompanying intervention through rapid, efficient, and precise multifunctional autonomic neuromodulation (Fig. 1b)."  
+
+Comment 3: Similarly, the results section offers very little narrative or explanation of the results, which makes it difficult to follow the rationale of each experiment. For example, Vacht and c- fos staining are introduced with no explanation of what those are, why they are being stained, or what those results actually mean.  
+
+Author reply: Thank you for your kind reminding. We have shown immunofluorescence staining in more details:  
+
+"Furthermore, immunofluorescence staining for vesicular acetylcholine transporter protein (VACht), c- Fos, and TRPV1 on histopathological sections of photothermally modulated NGs served to localize parasympathetic neurons and reflect neuronal activity as well as TRPV1 protein expression, respectively (Fig. 4i)."  
+
+"Furthermore, immunofluorescence staining was conducted on LSG tissues to examine the expression of tyrosine hydroxylase (TH), c- Fos, and TREK1 (Fig. 6i). Localization of sympathetic neurons by TH proteins and reflection of neuronal activity as well as TREK1 protein expression."
+
+<--- Page Split --->
+
+Comment 4: The work appears to be original. The authors have previously used the same canine I/R models and stimulation of the NG and LSG by other means, but I do not find this NP formulation described previously. Statistical tests, sample sizes and \(P\) values are mostly clearly annotated and described, and seem appropriate for the types of data being analysed. Sample sizes also seem reasonable, though it not always clear which data points are replicates, independent biological samples, or actual experimental repeats.  
+
+Author reply: To ensure the reproducibility and accuracy of our study, we repeated the experiment with independent biological samples in vitro and in vivo experiments to obtain the statistical information, including Fig 3d- j, Fig 4f- h, Fig 5e- k, Fig 6f- h, and Fig 7d- i, as well as the corresponding supplemental data.  
+
+Comment 5: I do not find any major flaws with the NP synthesis or characterisation aspects, though this is not my specialty. However, I do have a few questions about the canine I/R model: The text mentions that "The NG was subsequently exposed to NIR- II laser irradiation for a duration of 5 minutes prior to occlusion of the left anterior descending (LAD) coronary artery for reperfusion therapy." This text, and the diagram in Figures 4B, 5B and 6B, explain that the treatment was done before the I/R was induced. I am curious about why the authors choose a pre- treatment experimental design, rather than initiating the treatment after reperfusion. Obviously investigating only pre- treatment greatly lowers the clinical/therapeutic relevance of the research.  
+
+Author reply: Thank you for the comment. The clinical management of acute myocardial infarction primarily involves transvascular interventions to restore blood flow in occluded vessels. However, reperfusion during this process can lead to acute ventricular arrhythmic events (Mediat. Inflamm. 2017, 2017, 14). To mitigate these adverse effects through neuromodulation, we performed NIR- II irradiation intervention prior to reperfusion injury simulation, mimicking the clinical practice scenario. Furthermore, photothermal activation or inhibition of nerves exhibits sustained efficacy. This experimental protocol aligns more closely with the clinical significance of reducing reperfusion injury in patients undergoing myocardial infarction treatment.  
+
+Comment 6: Related to this, why do the authors think there is a reduction of serum troponin and myoglobin? (It is also not clear at what time point these samples were taken. Does this reflect acute cardio protection?) In my opinion, if the authors want to say that the system is cardioprotective (line 292) or protects against cardiac damage (line 350), additional metrics (echocardiography, infarct volume etc) would be required to support this claim.  
+
+Author reply: Thank you for the comment. Serum troponin and myoglobin are the primary and crucial clinical indicators of myocardial injury. Myoglobin levels typically increase within 1 to 2
+
+<--- Page Split --->
+
+hours following myocardial injury, while troponin elevation usually occurs 2 to 3 hours later. These two serum markers of cardiac injury can remain elevated for approximately 12 hours (Int. J. Cardiol. 2005, 98, 285). By implementing this experimental protocol, which involves collecting and testing blood samples at a time interval of 4 to 5 hours after modeling cardiac injury, accurate detection of myocardial injury in diverse individuals is facilitated.  
+
+Furthermore, significant differences in infarct area detection or ultrasound cardiac function testing can be observed in long- term myocardial ischemia models (Adv. Sci. 2023, 10, 2205551; Basic Res. Cardiol. 2022, 117, 34). Previous studies have usually indicated the extent of myocardial injury through acute indices, such as biomarkers of myocardial injury, in acute myocardial ischemia models (Adv. Mater. 2023, 35, 2304620). We utilized serum markers of myocardial injury to reflect the severity of acute myocardial infarction or acute reperfusion injury.  
+
+Comment 7: Figure 4f and 4g are a little difficult to understand. The Y axis is different between the two graphs, but it seems that the effect on max HR is quite mild? I think it would make sense to put the baseline and laser irradiation groups on the same graph and statistically compare those too.  
+
+Author reply: Thank you for the comment. We adjusted the Y axis of Fig. 4f (Fig. R2a) to align with Fig. 4g (Fig. R2b), facilitating a direct comparison of neural function between baseline and laser irradiation conditions. Considering our aim to demonstrate the neural functions of both the PtNP- shell group and control group under these two conditions, presenting the data separately would enhance clarity rather than combining them in one graph. This separate presentation allows for visualization that there is no significant difference between the two groups in the baseline condition, while highlighting that after laser irradiation, PtNP- shell significantly enhances neural activity across all levels of stimulation.  
+
+![](images/Figure_unknown_2.jpg)
+
+<center>Fig. R2 | Maximal HR changes of beagle treatment with PtNP-shell or control a, before and b, after NIR-II exposure, \(n = 6\) . </center>
+
+<--- Page Split --->
+
+Comment 8: I am curious about the overall delivery efficiency, and biodistribution of the NPs and whether direct injection into the NG/LSG is clinically applicable.  
+
+Author reply: The ganglion's surface is enveloped by a dense connective tissue matrix, facilitating our direct microinjection of PtNP- shell into the ganglion with exceptional efficiency and minimal loss. Long- term biosafety monitoring revealed an absence of NP distribution in histologic examinations of vital metabolic- immune organs such as the heart, liver, spleen, lungs, and kidneys, while liver and kidney functions remained unaffected (Fig. R3 and R4).  
+
+In clinical practice, ultrasound- guided injection of nerve blocking drugs into the ganglion enables local ganglion blockage (Curr. Pain Headache Rep. 2014, 18, 424). Our experimental approach aligns with established techniques for ganglion blocks and thus holds potential applicability in clinical settings.  
+
+![](images/Figure_unknown_3.jpg)
+
+<center>Fig. R3 | Ganglion biocompatibility of targeted injections of PtNP or PBS after NIR-II irradiation and after 30 days of follow-up. a, Representative images of H&E and TUNEL staining of NG from different treatment groups immediately after NIR-II irradiation or after 30 days of follow-up. b, Representative images of H&E and TUNEL staining of LSG from different treatment groups immediately after NIR-II irradiation or after 30 days of follow-up. </center>
+
+<--- Page Split --->
+![](images/Figure_unknown_4.jpg)
+
+<center>Fig. R4 | Long term biosafety of PtNP-shell microinjection. Long-term in vivo biosafety was assessed by local injection of PtNP-shell into the ganglion of Beagle or by injection of equal doses of PtNP-shell into the tail vein of Sprague-Dawley rats. a, Representative H&E staining of major organs of beagles following different treatments. Blood biochemical analyses including b, ALT, c, AST, d, Urea, e, Crea, f, LDH1, g, TNF- \(\alpha\) , and h, IL-6 were performed on Beagles in different treatment groups. i, Representative H&E staining of major organs of rats following different treatments. Blood biochemical analyses including j, ALT, k, AST, l, Urea, m, Crea, n, LDH1, o, TNF- \(\alpha\) and p, IL-6 were performed on rats in different treatment groups. </center>  
+
+Comment 9: I also note that the in vivo comparisons are simply nanoparticles vs. PBS. This can clearly demonstrate that the NPs have some activity; but how do they compare to other methods of stimulating the NG/LSG, or drugs which stimulate the sympathetic/parasympathetic nervous system? Without these sorts of comparison, it is difficult to put the significance of authors' findings into context. If the authors can demonstrate that their approach is better than other approaches, this could be a lot more convincing.  
+
+Author reply: Thank you for the comment. Currently, \(\beta\) - blockers are the primary pharmacological drugs employed in clinical practice for arrhythmia treatment (J. Am. Heart Assoc. 2018, 7, e007567; Eur. Heart J. 2023, 44, 3720). However, their administration during acute myocardial ischemia remains unclear and is contraindicated in patients with heart failure. Additionally,
+
+<--- Page Split --->
+
+our previous research investigated the local ganglion blockade using botulinum toxin A to protect the heart (Heart Rhythm 2022, 19, 2095). Nevertheless, its prolonged blocking effect renders it unsuitable for acute myocardial ischemia management. Conversely, PtNP- shell- based photothermal regulation offers reversible modulation within a short timeframe, exhibiting superior efficacy and controllability.  
+
+Cardiac sympathetic denervation (CSD) is a clinical procedure aimed at targeting the autonomic ganglia for refractory ventricular arrhythmias treatment. However, ganglion removal can be traumatic for patients and may lead to complications due to the loss of original physiological function (Eur. Heart J. 2022, 43, 2096).  
+
+Therefore, we aim to investigate the precise photothermal neuromodulation strategy facilitated by PtNP- shell as a reversible intervention approach for inhibiting ventricular arrhythmia associated with ganglion dysfunction. Notably, modulation specifically targeting NG has not been reported thus far, while non- invasive reversible neuromodulation strategies against LSG have primarily focused on photothermal modulation. Consequently, our study mainly established control and NP photothermal modulation groups similar to previous investigations (Adv. Mater. 2023, 35, 2304620; Adv. Funct. Mater. 2019, 29, 1902128).  
+
+Comment 10: In terms of overall interest, I think is a good technical demonstration of clever system; but what is the real- world application? Could the authors envisage a way in which this technology can actually be applied to MI patients?  
+
+Comment 10: In terms of overall interest, I think is a good technical demonstration of clever system; but what is the real- world application? Could the authors envisage a way in which this technology can actually be applied to MI patients?Author reply: Thank you for the comment. Both myocardial ischemia and reperfusion injury- induced malignant arrhythmic events pose challenges in the treatment of acute myocardial infarction. Photothermal neuromodulation based on PtNP- shell offers a precise, transient, and reversible approach that may serve as an adjuvant therapy to improve patient prognosis. Additionally, ultrasound- guided targeted ganglionic microinjection is suitable for clinical applications. However, it is undeniable that despite the excellent tissue penetration depth of NIR- II photons (5–20 mm) (Nat. Nanotech. 2009, 4, 710; Nat. Med. 2012, 18, 1841; Nat. Biomed. Eng. 2017, 1, 0010), photothermal regulation still encounters challenges in intervening deeper tissues. Considering the presence of blood vessels surrounding the ganglion, which offers an opportunity to reduce the propagation path of photons in tissues, approaching the NIR- II fiber to the ganglion for photothermal modulation through a transvascular route during interventional therapy could be a potential solution for direct clinical translational application.  
+
+Comment 11: The abstract mentions that the NPs conferred protection against ventricular arrhythmias following MI. However, supplementary figure 25 seems to show that there was no difference in overall VA events.
+
+<--- Page Split --->
+
+**Author reply:** Thank you for the comment. The statistics of ventricular arrhythmia events encompass various forms, including ventricular premature beats (VPBs), ventricular tachycardia (VT), and ventricular fibrillation (VF). VT is defined as the occurrence of three or more consecutive VPBs. As depicted in Fig. R5 (Fig. 7i), the PtNP-shell group exhibited a significantly lower number of VPBs compared to the control group (p<0.05).  
+
+![](images/Figure_unknown_5.jpg)
+
+<center>Fig. R5 | Quantitative analysis of VF threshold in different groups. Data are shown as the mean \(\pm\) S.E.M. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001. </center>  
+
+Fig. R6 (Supplementary Fig. 28) presents a comparison between groups regarding the frequency and duration of VT occurrences. Although not statistically significant, there was a notable trend towards VT suppression in the NP group when compared to the control group. Supplementary Fig. 25 has been updated to Supplementary Fig. 28.  
+
+![](images/Figure_unknown_6.jpg)
+
+<center>Fig. R6 | Statistical analysis of recorded ventricular arrhythmia events post myocardial ischemia. Quantitative analysis the number of a, VTs and b, the duration of sVT of beagles with MI. Data are shown as the mean \(\pm\) S.E.M. ns means that the difference is not statistically significant. </center>  
+
+**Comment 12:** Figure 3i is not very easy to read or understand.  
+
+**Author reply:** Thank you for the comment. We conducted a repeated experiment and made modifications to fig3i, transforming the 3D diagram into two 2D diagrams for enhanced
+
+<--- Page Split --->
+
+comprehensibility. Fig. 3i (Fig. R7a) and Fig. 3j (Fig. R7b) depict cell viability following two different power density (0.75 W·cm⁻¹ and 1 W·cm⁻¹) NIR-II laser irradiations at varying time intervals, respectively.  
+
+![](images/Figure_unknown_7.jpg)
+
+<center>Fig. R7 | Effect of NIR-II laser irradiation with varying durations on the viability of HT-22 cells treated with PtNP-shell (50 µg·mL⁻¹) (Power densities: a, 0.75 W·cm⁻² and b, 1 W·cm⁻²). The error bar indicates S.E.M. </center>  
+
+Comment 13: Line 373, I think that more than a few blood tests and some organ histologically is required to make such a strong claim of "unequivocally demonstrate" long- term safety.  
+
+Author reply: Thank you for the comment. In supplementary Figure 27, we first verified neuronal safety by histologic examination of the microinjected ganglion tissue as well as tunnel staining. In addition, we monitored the biosafety of both local tissue- injected beagles and tail vein- injected rats for 1 month. The major organs of these individuals were examined histologically, including the heart, liver, spleen, lungs, and kidneys. The most important routes of elimination, such as liver function and kidney function, were also examined by serology. In addition, we analyzed the effect of PtNP- shell on the inflammatory response in vivo by ELISA assay (Fig. R8). These detailed tests demonstrated the long- term biosafety of the NPs. The corresponding results have been included in the Supplementary Fig. 31g,h,o,p.
+
+<--- Page Split --->
+![](images/Figure_unknown_8.jpg)
+
+<center>Fig. R8 | Blood biochemical analyses including a, TNF-α, and b, IL-6 were performed on Beagles in different treatment groups. Blood biochemical analyses including c, TNF-α and d, IL-6 were performed on rats in different treatment groups. </center>  
+
+Comment 14: Supplementary figure 6, 13. I think these would be more readable as tables rather than bar charts.  
+
+Author reply: Thank you for the comment. We have changed Supplementary Fig. 6 and 13 into Supplementary Table 1 (Table R2) and Supplementary Table 2 (Table R3), respectively.  
+
+Table R2 | \(\xi\) potential of bare PtNP-shell, PtNP-shell + KOH and PtNP-shell@PEG.   
+
+<table><tr><td></td><td>Zata Potential (mV)</td><td>St Dev(mV)</td></tr><tr><td>PtNP-shell</td><td>45.8</td><td>5.8</td></tr><tr><td>PtNP-shell + KOH</td><td>-25.7</td><td>4.64</td></tr><tr><td>PtNP-shell@PEG</td><td>-19.9</td><td>3.69</td></tr></table>  
+
+Table R3 | Comparison of photothermal conversion efficiency.   
+
+<table><tr><td></td><td>Photothermal conversion efficiency (%)</td></tr><tr><td>This work</td><td>73.70</td></tr><tr><td>PEDOT:ICG@PEG-GTA¹</td><td>71.10</td></tr><tr><td>MINDS²</td><td>71.00</td></tr><tr><td>PTG NPs³</td><td>67.60</td></tr><tr><td>RBC@Cu2-xSeNPs⁴</td><td>67.20</td></tr><tr><td>AuDAg₂S⁵</td><td>67.10</td></tr></table>
+
+<--- Page Split --->
+
+<table><tr><td>MAPSULES⁶</td><td>67.00</td></tr><tr><td>Fe₃O₄@PPy@GOD NCs⁷</td><td>66.40</td></tr><tr><td>NPPBTPBF-BT⁸</td><td>66.40</td></tr><tr><td>AS1064⁹</td><td>65.92</td></tr><tr><td>Gold Nanoraspberry¹⁰</td><td>65.00</td></tr><tr><td>P-Pc-HSA¹¹</td><td>64.70</td></tr><tr><td>Ultrathin polypyrrole nanosheets¹²</td><td>64.60</td></tr><tr><td>HₓMoO₃¹³</td><td>60.90</td></tr><tr><td>NiP PHNPs¹⁴</td><td>56.80</td></tr><tr><td>FP NRs¹⁵</td><td>56.60</td></tr><tr><td>COF¹⁶</td><td>55.20</td></tr><tr><td>2MPT⁺-CB¹⁷</td><td>54.60</td></tr><tr><td>SPN-PT¹⁸</td><td>53.00</td></tr><tr><td>Pdots¹⁹</td><td>53.00</td></tr><tr><td>Pt Spirals²⁰</td><td>52.50</td></tr><tr><td>TBDOPV-DT²¹</td><td>50.50</td></tr><tr><td>Ti₂O₃@HA NPs²²</td><td>50.20</td></tr><tr><td>TBDOPV-DT NP²³</td><td>50.00</td></tr><tr><td>DPP-IID-FA NPs²⁴</td><td>49.50</td></tr><tr><td>SPN-DT¹⁸</td><td>49.00</td></tr><tr><td>NP²⁵</td><td>49.00</td></tr><tr><td>FTQ nanoparticles²⁶</td><td>49.00</td></tr><tr><td>CNPs²⁷</td><td>49.00</td></tr><tr><td>H-SiOₓ NPs²⁸</td><td>48.60</td></tr><tr><td>BETA NPs²⁹</td><td>47.60</td></tr><tr><td>CN-NPs³⁰</td><td>47.60</td></tr><tr><td>Pt-NDs³¹</td><td>46.90</td></tr><tr><td>MoO₃-x nanobelts³²</td><td>46.90</td></tr><tr><td>P3 NPs³³</td><td>46.00</td></tr><tr><td>Ni₉S₈³⁴</td><td>46.00</td></tr><tr><td>PtAg nanosheets³⁵</td><td>45.70</td></tr><tr><td>Nb₂C NSs³⁶</td><td>45.65</td></tr><tr><td>V₂C-TAT@Ex-RGD³⁷</td><td>45.10</td></tr><tr><td>PEG-TONW NRs³⁸</td><td>43.60</td></tr><tr><td>SPNI-II³⁹</td><td>43.40</td></tr><tr><td>1T-MoS₂⁴⁰</td><td>43.30</td></tr><tr><td>Bi@C NPs⁴¹</td><td>43.20</td></tr><tr><td>Au NPL@TiO₂⁴²</td><td>42.10</td></tr><tr><td>CT NPs⁴³</td><td>42.00</td></tr><tr><td>PPy-PEG NPs⁴⁴</td><td>41.97</td></tr><tr><td>AuPt@CuS NSs⁴⁵</td><td>41.56</td></tr><tr><td>Bi₁₉S₂₇I₃ nanorods⁴⁶</td><td>41.50</td></tr><tr><td>Cu₃BiS₃ NR⁴⁷</td><td>40.70</td></tr><tr><td>MPAE-NPS⁴⁸</td><td>40.07</td></tr></table>
+
+<--- Page Split --->
+
+Comment 15: Supplementary figure 7; this is quite a broad range of nanoparticle sizes. What makes up the smaller (100 nm) particles? Is there aggregation to produce larger particles?  
+
+Author reply: Thank you for the comment. As show in Fig. R9, the smaller PtNP- shell (100 nm) is composed of ultra- small Pt nanoparticles (2–5 nm), similar to the larger PtNP- shell (200 nm).  
+
+![](images/Figure_unknown_9.jpg)
+
+<center>Fig. R9 | Characterization of the smaller PtNP-shell (100 nm). a, TEM image and b, element mapping of the smaller PtNP-shell (100 nm). </center>  
+
+PtNP- shell will not aggregate to produce larger particles. We surface- modified the PtNP- shell with PEG to enhance its biocompatibility and maintain its dispersibility in PBS. Additionally, dynamic light scattering analysis confirmed that the PtNP- shell exhibited little aggregation behavior after being undisturbed for 14 days (Fig. R10). The corresponding results have been included in the Supplementary Fig. 8.  
+
+![](images/Figure_unknown_10.jpg)
+
+<center>Fig. R10 | Hydrodynamic size size of PtNP-shell after a, 1, b, 4, c, 7, and d, 14 days of standing (Inset: digital photograph). </center>
+
+<--- Page Split --->
+
+## To Referee #3  
+
+First of all, we really appreciate your constructive comments. According to your suggestions, we have made corresponding revisions to our manuscript as listed below.  
+
+Overall remark: The manuscript describes photothermal neuromodulation via Pt nano- shell nanoparticles. Ga nanoparticles are used as a template for electrocoupling substitution- based synthesis of the Pt nano- shell. Using KOH wet etching, Ga core is etched and a Pt nano- shell structure is obtained. The rough surface topography of the Pt nano- shell structure allows the particles to exhibit high optical absorbance. It is claimed that these particles have one of the highest photothermal energy conversion efficiencies. The photothermal conversion of optical irradiation is then utilized for stimulation of target cells and tissues via temperature activated ion channels- TRPV1 and TREK1. The potential application of such photothermal modulation technique in regulating cardiac pulsing is demonstrated with regards to protecting against acute ventricular arrhythmias. However, the manuscript does not include proper controls to demonstrate that in- vivo photothermal modulation is achieved exclusively through the Pt nanoparticles. In addition, there are certain claims and results that need to be better corroborated to reach the scientific requirements of the journal. Therefore, I cannot recommend that this manuscript be accepted at Nature Communications in its current form.  
+
+Author reply: Following your suggestion, we have added appropriate controls to demonstrate that in- vivo photothermal modulation is achieved exclusively through PtNP- shell. Furthermore, we have meticulously validated various propositions and findings with enhanced detail. These comments have been systematically addressed and resolved. For further information, please refer to the one- to- one reply corresponding to each comment.  
+
+Comment 1: Abstract: It is claimed that "the autonomic nervous system plays a pivotal role in the pathophysiology of cardiovascular diseases." This sentence is misleading since the dysregulation of the autonomic nervous system can contribute to cardiovascular diseases. However, it is not the primary contributor to the diseases, autonomous nervous system in fact regulates normal functioning of the cardiovascular system. (see: Purves D, Augustine GJ, Fitzpatrick D, LaMantia AS, McNamara JO, Williams SM. Autonomic regulation of cardiovascular function. Neuroscience. 2001:491- 3 AND Gordan R, Gwathmey JK, Xie LH. Autonomic and endocrine control of cardiovascular function. World journal of cardiology. 2015 Apr 4;7(4):204. )  
+
+Author reply: Thank you for the comment. We have made a modification: "Autonomic nervous system disorders play a pivotal role in the pathophysiology of cardiovascular diseases."
+
+<--- Page Split --->
+
+Comment 2: The authors claim that bi- directional reversible autonomic modulation is achieved via NIR- II photothermal modulation using Pt nano- shell nanoparticles. The manuscript presents unidirectional modulation where the target tissues are stimulated. Bi- directionality isn't demonstrated since in terms of neural interfaces, bi- directionality refers to the ability to record neural activity as well as stimulate (see: Song KI, Seo H, Seong D, Kim S, Yu KJ, Kim YC, Kim J, Kwon SJ, Han HS, Youn I, Lee H. Adaptive self- healing electronic epineurium for chronic bidirectional neural interfaces. Nature communications. 2020 Aug 21;11(1):4195. AND Hughes C, Herrera A, Gaunt R, Collinger J. Bidirectional brain- computer interfaces. Handbook of clinical neurology. 2020 Jan 1;168:163- 81. ).  
+
+Author reply: Targeting the autonomic nervous system, we achieve nerve inhibition and activation through PtNP- shell- mediated photothermal effect, enabling adjustment of autonomic nervous imbalance. Hence, in this sense, we define it as a "bidirectional" neuromodulation.  
+
+Comment 3: It is unclear why the NIR- II range was utilized in this work. This is important for selecting the right materials, models, and experiments. (see: nature.com/articles/s44222- 023- 00022- y AND nature.com/articles/s41551- 022- 00862- w).  
+
+Author reply: Thank you for the comment. In comparison to the first near- infrared (NIR- I, 650–900 nm) and visible window, the photons in the second near- infrared window (NIR- II, 900–1700 nm) exhibits reduced tissue scattering and absorption, thereby increasing the maximum allowable exposure (MPE) of biological tissues. Consequently, photons within the NIR- II window exhibit significantly enhanced tissue penetration depths (up to 5–20 mm) (Nat. Nanotech. 2009, 4, 710; Nat. Med. 2012, 18, 1841; Nat. Biomed. Eng. 2017, 1, 0010). To achieve deep photothermal nerve regulation for cardioprotection, we developed PtNP- shell and validated its photothermal neuromodulation efficacy in the NIR- II window both in vivo and in vitro. Given its wavelength independence, further investigations may facilitate selection of a more suitable laser for achieving deeper tissue penetration while adhering to the MPE range. The exceptional potential of PtNP- shell makes it highly promising for precise neural regulation in deeper tissue and holds significant clinical translational value.  
+
+Comment 4: Instead of using terminologies like "nearly perfect blackbody absorption", the actual optical properties and metrics should be presented.  
+
+Author reply: Thank you for the comment. Referring to the study (Nat. Nanotech. 2016, 11, 60), we have obtained the absorption spectra of PtNP- shell, which demonstrate its exceptional blackbody absorption characteristics. In the range of 250–1300 nm, the PtNP- shell exhibits an absorbance close to 1 at 75 μg·mL<sup>- 1</sup>, which is significantly enhanced in the range of 400–1300 nm compared to GaNPs and Ga@Pt NPs (Fig. R11). Additionally, optical images were acquired for comparison purposes.
+
+<--- Page Split --->
+
+Comparing with GaNPs and Ga@Pt NPs, the grayscale feature of PtNP- shell closely approximates the darkest point on the RGB spectrum, providing evidence that PtNP- shell exhibits a strong tendency towards perfect blackbody behavior (Fig. R12).  
+
+![](images/Figure_unknown_11.jpg)
+
+<center>Fig. R11 | UV-vis-NIR absorption spectrum of GaNPs, Ga@Pt NPs and PtNP-shell (75 \(\mu \mathrm{g}\cdot \mathrm{mL}^{-1}\) ). </center>  
+
+![](images/Figure_unknown_12.jpg)
+
+<center>Fig. R12 | Measurement of PtNP-shell blackness. a, Visual appearance of GaNPs, Ga@Pt NPs and PtNP-shell at different concentrations. b, Position of each color in the RGB cube, obtained by extracting the relative components of red, green and blue from Fig. R8a. </center>  
+
+Comment 5: Figure 1 presents how the nanoparticles will interact with the biological systems, however it does not show how light pulses/irradiation will be delivered to the target tissues/sites. This should be discussed in the figure and the manuscript since it is important for clinical translation.  
+
+Author reply: Thank you for the comment. In Fig. 1 (Fig. R13), we have added the laser transmission path towards the target sites. Furthermore, the methodology section of the manuscript has a comprehensive account of the laser transmission process at the target sites.  
+
+"Initial vertical irradiation of NIR- II laser (1064 nm) at \(0.80\mathrm{W}\cdot \mathrm{cm}^{- 2}\) was performed on NG and LSG surfaces. The power density of the NIR- II laser was reduced to \(0.45\mathrm{W}\cdot \mathrm{cm}^{- 2}\) for continuous irradiation when the temperature of the NG reached \(42.0^{\circ}\mathrm{C}\) , and was reduced to \(0.6\mathrm{W}\cdot \mathrm{cm}^{- 2}\) for continuous irradiation when the temperature of the LSG reached \(46.0^{\circ}\mathrm{C}\) . The NIR- II laser irradiation remains stable with a spot size maintained at \(1.0\mathrm{cm}^{- 2}\) ."
+
+<--- Page Split --->
+![](images/Figure_unknown_13.jpg)
+
+<center>Fig. R13 | The synthesis steps of the PtNP-shell and the concept of mediating precise photothermal effects for cardioprotection. a, The synthesis steps of PtNP-shell and schematic diagram of photothermal effect. b, Schematic diagram of multifunctional autonomic modulation mediated by photothermal effect of PtNP-shell for precise cardioprotection against myocardial I/R injury and MI-induced VAs. </center>  
+
+Comment 6: Adequate controls should be provided to better compare the physical properties of PtNP- shells. That is, please provide the optical absorbance of GaNPs, Pt coated GaNPs for Figure 2. d; similar controls should be provided for Figure 2. e (including the thermal transients of such the solvent under irradiation.  
+
+Author reply: Following your suggestion, we supplemented the absorption spectra of GaNPs and Ga@Pt NPs in Fig. 2d (Fig. R14) and compared them with PtNP- shell. It was observed that the absorption of PtNP- shell in the range of 400–1300 nm was significantly higher than that of GaNPs and Ga@Pt NPs at equivalent concentrations. We made corresponding changes in the manuscript:  
+
+"In the range of 250–1300 nm, the PtNP-shell exhibits an absorbance close to 1 at 75 μg·mL<sup>- 1</sup>, which is significantly enhanced in the range of 400–1300 nm compared to GaNPs and Pt-coated Ga nanoparticles (Ga@Pt NPs) (Fig. 2d)."
+
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+![](images/Figure_unknown_14.jpg)
+
+<center>Fig. R14 | UV-vis-NIR absorption spectrum of GaNPs, Ga@Pt NPs and PtNP-shell (75 \(\mu \mathrm{g}\cdot \mathrm{mL}^{-1}\) ). </center>  
+
+In addition, the thermal transient curves of PBS, GaNPs, Ga@Pt NPs and PtNP- shell are supplemented in Fig. 2e (Fig. R15). The results demonstrate that the rate at which PtNP- shell reaches the target temperature is significantly higher compared to that of GaNPs and Ga@Pt NPs. We made corresponding changes in the manuscript:  
+
+"Even in vitro, PtNP- shell (50 \(\mu \mathrm{g}\cdot \mathrm{mL}^{- 1}\) ) exhibited rapid temperature elevation, achieving a rise from room temperature to \(41.0^{\circ}\mathrm{C}\) and \(45.0^{\circ}\mathrm{C}\) within only 96 s and 133 s, respectively. However, for GaNPs (409 s and over 600 s) and GaIn@Pt NPs (317 s and over 600 s), it took significantly longer time to reach the same temperatures (Fig. 2e)."  
+
+![](images/Figure_unknown_15.jpg)
+
+<center>Fig. R15 | Temperature elevation curves of PBS, GaNPs, Ga@Pt NPs and PtNP-shell (50 \(\mu \mathrm{g}\cdot \mathrm{mL}^{-1}\) ) under NIR-II laser irradiation (1 \(\mathrm{W}\cdot \mathrm{cm}^{-2}\) ). </center>  
+
+Comment 7: For the XPS characterization, a survey scan of presentative sample should be presented along with the detailed XPS characterization of oxygen (O1s) and potassium (K2p). The elemental composition of the PtNP-shells, Pt coated GaNPs, and GaNPs should be compared as well. This will better elucidate the composition of effectiveness of the synthesis protocols.  
+
+Author reply: Following your suggestion, we supplemented the XPS survey spectrum of GaNPs, Ga@Pt NPs and PtNP- shell (Fig. R16), and detailed XPS characterization of oxygen (O1s) and potassium (K2p) (Fig. R17). The elemental compositions of GaNPs, Ga@Pt NPs and PtNP- shell were
+
+<--- Page Split --->
+
+compared by high- resolution XPS spectra (Fig. R17). We made corresponding changes in the manuscript and the results have been included in the Supplementary Fig. 5 and Supplementary Fig. 6.  
+
+![](images/Figure_unknown_16.jpg)
+
+<center>Fig. R16 | The XPS survey spectrum of a, GaNPs, b, Ga@Pt NPs and c, PtNP-shell. </center>  
+
+![](images/Figure_unknown_17.jpg)
+
+<center>Fig. R17 | High-resolution XPS spectra and fitting results of a, GaNPs, b, Ga@Pt NPs and c, PtNP-shell. </center>  
+
+"X- ray photoelectron spectroscopy (XPS) analysis reveals the presence of Ga and O in GaNPs, while Ga@Pt NPs and PtNP- shell exhibit the coexistence of Ga, O, and Pt (Supplementary Fig. 5). As depicted in the Supplementary Fig. 6, despite treatment with KOH, no presence of K element was detected in the PtNP- shell. The strong signals of Pt 4f5/2 and Pt 4f7/2 indicate that the Pt in Ga@Pt NPs and PtNP- shell exists in a metallic state (Nat. Commun. 2017, 8, 15802). In GaNPs, the peak centered at 1118.11 eV is attributed to Ga3+ 2p3/2, while the peak centered at 1115.89 eV corresponds
+
+<--- Page Split --->
+
+to Ga \(2\mathrm{p}_{3 / 2}\) . In Ga@Pt NPs, the peak centered at \(1118.80\mathrm{eV}\) is assigned to \(\mathrm{Ga}^{3 + }2\mathrm{p}_{3 / 2}\) , and the peak centered at \(1116.52\mathrm{eV}\) corresponds to Ga \(2\mathrm{p}_{3 / 2}\) . As for PtNP- shell, the presence of a peak around \(1118.56\mathrm{eV}\) indicates complete oxidation of Ga in PtNP- shell into \(\mathrm{Ga}^{3 + }\) (Adv. Funct. Mater. 2023, 34, 2302172). The O 1s spectrum was fitted using two peak functions, which were assigned to Ga- O at \(530.44\mathrm{eV}\) (GaNPs), \(530.98\mathrm{eV}\) (Ga@Pt NPs), \(531.74\mathrm{eV}\) (PtNP- shell) and Ga- OH at \(531.71\mathrm{eV}\) (GaNPs), \(532.08\mathrm{eV}\) (Ga@Pt NPs), \(532.74\mathrm{eV}\) (PtNP- shell) (ACS Appl. Mater. Interfaces 2024, 16, 4212). Compared to GaNPs and Ga@Pt NPs, the binding energy of the Ga- O and Ga- OH peaks in the PtNP- shell is shifted towards higher values, indicating a lower oxidation degree of the PtNP- shell."  
+
+Comment 8: The stability of the Pt- nanoshell suspensions should be evaluated as a function of time. Do the nanoparticle aggregate over time? Will this be a concern when the Pt- nanoshells are injected into biological systems.  
+
+Author reply: Thank you for the comment. PtNP- shell exhibit long- term stability without aggregation, ensuring it compatibility for injection into biological systems. After standing for 1, 4, 7 and 14 days respectively, the statistically averaged hydrated nanoparticle size of PtNP- shell was determined using dynamic light scattering (Fig. R18). It is worth noting that the change of the average hydrated nanoparticle size of PtNP- shell remains negligible after 14 days, indicating its excellent stability. The corresponding results have been included in the Supplementary Fig. 8.  
+
+![](images/Figure_unknown_18.jpg)
+
+<center>Fig. R18 | Hydrodynamic size of PtNP-shell after a, 1, b, 4, c, 7, and d, 14 days of standing (Inset: digital photograph). </center>
+
+<--- Page Split --->
+
+Comment 9: How does the addition of mPEG- SH5000 effect the photothermal properties of the nanoparticles?  
+
+Comment 9: How does the addition of mPEG- SH5000 effect the photothermal properties of the nanoparticles?Author reply: Thank you for the comment. The current research indicates that the photothermal effect of mPEG- SH5000 itself can be disregarded (ACS Nano 2020, 14, 2265; ACS Appl. Energ. Mater. 2021, 4, 7710). We supplemented the temperature elevation curves of mPEG- SH5000 modified and unmodified PtNP- shell (Fig. R19a). The heating rate of the mPEG- SH5000 modified PtNP- shell is significantly higher compared to that of the unmodified PtNP- shell, potentially attributed to the agglomeration tendency of unmodified PtNP- shell at elevated temperatures, leading to a reduction in photothermal performance. Following 600 s of laser irradiation at 1064 nm, the statistically averaged hydrodynamic size for mPEG- SH5000- modified PtNP- shell was measured as 206.5 nm (Fig. R19b), whereas unmodified PtNP- shell exhibited a size of 1517 nm (Fig. R19c). TEM analysis further confirmed the observed agglomeration behavior in unmodified PtNP- shell subsequent to laser irradiation (Fig. R19d). The corresponding results have been included in the Supplementary Fig. 13.  
+
+![](images/Figure_unknown_19.jpg)
+
+<center>Fig. R19 | The impact of PEG on the photothermal properties of PtNP-shell. a, Temperature elevation curves of SH-PEG modified and unmodified PtNP-shell. The hydrodynamic size of PtNP-shell b, before and c, after SH-PEG modification (after 600 s of 1064 nm laser irradiation). d, TEM image of PtNP-shell before SH-PEG modification (after 600 s of 1064 nm laser irradiation). </center>  
+
+Comment 10: Critical information from the methods section is missing- for example, details regarding the cell culture protocol and photothermal stimulation (such as power and pulse duration of optical irradiation are missing). How long was the ECG data recorded for? What were the exact stimulation conditions for all in- vivo experiments?
+
+<--- Page Split --->
+
+**Author reply:** Thank you for your kind reminding. We provide a more detailed description of the Method:  
+
+"Cell- specific medium was prepared by mixing Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum and penicillin-streptomycin mixture at \(89\%\) , \(10\%\) and \(1\%\) , respectively.”  
+
+“To induce activation of TRPV1 and TREK1 ion channels, which had been previously studied, (Science 2003, 300, 1284; EMBO J. 2000, 19, 2483) the culture dish was exposed to 1064 nm laser (0.75 W·cm\(^{- 2}\), TRPV1: 50 s, TREK1: 80 s), resulting in an elevation of temperature.”  
+
+“The same grouping pattern as in part1 was used, with NIR-II irradiation (Heating stage: 0.8 W·cm\(^{- 2}\), 12±3 s; Equilibrium stage: 0.45 W·cm\(^{- 2}\), 5 min) of the NG before opening the occluded LAD coronary vessel.”  
+
+“The in vivo effects of precise photothermal stimulation of the sympathetic nervous system by PtNP-shell under NIR-II irradiation (Heating stage: 0.8 W·cm\(^{- 2}\), 25±5 s; Equilibrium stage: 0.6 W·cm\(^{- 2}\), 5 min) were explored.”  
+
+“Malignant arrhythmic events occurring within 1 hour of MI and I/R injury were assessed by electrocardiographic recordings in a canine model using Lead 7000 Computerized Laboratory System.”  
+
+**Comment 11:** For the in-vitro experiments, are the Pt nanoparticles engulfed by the target cells or are they localized in the vicinity of the cell membrane?  
+
+**Author reply:** The PtNP-shell is partially localized within the target cells, while the remaining portion exhibits distribution around the cell membrane. We supplemented TEM and SEM images of PtNP-shell (50 \(\mu\) g·ml\(^{- 1}\)) co-cultured with cells for 24 hours. Cross-sectional TEM and SEM images showed that PtNP-shell was randomly distributed inside or on the surface of the cells (Fig. R20). This is attributed to the fact that the PtNP-shell exhibits a particle size of approximately 200 nm, enabling smaller particles to traverse the cell membrane. The corresponding results have been included in the Supplementary Fig.15.
+
+<--- Page Split --->
+![](images/Figure_unknown_20.jpg)
+
+<center>Fig. R20 | PtNP-shell co-cultured with neurons. a and b, Cross-sectional TEM and c, SEM of the neurons incubated with PtNP-shell particles for \(24\mathrm{h}\) . </center>  
+
+Comment 12: Both in- vivo and in- vitro photothermal stimulation experiments require the cells' microenvironment to reach temperatures greater than \(42^{\circ}\mathrm{C}\) . Does repeated photothermal stimulation using such high temperatures adversely affect cellular health by disrupting the cell membrane or trigger heat shock response?  
+
+Author reply: The discovery of temperature- sensitive ion channels has given rise to a boom in the modulation of neuronal activity by temperature. Numerous experiments on temperature modulation of neuronal activity have shown that temperatures of \(42^{\circ}\mathrm{C}\) or even above can safely achieve reversible modulation of neural activity (Nano Converg. 2022, 9, 13; Brain Stimul. 2021, 14, 790). In addition, our results also indicate that the NPs- mediated photothermal modulation strategy is biologically safe, both at the cellular (Fig R21) and tissue levels (Fig R22 and R23).
+
+<--- Page Split --->
+![](images/Figure_unknown_21.jpg)
+
+<center>Fig. R21 | a, Cell viability of HT-22 treated with different concentrations of PtNP-shell for \(24\mathrm{h}\) . Effect of NIR-II laser irradiation with varying durations on the viability of HT-22 cells treated with PtNP-shell (50 \(\mu \mathrm{g}\cdot \mathrm{mL}^{-1}\) ) (Power densities: b, \(0.75\mathrm{W}\cdot \mathrm{cm}^{-2}\) and c, \(1\mathrm{W}\cdot \mathrm{cm}^{-2}\) ). The error bar indicates S.E.M. </center>  
+
+![](images/Figure_unknown_22.jpg)
+
+<center>Fig. R22 | Ganglion biocompatibility of targeted injections of PtNP or PBS after NIR-II irradiation and after 30 days of follow-up. a, Representative images of H&E and TUNEL staining of NG from different treatment groups immediately after NIR-II irradiation or after 30 days of follow-up. b, Representative images of H&E and TUNEL staining of LSG from different treatment groups immediately after NIR-II irradiation or after 30 days of follow-up. </center>
+
+<--- Page Split --->
+![](images/Figure_unknown_23.jpg)
+
+<center>Fig. R23 | Long term biosafety of PtNP-shell microinjection. Long-term in vivo biosafety was assessed by local injection of PtNP-shell into the ganglion of Beagle or by injection of equal doses of PtNP-shell into the tail vein of Sprague-Dawley rats. a, Representative H&E staining of major organs of beagles following different treatments. Blood biochemical analyses including b, ALT, c, AST, d, Urea, e, Crea, f, LDH1, g, TNF- \(\alpha\) , and h, IL-6 were performed on Beagles in different treatment groups. i, Representative H&E staining of major organs of rats following different treatments. Blood biochemical analyses including j, ALT, k, AST, l, Urea, m, Crea, n, LDH1, o, TNF- \(\alpha\) and p, IL-6 were performed on rats in different treatment groups. </center>  
+
+Comment 13: The claim that Pt- NP shell does not induce significant damage to neurons under controlled NIR- II laser irradiation is incorrect since there is \(\sim 10\%\) loss in cellular viability.  
+
+Author reply: Thank you for your kind reminding. We conducted a repeated experiment. As illustrated in Fig. R24, although there was a slight decrease in cell activity across all laser irradiation groups compared to the control group (laser duration time of 0 s), no statistically significant differences were observed (all \(\mathrm{P} > 0.05\) ). Hence, we conclude that "Pt- NP shell does not induce significant damage to neurons under controlled NIR- II laser irradiation".
+
+<--- Page Split --->
+![](images/Figure_unknown_24.jpg)
+
+<center>Fig. R24 | Effect of NIR-II laser irradiation with varying durations on the viability of HT-22 cells treated with PtNP-shell (50 \(\mu \mathrm{g}\cdot \mathrm{mL}^{-1}\) ) (Power densities: i, \(0.75\mathrm{W}\cdot \mathrm{cm}^{-2}\) and j, \(1\mathrm{W}\cdot \mathrm{cm}^{-2}\) ). The error bar indicates S.E.M. </center>  
+
+Comment 14: It will be recommended that the data presentation in Figure 3. i be changed since the details of the data are difficult to comprehend through a 3- D plot.  
+
+Author reply: Thank you for your kind reminding. We conducted a repeated experiment and made modifications to Fig. 3i, transforming the 3D diagram into two 2D diagrams for enhanced comprehensibility. Fig. 3i (Fig. R25a) and Fig. 3j (Fig. R25b) depict cell viability following two different power density (0.75 \(\mathrm{W}\cdot \mathrm{cm}^{- 1}\) and \(1\mathrm{W}\cdot \mathrm{cm}^{- 1}\) ) NIR- II laser irradiations at varying time intervals, respectively.  
+
+![](images/Figure_unknown_25.jpg)
+
+<center>Fig. R25 | Effect of NIR-II laser irradiation with varying durations on the viability of HT-22 cells treated with PtNP-shell (50 \(\mu \mathrm{g}\cdot \mathrm{mL}^{-1}\) ) (Power densities: i, \(0.75\mathrm{W}\cdot \mathrm{cm}^{-2}\) and j, \(1\mathrm{W}\cdot \mathrm{cm}^{-2}\) ). The error bar indicates S.E.M. </center>  
+
+Comment 15: For the in- vivo photothermal stimulation experiments, can similar affects be achieved without the presence of the Pt- nanoshell particles? Figure 4. d presents high temperature gradients for the surrounding tissue as well. Stimulation using infra- red radiation has been demonstrated previously, see: doi.org/10.1364/OL.30.000504 and doi.org/10.1117/1.2121772.  
+
+Author reply: Thank you for the comment. First of all, the aim of our study was to achieve precise, rapid, and reversible neuromodulation for cardioprotection through NPs mediated conversion of light
+
+<--- Page Split --->
+
+energy into thermal energy. Non- invasive intervention was also the goal we pursued, so we chose to use NIR- II, which has a deeper penetration depth, as the light source. Due to the limitations of NIR itself and its inability to radiate energy to deep tissues, the effects of NIR on nerves are not clinically significant when viewed in isolation. The high- temperature gradient observed in Fig. 4d is only the temperature transfer to the fat, muscle, and other tissues around the ganglion, and does not affect the changes in nerve activity and function.  
+
+For experimental rigor, we also added changes in ganglion local temperature and neural function before and after NIR irradiation alone at the same parameters. We found limited local temperature elevation ( \(< 2^{\circ}\mathrm{C}\) ) and no significant changes in nerve function under NIR irradiation alone, including NG (Fig. R26) and LSG (Fig. R27). The corresponding results have been included in the Supplementary Fig.19 and 25.  
+
+![](images/Figure_unknown_26.jpg)
+
+<center>Fig. R26 | Direct effect of NIR irradiation of NG. a, Local temperature curve of NG under NIR-II irradiation. b, Neural function of NG before and after NIR-II irradiation. </center>  
+
+![](images/Figure_unknown_27.jpg)
+
+<center>Fig. R27 | Direct effect of NIR irradiation of LSG. a, Local temperature curve of LSG under NIR-II irradiation. b, Neural function of LSG before and after NIR-II irradiation. </center>  
+
+Comment 16: The biosafety of Pt- nanoshell particles was evaluated after a rapid excision of the LSG and NG tissues. Can the authors comments on the long- term biosafety of the nanoparticles in passive (without photothermal stimulation) and active (with photothermal stimulation) states?
+
+<--- Page Split --->
+
+**Author reply:** Thank you for the comment. In Fig. R28, we first verified neuronal safety by histologic examination of the microinjected ganglion tissue as well as Tunel staining. We also showed by neurofunctional testing that PtNP photothermal stimulation is safe and reversible (Fig. R29 and R30). In addition, we monitored the biosafety of both local tissue-injected beagles for 1 month. These detailed tests demonstrated the long-term biosafety of the NPs (Fig. R31). 
+
+![](images/Figure_unknown_28.jpg)
+
+ 
+
+<center>**Fig. R28 | Ganglion biocompatibility of targeted injections of PtNP or PBS after NIR-II irradiation and after 30 days of follow-up. a,** Representative images of H&E and TUNEL staining of NG from different treatment groups immediately after NIR-II irradiation or after 30 days of follow-up. **b,** Representative images of H&E and TUNEL staining of LSG from different treatment groups immediately after NIR-II irradiation or after 30 days of follow-up.</center> 
+
+![](images/Figure_unknown_29.jpg)
+
+ 
+
+<center>**Fig. R29 | Effect of PtNP-shell photothermal stimulation of NG.** Maximal HR changes of beagles treatment with PtNP-shell or control from 1 to 3 hours after NIR irradiation, \(n = 6\). Data are shown as the mean \(\pm\) S.E.M. *\(P < 0.05\), **\(P < 0.01\), ns means that the difference is not statistically significant.</center>
+
+<--- Page Split --->
+![](images/Figure_unknown_30.jpg)
+
+<center>Fig. R30 | Effect of PtNP-shell photothermal inhibition of LSG. Maximal SBP changes of beagles treatment with PtNP-shell or control from 1 to 3 hours after NIR irradiation, \(\mathrm{n} = 6\) . Data are shown as the mean \(\pm\) S.E.M. \(^\ast \mathrm{P}< 0.05\) , \(^{**}P< 0.01\) , ns means that the difference is not statistically significant. </center>  
+
+![](images/Figure_unknown_31.jpg)
+
+<center>Fig. R31 | Long term biosafety of PtNP-shell microinjection. Long-term in vivo biosafety was assessed by local injection of PtNP-shell into the ganglion of Beagle or by injection of equal doses of PtNP-shell into the tail vein of Sprague-Dawley rats. a, Representative H&E staining of major organs of beagles following different treatments. Blood biochemical analyses including b, ALT, c, AST, d, Urea, e, Crea, f, LDH1, g, TNF- \(\alpha\) , and h, IL-6 were performed on Beagles in different treatment groups. i, Representative H&E staining of major organs of rats following different treatments. Blood biochemical analyses including j, ALT, k, AST, l, Urea, m, Crea, n, LDH1, o, TNF- \(\alpha\) and p, IL-6 were performed on rats in different treatment groups. </center>
+
+<--- Page Split --->
+
+Comment 17: Page 2, line 21: Please include examples and appropriate references for "conventional international procedures for MI."  
+
+Author reply: We have attached the reference after "Conventional international procedures for MI", and the reference number is "3".  
+
+Comment 18: Page 4, line 16: Please change the word "encapsulated on" since Pt is not encapsulated on the surface of GaNPs. Pt is deposited onto of GaNP core then it encapsulates GaNP core.  
+
+Author reply: Thank you for your kind reminding. We have changed the word "encapsulated on" in the manuscript to "deposited on".  
+
+Finally, we want to thank the referees again for their constructive comments on our work, and we hope our newly revised manuscript can reach the quality expectation to be published in Nature Communications. Please find our revisions marked in red copy of the revised manuscript.
+
+<--- Page Split --->
+
+Reviewers' Comments:  
+
+Reviewer #1: Remarks to the Author: I have no additional comments.  
+
+Reviewer #2: Remarks to the Author:  
+
+The authors have addressed many of the comments I gave, such as improving explanations, results narrative, adding supplementary tables and revising some figure layouts for clarity. I also appreciate the new NP characterisation data. However, I still think there are a few points which need to be addressed further:  
+
+Comment 5: Please clarify the exact timing of NP and NIR treatment in relation to the surgery. The text says "The NG was subsequently exposed to NIR- II laser irradiation for a duration of 5 minutes prior to occlusion of the left anterior descending (LAD) coronary artery for reperfusion therapy." This means the treatment is given before artery occlusion. However, the authors' response to my comment is talking about the importance of reducing reperfusion injury (which I agree is very important). However, if preventing reperfusion injury is the goal, why not induce the MI, then give the NIR treatment at the time of artery re- opening and reperfusion? This would simulate the clinical reality where an intervention could be given before, or during reperfusion. If the experimental design indeed is treating the animals before MI, this limitation needs to be clearly mentioned.  
+
+Comment 6: The time point of blood sampling for myoglobin and c- TnI measurement is still not clear in the manuscript. The text simply says "after MI and myocardial I/R injury" (line 736). Please specify the exact time points used for data in 5j and 5k.  
+
+I also disagree that reductions in these biomarkers is strong enough evidence to demonstrate cardioprotection, which is claimed multiple times throughout the text. This claim can only be made if there are functional tests or at least histological findings (i.e. reduced infarct size). I think claiming the reduction in acute VAs is fair enough, but "cardiac protection" strongly implies preservation of tissue and corresponding functional changes.  
+
+Lastly, the authors also did not provide any explanation for \\*how or why\\* these markers would be reduced by the NP/NIR treatment. The implication of lower circulating damage markers would be that there is less cardiac muscle loss - but there are no data to support this. Again, this is where functional metrics would be very useful. Still, as a principle, it's not exactly clear to me how the NP/NIR- II intervention would lower myoglobin/c- TnI release.  
+
+Comment 9- 10: I think the responses to my comments are fine, but some of these points should go into the manuscript discussion section.  
+
+Reviewer #3:  
+
+Remarks to the Author:  
+
+The authors have addressed most of the reviewer comments. Adequate control experiments and results have also been provided.  
+
+However, there are a couple more concerns regarding the revised manuscript1. For neural interfaces, bidirectionality is defined as the ability and record and stimulate neural activity. Therefore, stimulating and inhibiting neural activity should not be defined as bidirectionality of neuromodulation. A more appropriate term will be multi- modal neuromodulation.
+
+<--- Page Split --->
+
+2. All peaks in the XPS spectra should be identified. For example, there is an emergence of peaks (at ca. 400 eV) other than Ga and O in the GaNP samples. The elemental composition should be assessed to better elucidate the chemical composition of each sample.
+
+<--- Page Split --->
+
+## Reply to the referees  
+
+## To Referee #1  
+
+Overall remark: I have no additional comments.  
+
+Author reply: We appreciate your positive evaluation on our manuscript.
+
+<--- Page Split --->
+
+## To Referee #2  
+
+Overall remark: The authors have addressed many of the comments I gave, such as improving explanations, results narrative, adding supplementary tables and revising some figure layouts for clarity. I also appreciate the new NP characterisation data. However, I still think there are a few points which need to be addressed further.  
+
+Author reply: We appreciate your positive evaluation on our manuscript. We have made a pointby- point response to your comments and carefully revised the manuscript as you suggested. For your reference, please find our revisions marked in red color.  
+
+Comment 1: Please clarify the exact timing of NP and NIR treatment in relation to the surgery. The text says "The NG was subsequently exposed to NIR- II laser irradiation for a duration of 5 minutes prior to occlusion of the left anterior descending (LAD) coronary artery for reperfusion therapy." This means the treatment is given before artery occlusion. However, the authors' response to my comment is talking about the importance of reducing reperfusion injury (which I agree is very important). However, if preventing reperfusion injury is the goal, why not induce the MI, then give the NIR treatment at the time of artery re- opening and reperfusion? This would simulate the clinical reality where an intervention could be given before, or during reperfusion. If the experimental design indeed is treating the animals before MI, this limitation needs to be clearly mentioned.  
+
+Author reply: Thank you for the comment. Previously for the timing of the intervention in the reperfusion injury model we led to misunderstandings in the text and picture descriptions. In fact, the time point of neuromodulation by NIR irradiation was just 5 min before reperfusion injury, which is consistent with the timing of interventions for clinical application in ischemia- reperfusion injury.  
+
+We first performed microinjections of PtNP- shell in ganglia and occluded the left anterior descending (LAD) coronary artery for 1 h to induce myocardial ischemia. Subsequently, NIR- II laser irradiation was applied to NG for 5 min, followed by reperfusion treatment achieved by opening the ligated knot. Consequently, we further refined the corresponding content and Fig. 5b (Fig. R1) to provide a clearer and more direct representation of the treatment time.  
+
+![](images/Figure_unknown_32.jpg)
+
+<center>Fig. R1 | Flowchart of regulating NG to protect against myocardial I/R injury and associated VAs. </center>
+
+<--- Page Split --->
+
+"The left anterior descending (LAD) coronary artery was occluded for 1 h to induce myocardial ischemia. Subsequently, NIR- II laser irradiation was applied to NG for 5 min, followed by reperfusion treatment achieved by opening the ligated knot."  
+
+Comment 2: The time point of blood sampling for myoglobin and c- TnI measurement is still not clear in the manuscript. The text simply says "after MI and myocardial I/R injury" (line 736). Please specify the exact time points used for data in 5j and 5k.  
+
+Author reply: Thank you for your kind reminding. Blood samples for myoglobin and c- TnI measurement were collected via jugular vein of each beagle 4- 5 h after coronary artery ligation (3- 4 h after reperfusion treatment). We have made corresponding changes in the manuscript.  
+
+"Serum Elisa assay revealed significantly lower levels of markers of myocardial injury (MYO and c- TnI) at 4- 5 h post- infarction in the PtNP- shell group compared to the control group (all \(\mathrm{p}<\) 0.05, Fig. 5j and k)."  
+
+In Methods:  
+
+"In myocardial I/R model experiments, \(5\mathrm{mL}\) of venous blood was obtained from the jugular vein of each beagle 4- 5 hours after ligation of the coronary vessels (3- 4 h after reperfusion treatment)."  
+
+Comment 3: I also disagree that reductions in these biomarkers is strong enough evidence to demonstrate cardioprotection, which is claimed multiple times throughout the text. This claim can only be made if there are functional tests or at least histological findings (i.e. reduced infarct size). I think claiming the reduction in acute VAs is fair enough, but "cardiac protection" strongly implies preservation of tissue and corresponding functional changes.  
+
+Author reply: Thank you for the comment. We totally agree with your insightful suggestion, and have substituted the term "cardioprotection" with "reduction in the occurrence of ventricular arrhythmias induced by myocardial ischemia or reperfusion injury" in the manuscript.  
+
+In this study, we investigated the role of PtNP- shell- mediated photothermal neuromodulation in a myocardial infarction (MI) model and a myocardial ischemia/reperfusion (I/R) injury model. Our findings not only demonstrated a reduction in myocardial injury biomarkers but also revealed that the neuromodulation technique effectively improved cardiac electrophysiological stability, suppressed the occurrence of MI or I/R- induced VAs.  
+
+Indeed, serum markers of myocardial injury in acute infarction models and acute reperfusion injury models do not fully reflect cardioprotective effects. Comprehensive cardioprotective effects should be further assessed in the long- term myocardial injury model through its evaluation of cardiac function and infarct area indexes. The relevant content has been incorporated into the discussion
+
+<--- Page Split --->
+
+section of the manuscript.  
+
+"In this study, we validated the protective efficacy of PtNP- shell photothermal neuromodulation strategy in models of acute myocardial infarction and acute reperfusion injury to mitigate ventricular arrhythmia incidence. However, further evaluation through experiments such as assessment of cardiac function and infarct area is required to determine the cardioprotective potential of this strategy in chronic myocardial injury models."  
+
+Comment 4: Lastly, the authors also did not provide any explanation for \*how or why\* these markers would be reduced by the NP/NIR treatment. The implication of lower circulating damage markers would be that there is less cardiac muscle loss - but there are no data to support this. Again, this is where functional metrics would be very useful. Still, as a principle, it's not exactly clear to me how the NP/NIR- II intervention would lower myoglobin/c- TnI release.  
+
+Author reply: Thank you for the comment. In the case of myocardial cell injury, biomarkers such as troponin, indicative of myocardial damage, are released into the bloodstream (JAMA. 2013, 309, 2262). In the guidelines for cardiovascular disease published by the European Society of Cardiology and others, testing for cardiac injury biomarkers is also an important indicator for clinical detection of myocardial injury in patients (Eur. Heart J. 2012, 33, 2551; Eur. Heart J. 2023, 44, 3720). Therefore, we validate the protective efficacy of PtNP- shell mediated photothermal neuromodulation strategy against ischemia and reperfusion injury by assessing the levels of serum myocardial injury markers.  
+
+Neurotransmitters released by sympathetic nerves can bind adrenergic receptors in cardiomyocytes to control cardiomyocyte contraction (Annu. Rev. Physiol. 2022, 84, 285). Myocardial ischemia causes activation of sympathetic nerves, releasing large amounts of sympathetic neurotransmitters (Eur. Heart J. 2024, 45, 669). Subsequently, sympathetic activation of adrenergic receptors promotes sarcoplasmic reticulum calcium release from cardiomyocytes, exacerbating calcium overload and causing cardiac electrophysiologic disturbances (J. Am. Coll. Cardiol. 2010, 56, 805). Acute adrenergic receptor activation results in rapid activation of cardiomyocyte- specific inflammatory vesicles, which induces IL- 18 activation, promotes cardiac cytokine waterfall response and macrophage infiltration, and leads to myocardial injury and reduced cardiac function (Fig R2) (Eur. Heart J. 2018, 39, 60). Additionally, parasympathetic nerve stimulation can elicit the activation of \(\alpha - 7\) nicotinic acetylcholine receptor ( \(\alpha 7nAChR\) ), leading to a reduction in inflammatory response and oxidative stress (J. Am. Heart Assoc. 2023, 12, e030539). The activation of \(\alpha 7nAChR\) has been shown to reverse the up- regulation of myocardial arginase induced by ischemia- reperfusion injury and reduce infarct size (Fig R3) (Acta Physiol. 2017, 221, 174).
+
+<--- Page Split --->
+![](images/Figure_unknown_33.jpg)
+
+<center>Fig. R2 | Take home figure working model for b-adrenergic activation induced cardiac inflammatory cascade which finally results in cardiac remodeling (left) and therapeutic strategy (right) (Eur. Heart J. 2018, 39, 60). </center>  
+
+![](images/Figure_unknown_34.jpg)
+
+<center>Fig. R3 | Myocardial area at risk and infarct size. a, Area at risk expressed as \(\%\) of left ventricle and b, infarct size (with representative images of infarcted myocardium) expressed as \(\%\) of the area at risk following \(30\mathrm{min}\) ischaemia and 2-h reperfusion in control animals (Control IR; \(\mathrm{n} = 14\) ), after vagal nerve stimulation (VNS + IR; \(\mathrm{n} = 13\) ), \(\alpha 7\mathrm{nAChR}\) blockade and VNS (MLA + VNS + IR; \(\mathrm{n} = 7\) ), the arginase inhibitor nor-NOHA and IR (nor-NOHA + IR, \(\mathrm{n} = 5\) ), nor-NOHA+VNS+IR ( \(\mathrm{n} = 6\) ) and MLA alone ( \(\mathrm{n} = 5\) ). Data are shown as mean \(\pm\) SEM. Significant differences between groups are shown; \(*P< 0.05\) and \(\ddagger P< 0.001\) (Acta Physiol. 2017, 221, 174). </center>
+
+<--- Page Split --->
+
+In summary, neuromodulation (inhibition of sympathetic nerve or activation of parasympathetic nerve) may reduce myocardial injury and decrease serum levels of markers of myocardial injury through mechanisms such as reduction of calcium overload, inflammatory response, and oxidative stress. The corresponding content have been included in the manuscript.  
+
+"Serum Elisa assay revealed significantly lower levels of markers of myocardial injury (MYO and c- TnI) at 4–5 h post- infarction in the PtNP- shell group compared to the control group (all p < 0.05, Fig. 5j and k), indicating an improvement in myocardial injury (JAMA. 2013, 309, 2262). This may be attributed to the activation of \(\alpha - 7\) nicotinic acetylcholine receptor by stimulating parasympathetic nerves, thereby alleviating inflammatory reactions and oxidative stress (J. Am. Heart Assoc. 2023, 12, e030539; Acta Physiol. 2017, 221, 174)."  
+
+Comment 5: Comment 9- 10: I think the responses to my comments are fine, but some of these points should go into the manuscript discussion section.  
+
+Author reply: Thank you for your kind reminding. Your valuable suggestion has significantly enhanced the depth of our research. The relevant content has been incorporated into the discussion section of the manuscript.  
+
+"Cardiac sympathetic denervation (CSD) is a clinical procedure aimed at targeting the autonomic ganglia for refractory ventricular arrhythmias. However, ganglion removal can be traumatic for patients and may lead to complications due to the loss of original physiological function (Eur. Heart J. 2022, 43, 2096). Currently, \(\beta\) - blockers are the primary pharmacological drugs employed in clinical practice for arrhythmia treatment (J. Am. Heart Assoc. 2018, 7, e007567; Eur. Heart J. 2023, 44, 3720). However, their administration during acute myocardial ischemia remains unclear and is contraindicated in patients with heart failure. Additionally, previous research investigated the local ganglion blockade using botulinum toxin A to protect the heart (Heart Rhythm 2022, 19, 2095). Nevertheless, its prolonged blocking effect renders it unsuitable for acute myocardial ischemia management. Conversely, PtNP- shell- based photothermal neuromodulation offers reversible modulation within a short timeframe, exhibiting superior efficacy and controllability."  
+
+"Simultaneously, exploiting the presence of blood vessels surrounding the ganglion presents an opportunity to minimize photon propagation within tissues. Consequently, photothermal modulation of NIR- II fibers in proximity to the ganglion through vascular routes during interventional therapy emerges as a promising avenue for direct clinical translation."
+
+<--- Page Split --->
+
+## To Referee #3  
+
+Overall remark: The authors have addressed most of the reviewer comments. Adequate control experiments and results have also been provided. However, there are a couple more concerns regarding the revised manuscript.  
+
+Author reply: We appreciate your positive evaluation on our manuscript. We have made a pointby- point response to your comments and carefully revised the manuscript as you suggested. For your reference, please find our revisions marked in red color.  
+
+Comment 1: For neural interfaces, bidirectionality is defined as the ability and record and stimulate neural activity. Therefore, stimulating and inhibiting neural activity should not be defined as bidirectionality of neuromodulation. A more appropriate term will be multi- modal neuromodulation.  
+
+Author reply: Thank you for the comment. We have substituted the term "bi- directional" with "multimodal".  
+
+Comment 2: All peaks in the XPS spectra should be identified. For example, there is an emergence of peaks (at ca. 400 eV) other than Ga and O in the GaNP samples. The elemental composition should be assessed to better elucidate the chemical composition of each sample.  
+
+Author reply: Following your suggestion, we identified all peaks in the XPS survey spectra (Fig. R4). The peaks at about 400 eV can be identified to Auger peaks of Ga. The corresponding results have been included in the Supplementary Fig. 5.  
+
+![PLACEHOLDER_46_0]
+
+<center>Fig. R4 | The XPS survey spectra of a, GaNPs, b, Ga@Pt NPs and c, PtNP-shell. </center>  
+
+Finally, we want to thank the referees again for their constructive comments on our work, and we hope our newly revised manuscript can reach the quality expectation to be published in Nature Communications. Please find our revisions marked in red copy of the revised manuscript.
+
+<--- Page Split --->
+
+Reviewers' Comments:  
+
+Reviewer #2:  Remarks to the Author:  The authors have addressed all comments in the latest version of the manuscript.  
+
+Reviewer #3:  Remarks to the Author:  The authors have addressed all comments.
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__012c200754316231695b8af56a30435ab233e96b827c0370265adf0c1c9cfda9/images_list.json

@@ -0,0 +1 @@
+[]

peer_reviews/supplementary_0_Peer Review File__012c200754316231695b8af56a30435ab233e96b827c0370265adf0c1c9cfda9/supplementary_0_Peer Review File__012c200754316231695b8af56a30435ab233e96b827c0370265adf0c1c9cfda9.mmd

@@ -0,0 +1,83 @@
+
+# nature portfolio  
+
+Peer Review File  
+
+# A Nucleic Acid Binding Protein Map of Germline Regulation in Caenorhabditis elegans  
+
+![PLACEHOLDER_0_0]
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+Editorial Note: This manuscript has been previously reviewed at another journal that is not operating a transparent peer review scheme. This document only contains reviewer comments and rebuttal letters for versions considered at Nature Communications.
+
+<--- Page Split --->
+
+Reviewer #1 (Remarks to the Author):  
+
+The changes made in response to the reviews have improved the clarity of the manuscript and the accessibility of the data, especially the data in the supplemental files. It is still not easy to access the images, and it would have been much better to have a searchable online database available for this purpose. However, overall the data are much better organized, which is critical for a paper with the goal of presenting a large amount of screen data.  
+
+Reviewer #2 (Remarks to the Author):  
+
+The authors have done a good job addressing the points raised in the reviews. I just have two minor points:  
+
+The experiment assessing proliferation rates (Figure 2f) is still a little unclear. First, the legend says that " \(n = 9 - 13\) for each experiment." but I can only see three dots for each RNAi condition in the figure and from the table it appears that only three experiments were done. Second, why was xnd- 1 not included in Figure 2f? There are data in the table for this RNAi.  
+
+With respect to the lack of sterility in the distal screen for 10 genes found in the initial screen, the authors hypothesize that this was due to a synthetic effect with the reporters. An alternative (which has been observed by other for different germline transgenes) is that the transgenic strain is more sensitive to RNAi than the strain used in the distal screen. If the authors do not want to test this (i.e., by incorporating rde- 1 and the germline rde- 1 rescue into the rrf- 3 strain), they should suggest enhanced RNAi sensitivity as a second possible explanation. They might get an inkling about this by comparing the strength of phenotypes in the distal screen to those done in the reporter background for all genes where RNAi was done in both.  
+
+Reviewer #3 (Remarks to the Author):  
+
+The revised manuscript from Cao et al. titled "A Nucleic Acid Binding Protein Map of Germline Regulation" examines the role of nucleic acid binding proteins in the germline of C. elegans. The results provide an extremely useful atlas of transcription factor function throughout the different stages of germ cell development in C. elegans. This work should be relevant to researchers interested in mitotic and meiotic cell divisions and germ cell development and differentiation. I expressed 3 major concerns in my review of a previous version of this manuscript.  
+
+These concerns were relatively minor, and the authors adequately address each of them in this revision. My first two concerns focused on the fact that their RNA was isolated from whole gonads and appears to have some contamination from the somatic gonad and other non- gonadal tissues. They have restated the description of the genes they identify as "germline enriched" rather than more specifically "germline- expressed" throughout the manuscript and have mentioned the possibility of contamination from somatic tissues in the methods. My third concern was that it was not entirely clear how they selected NBP genes for further analysis when combining their RNAseq results with results form earlier work. They point out how they
+
+<--- Page Split --->
+
+explained this selection in the original manuscript and have revised this slightly in the current manuscript to indicate they apparently chose genes that were detected in all three of their RNAseq samples and in in both gonads analyzed by Tzur et al. In addition, they included any genes that detected \(>10\) CPM in the analyses by Cao et al (2017). This concern was also shared by another reviewer, so I hope this reviewer is also satisfied with the revised description.  
+
+The authors also addressed my more minor comments. In particular, I found it much easier to access information in the very useful Supplementary Tables.
+
+<--- Page Split --->
+
+## REVIEWERS' COMMENTS  
+
+Reviewer #1 (Remarks to the Author):  
+
+The changes made in response to the reviews have improved the clarity of the manuscript and the accessibility of the data, especially the data in the supplemental files. It is still not easy to access the images, and it would have been much better to have a searchable online database available for this purpose. However, overall the data are much better organized, which is critical for a paper with the goal of presenting a large amount of screen data.  
+
+No response required  
+
+Reviewer #2 (Remarks to the Author):  
+
+The authors have done a good job addressing the points raised in the reviews. I just have two minor points:  
+
+The experiment assessing proliferation rates (Figure 2f) is still a little unclear. First, the legend says that " \(n = 9 - 13\) for each experiment." but I can only see three dots for each RNAi condition in the figure and from the table it appears that only three experiments were done.  
+
+The triplicate data are shown in the figure. We have now amended the figure legend to make it clearer: Proliferation rates of the C. elegans germ line after GNBPR RNAi. RNAi was performed in triplicate from the L1 stage for 66 hrs before commencing EdU labelling, and germ lines were collected and imaged after 4 and 10 hrs. The 3xflag::syg1; sun- 1p::rde- 1; rde- 1(mkc36) strain was used in this experiment. Data for each replicate shown; \(n = 63\) , 61, 63, 61, 62, 61, 58, 61, 63, 62 and 61 germlines for each condition. \(P\) values assessed by unpaired t- test.  
+
+Second, why was xnd- 1 not included in Figure 2f ? There are data in the table for this RNAi.  
+
+These xnd- 1 data are in Fig S4g as the experiment was performed independently.  
+
+With respect to the lack of sterility in the distal screen for 10 genes found in the initial screen, the authors hypothesize that this was due to a synthetic effect with the reporters. An alternative (which has been observed by other for different germline transgenes) is that the transgenic strain is more sensitive to RNAi than the strain used in the distal screen. If the authors do not want to test this (i.e., by incorporating rde- 1 and the germline rde- 1 rescue into the rrf- 3 strain), they should suggest enhanced RNAi sensitivity as a second possible explanation. They might get an inkling about this by comparing the strength of phenotypes in the distal screen to those done in the reporter background for all genes where RNAi was done in both.  
+
+OK - we have also suggested increased RNAi sensitivity as a possibility.  
+
+Here is the new sentence "We hypothesize that the sterility phenotype of the 10 genes identified only in the proximal screen is either due to increased RNAi sensitivity or a synthetic effect of the RNAi silencing and the transgenes used (mCherry- histone H2B and plasma membrane GFP- PH(PLC161)), as we did not observe sterility in the distal screen (Supplementary Table 4)."  
+
+Reviewer #3 (Remarks to the Author):  
+
+The revised manuscript from Cao et al. titled "A Nucleic Acid Binding Protein Map of Germline Regulation" examines the role of nucleic acid binding proteins in the germline of C. elegans. The results provide an extremely useful atlas of transcription factor function throughout the different stages of germ cell development in C. elegans. This work should be relevant to researchers interested in mitotic and meiotic cell divisions and germ cell development and differentiation. I expressed 3 major concerns in my review of a previous version of this manuscript.  
+
+These concerns were relatively minor, and the authors adequately address each of them in this revision. My first two concerns focused on the fact that their RNA was isolated from whole gonads and appears to have some contamination from the somatic gonad and other non- gonadal tissues. They have restated the description of the genes they identify as "germline enriched" rather than more specifically "germline- expressed" throughout the manuscript and have mentioned the possibility of contamination from somatic tissues in the methods. My third concern was that it was not entirely clear how they selected NBP genes for further analysis when combining their RNAseq results with results form earlier work. They point out how they explained this selection in the original manuscript and have revised this slightly in the current manuscript to indicate they apparently chose genes that were detected in all three of their RNAseq samples and in in both gonads analyzed by Tzur et al. In addition, they included any genes that detected \(>10\) CPM in the analyses by Cao et al (2017). This concern was also shared by another reviewer, so I hope this reviewer is also satisfied with the revised description.  
+
+The authors also addressed my more minor comments. In particular, I found it much easier to access information in the very useful Supplementary Tables.
+
+<--- Page Split --->
+
+No response required
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__012c200754316231695b8af56a30435ab233e96b827c0370265adf0c1c9cfda9/supplementary_0_Peer Review File__012c200754316231695b8af56a30435ab233e96b827c0370265adf0c1c9cfda9_det.mmd

@@ -0,0 +1,110 @@
+<|ref|>title<|/ref|><|det|>[[60, 40, 508, 90]]<|/det|>
+# nature portfolio  
+
+<|ref|>text<|/ref|><|det|>[[68, 110, 362, 139]]<|/det|>
+Peer Review File  
+
+<|ref|>title<|/ref|><|det|>[[76, 154, 920, 211]]<|/det|>
+# A Nucleic Acid Binding Protein Map of Germline Regulation in Caenorhabditis elegans  
+
+<|ref|>image<|/ref|><|det|>[[56, 732, 240, 784]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[250, 732, 912, 785]]<|/det|>
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 83, 868, 133]]<|/det|>
+Editorial Note: This manuscript has been previously reviewed at another journal that is not operating a transparent peer review scheme. This document only contains reviewer comments and rebuttal letters for versions considered at Nature Communications.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[119, 85, 414, 100]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 118, 875, 204]]<|/det|>
+The changes made in response to the reviews have improved the clarity of the manuscript and the accessibility of the data, especially the data in the supplemental files. It is still not easy to access the images, and it would have been much better to have a searchable online database available for this purpose. However, overall the data are much better organized, which is critical for a paper with the goal of presenting a large amount of screen data.  
+
+<|ref|>text<|/ref|><|det|>[[119, 255, 414, 271]]<|/det|>
+Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 290, 848, 324]]<|/det|>
+The authors have done a good job addressing the points raised in the reviews. I just have two minor points:  
+
+<|ref|>text<|/ref|><|det|>[[118, 341, 877, 410]]<|/det|>
+The experiment assessing proliferation rates (Figure 2f) is still a little unclear. First, the legend says that " \(n = 9 - 13\) for each experiment." but I can only see three dots for each RNAi condition in the figure and from the table it appears that only three experiments were done. Second, why was xnd- 1 not included in Figure 2f? There are data in the table for this RNAi.  
+
+<|ref|>text<|/ref|><|det|>[[117, 427, 874, 565]]<|/det|>
+With respect to the lack of sterility in the distal screen for 10 genes found in the initial screen, the authors hypothesize that this was due to a synthetic effect with the reporters. An alternative (which has been observed by other for different germline transgenes) is that the transgenic strain is more sensitive to RNAi than the strain used in the distal screen. If the authors do not want to test this (i.e., by incorporating rde- 1 and the germline rde- 1 rescue into the rrf- 3 strain), they should suggest enhanced RNAi sensitivity as a second possible explanation. They might get an inkling about this by comparing the strength of phenotypes in the distal screen to those done in the reporter background for all genes where RNAi was done in both.  
+
+<|ref|>text<|/ref|><|det|>[[119, 616, 414, 632]]<|/det|>
+Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 650, 880, 755]]<|/det|>
+The revised manuscript from Cao et al. titled "A Nucleic Acid Binding Protein Map of Germline Regulation" examines the role of nucleic acid binding proteins in the germline of C. elegans. The results provide an extremely useful atlas of transcription factor function throughout the different stages of germ cell development in C. elegans. This work should be relevant to researchers interested in mitotic and meiotic cell divisions and germ cell development and differentiation. I expressed 3 major concerns in my review of a previous version of this manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[117, 771, 872, 909]]<|/det|>
+These concerns were relatively minor, and the authors adequately address each of them in this revision. My first two concerns focused on the fact that their RNA was isolated from whole gonads and appears to have some contamination from the somatic gonad and other non- gonadal tissues. They have restated the description of the genes they identify as "germline enriched" rather than more specifically "germline- expressed" throughout the manuscript and have mentioned the possibility of contamination from somatic tissues in the methods. My third concern was that it was not entirely clear how they selected NBP genes for further analysis when combining their RNAseq results with results form earlier work. They point out how they
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 84, 871, 169]]<|/det|>
+explained this selection in the original manuscript and have revised this slightly in the current manuscript to indicate they apparently chose genes that were detected in all three of their RNAseq samples and in in both gonads analyzed by Tzur et al. In addition, they included any genes that detected \(>10\) CPM in the analyses by Cao et al (2017). This concern was also shared by another reviewer, so I hope this reviewer is also satisfied with the revised description.  
+
+<|ref|>text<|/ref|><|det|>[[118, 187, 860, 220]]<|/det|>
+The authors also addressed my more minor comments. In particular, I found it much easier to access information in the very useful Supplementary Tables.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[93, 65, 280, 78]]<|/det|>
+## REVIEWERS' COMMENTS  
+
+<|ref|>text<|/ref|><|det|>[[94, 90, 348, 103]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[93, 114, 896, 166]]<|/det|>
+The changes made in response to the reviews have improved the clarity of the manuscript and the accessibility of the data, especially the data in the supplemental files. It is still not easy to access the images, and it would have been much better to have a searchable online database available for this purpose. However, overall the data are much better organized, which is critical for a paper with the goal of presenting a large amount of screen data.  
+
+<|ref|>text<|/ref|><|det|>[[93, 177, 240, 190]]<|/det|>
+No response required  
+
+<|ref|>text<|/ref|><|det|>[[93, 201, 348, 214]]<|/det|>
+Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[92, 225, 810, 238]]<|/det|>
+The authors have done a good job addressing the points raised in the reviews. I just have two minor points:  
+
+<|ref|>text<|/ref|><|det|>[[92, 248, 888, 288]]<|/det|>
+The experiment assessing proliferation rates (Figure 2f) is still a little unclear. First, the legend says that " \(n = 9 - 13\) for each experiment." but I can only see three dots for each RNAi condition in the figure and from the table it appears that only three experiments were done.  
+
+<|ref|>text<|/ref|><|det|>[[92, 299, 900, 362]]<|/det|>
+The triplicate data are shown in the figure. We have now amended the figure legend to make it clearer: Proliferation rates of the C. elegans germ line after GNBPR RNAi. RNAi was performed in triplicate from the L1 stage for 66 hrs before commencing EdU labelling, and germ lines were collected and imaged after 4 and 10 hrs. The 3xflag::syg1; sun- 1p::rde- 1; rde- 1(mkc36) strain was used in this experiment. Data for each replicate shown; \(n = 63\) , 61, 63, 61, 62, 61, 58, 61, 63, 62 and 61 germlines for each condition. \(P\) values assessed by unpaired t- test.  
+
+<|ref|>text<|/ref|><|det|>[[92, 372, 707, 386]]<|/det|>
+Second, why was xnd- 1 not included in Figure 2f ? There are data in the table for this RNAi.  
+
+<|ref|>text<|/ref|><|det|>[[93, 397, 641, 411]]<|/det|>
+These xnd- 1 data are in Fig S4g as the experiment was performed independently.  
+
+<|ref|>text<|/ref|><|det|>[[92, 421, 904, 508]]<|/det|>
+With respect to the lack of sterility in the distal screen for 10 genes found in the initial screen, the authors hypothesize that this was due to a synthetic effect with the reporters. An alternative (which has been observed by other for different germline transgenes) is that the transgenic strain is more sensitive to RNAi than the strain used in the distal screen. If the authors do not want to test this (i.e., by incorporating rde- 1 and the germline rde- 1 rescue into the rrf- 3 strain), they should suggest enhanced RNAi sensitivity as a second possible explanation. They might get an inkling about this by comparing the strength of phenotypes in the distal screen to those done in the reporter background for all genes where RNAi was done in both.  
+
+<|ref|>text<|/ref|><|det|>[[92, 520, 576, 533]]<|/det|>
+OK - we have also suggested increased RNAi sensitivity as a possibility.  
+
+<|ref|>text<|/ref|><|det|>[[92, 544, 875, 595]]<|/det|>
+Here is the new sentence "We hypothesize that the sterility phenotype of the 10 genes identified only in the proximal screen is either due to increased RNAi sensitivity or a synthetic effect of the RNAi silencing and the transgenes used (mCherry- histone H2B and plasma membrane GFP- PH(PLC161)), as we did not observe sterility in the distal screen (Supplementary Table 4)."  
+
+<|ref|>text<|/ref|><|det|>[[93, 617, 348, 630]]<|/det|>
+Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[92, 641, 896, 706]]<|/det|>
+The revised manuscript from Cao et al. titled "A Nucleic Acid Binding Protein Map of Germline Regulation" examines the role of nucleic acid binding proteins in the germline of C. elegans. The results provide an extremely useful atlas of transcription factor function throughout the different stages of germ cell development in C. elegans. This work should be relevant to researchers interested in mitotic and meiotic cell divisions and germ cell development and differentiation. I expressed 3 major concerns in my review of a previous version of this manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[92, 717, 896, 853]]<|/det|>
+These concerns were relatively minor, and the authors adequately address each of them in this revision. My first two concerns focused on the fact that their RNA was isolated from whole gonads and appears to have some contamination from the somatic gonad and other non- gonadal tissues. They have restated the description of the genes they identify as "germline enriched" rather than more specifically "germline- expressed" throughout the manuscript and have mentioned the possibility of contamination from somatic tissues in the methods. My third concern was that it was not entirely clear how they selected NBP genes for further analysis when combining their RNAseq results with results form earlier work. They point out how they explained this selection in the original manuscript and have revised this slightly in the current manuscript to indicate they apparently chose genes that were detected in all three of their RNAseq samples and in in both gonads analyzed by Tzur et al. In addition, they included any genes that detected \(>10\) CPM in the analyses by Cao et al (2017). This concern was also shared by another reviewer, so I hope this reviewer is also satisfied with the revised description.  
+
+<|ref|>text<|/ref|><|det|>[[92, 863, 890, 889]]<|/det|>
+The authors also addressed my more minor comments. In particular, I found it much easier to access information in the very useful Supplementary Tables.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[92, 66, 241, 78]]<|/det|>
+No response required
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__015d965c0cb0c74c387f9596e3f91c0197ff39a7333b1c0b5bef96993bcbbd18/images_list.json

@@ -0,0 +1,108 @@
+[
+  {
+    "type": "image",
+    "img_path": "images/Extended_Data_Figure_6a.jpg",
+    "caption": "Figure: a, b, Reversed Ti-displacement vector map (top) based on the atomically resolved plane-view HAADF-STEM image (bottom) of a single skyrmion bubble (marked by a white circle in Extended Data Fig. 6a), showing the hedgehog-like skyrmion structure. The sketch of the superlattice in b is overlaid with the planar-view dark-field TEM image and gives a top view of the superlattice. c, Ti-displacement vector map (front) based on the atomically resolved cross-sectional HAADF-STEM image (back), showing a cylindrical domain with anti-parallel (up–down) polarization. The sketch in b is overlaid with the cross-sectional dark-field TEM image and shows the cross sectional view of the superlattice. d, e, The 4D-STEM image of a [(PbTiO3)16/(SrTiO3)16]8 superlattice gives the ADF image (d) and maps of polar order using the probability current flow (e), which were reconstructed from the same 4D dataset. f, g, Multislice simulations of the beam propagation through the model structure from Fig. 2 show the ADF image (f) and the probability current flow (g), which were analyzed using the same process as the experimental data. The signals are not simple projections, but weighted by electron beam channelling towards the middle of the skyrmion bubble, where the polarization exhibits a Bloch-like character.",
+    "footnote": [],
+    "bbox": [],
+    "page_idx": 0
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_0.jpg",
+    "caption": "Figure S4: Dark field images corresponding disks 1-4.",
+    "footnote": [],
+    "bbox": [],
+    "page_idx": 6
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_1.jpg",
+    "caption": "Figure S1: Polarization vector maps overlaid on the drift-corrected HAADF-STEM images. The yellow vector indicates the direction of polarization. The underlying red/blue contrast is the curl of the displacement.",
+    "footnote": [],
+    "bbox": [
+      [
+        120,
+        551,
+        875,
+        811
+      ]
+    ],
+    "page_idx": 7
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_2.jpg",
+    "caption": "Figure S2: Strain maps (top two) and infinitesimal rotation (bottom) extracted by A-site fitting of atoms in Figure S1.",
+    "footnote": [],
+    "bbox": [
+      [
+        152,
+        255,
+        491,
+        616
+      ]
+    ],
+    "page_idx": 7
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_3.jpg",
+    "caption": "Figure S6: Virtual image, polarization, and helicity maps from different 4D STEM datasets showing the repeatability of different chiral/achiral boundaries in the PTO/STO trilayer. The presence of triple point topologies is evident whenever the chiral and achiral boundaries intersect one another. Scale bar: \\(20 \\mathrm{nm}\\) for all panels.",
+    "footnote": [],
+    "bbox": [
+      [
+        118,
+        110,
+        685,
+        400
+      ]
+    ],
+    "page_idx": 8
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_unknown_4.jpg",
+    "caption": "Figure S7: Virtual image, polarization, and helicity maps from different 4D STEM datasets showing the repeatability of different chiral/achiral boundaries in the PTO/STO trilayer. The presence of triple point topologies is evident whenever the chiral and achiral boundaries intersect one another. Scale bar: \\(30 \\mathrm{nm}\\)",
+    "footnote": [],
+    "bbox": [
+      [
+        118,
+        495,
+        567,
+        809
+      ]
+    ],
+    "page_idx": 9
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_1b.jpg",
+    "caption": "Figure 1b (ii) explaining the origin of net lateral polarization.",
+    "footnote": [],
+    "bbox": [
+      [
+        120,
+        115,
+        655,
+        243
+      ]
+    ],
+    "page_idx": 10
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_1c.jpg",
+    "caption": "Question 4. The scale is missing in Figure 1c. Response: The scale bar has been added now.",
+    "footnote": [],
+    "bbox": [
+      [
+        120,
+        357,
+        875,
+        616
+      ]
+    ],
+    "page_idx": 11
+  }
+]

peer_reviews/supplementary_0_Peer Review File__015d965c0cb0c74c387f9596e3f91c0197ff39a7333b1c0b5bef96993bcbbd18/supplementary_0_Peer Review File__015d965c0cb0c74c387f9596e3f91c0197ff39a7333b1c0b5bef96993bcbbd18.mmd

@@ -0,0 +1,268 @@
+
+# nature portfolio  
+
+# Peer Review File  
+
+The emergence of three- dimensional chiral domain walls in polar vortices  
+
+![](images/Extended_Data_Figure_6a.jpg)
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+## REVIEWER COMMENTS  
+
+## Reviewer #1 (Remarks to the Author):  
+
+This paper investigates the local polarization within tubular vortex topologies in STO/PTO/STO superlattice by 4D- STEM. Through quantifying the helicity based on polarization, they discussed the domain chirality separated by different domain walls. The authors discovered new pair of triple points with the opposite/same rotation at the junction of achiral and chiral domain walls. They further discussed that all possible configuration of three point topologies with reliable analysis of the origin of the triple points. This article presents an interesting study of topologically driven chiral domain walls in oxide superlattices by using specialized electron microscopy. I have some comments:  
+
+1. The polarization measurement based on 4D-STEM is an important basis of the article. The authors briefly mentioned in the article that 4D-STEM can precisely measure polarization in ferroelectrics due to the violation of Friedel's Law, I think it is necessary to further elaborate the principle and quantitative details, which is crucial for this article. For example, why choose the difference between 1, 2, 3, and 4 pairs to map the polarization, and can we get the same results by performing similar operations on other pairs?  
+
+2. In fact, 4D-STEM only maps the tetragonality (strain). Although it is usually positively correlated with the polarization, but sometimes it is not. For example, at the surface of PZT, the polariton is suppressed although the tetragonality (strain) is larger (Nature Communications | 7:11318 | DOI: 10.1038/ncomms11318). In the vortex system, I would expect this case is more complicated. I wonder how the author handle this. Related discussion is needed to avoid any misleading.  
+
+3. Vortex is a three-dimensional polar structure. Obviously, in the STEM image the polarization along the viewing direction is not uniform for this sample, i.e., near the core and far away from the core should have different polarization. For the conventional HAADF-STEM method without the depth resolution, it is very difficult to precisely measure the in-plane polarization to extract the chirality information. However, I donot see how the 4D-STEM solve this problem. Did the authors simply ignore this and extract the averaged polarization information? Detailed discussion is needed.  
+
+4. I wonder how to determine the position of vortex cores from the BF-STEM image in Fig. 1a, I can only distinguish the dark contrast of the wave shape. How to correlate the bright/dark contrast with the core positions. Besides, it would be better to remove a few red circles to show more details.  
+
+5. Both the virtual dark field image and the actual dark field image in Fig. 1c are constructed from diffraction information. Why are these domain walls invisible in virtual dark field images? In addition, the dark field image in Fig. 1c need to clarify the selected diffraction point.  
+
+6. Can different domain walls be distinguished by dark field images? e.g., through branching or stripe periodicity.  
+
+7. I found some problems in the cited refereces in this manuscript. For example, the authors mentioned the "The first experimental demonstration was in 25, where chirality switches...". In fact, at the same time another group published an article of switching chirality of polar vortex at the atomic scale STEM and dark-field TEM, which has been totally ingored in this manuscript. (Sci. China-Phys.Mech. Astron. 65, 237011 (2022), https://doi.org/10.1007/s11433-021-1820-4). Another one is the authors mentioned that "novel polarization textures in ferroelectrics such as merons, polar flux-closure domains,
+
+<--- Page Split --->
+
+vortices......in oxide superlattices", ignoring the representative polar antivortex (Nature Communications | (2021) 12:2054, https://doi.org/10.1038/s41467-021-22356-0), and threefold polar vertices (Nature Communications | (2022) 13:6340, https://doi.org/10.1038/s41467-022-33973-8)  
+
+8. Some minor suggestions: Fig. 1c lacks the scale bar; Fig. 3a lacks the axis; "Once the atoms were identified, the atomic planes were divided into different zone axis such as along [001]o and [001]o" seems to be a typo here.  
+
+## Reviewer #2 (Remarks to the Author):  
+
+S. Susarla et al. reported the chirality engineering of the topological polar vortex via atomic-scale symmetry-breaking operations. In this work, 4D-TEM results display the topology-driven three-dimensional domain walls, and the manuscript was well organized. As the author said, the chirality of the polar vortex is governed by the perpendicular and parallel polarization of the tubular vortex. In my opinion, polarization vector mapping of the vortex region in in-plane geometry will be a more intuitive way to demonstrate the vortex' chirality. Furthermore, the 4D-TEM data needs to be reanalysed, otherwise it is difficult to support the existing conclusions. Two main concerns are as follows:  
+
+1. In Figure 1b, it is difficult for the reader to locate the vortex center, which is consistent with the position marked by the author.  
+
+2. The combination of Figure 2 a, 2e and Figure 4e, there is a dislocation structure with respect to the tubular vortex across the \(\gamma\) -domain wall, in accordance with Figure 1b shows. However, in Fig. S3 and S4, the Virtual image has obviously the same boundary (in the middle of the image) as in Fig.2b, but the author does not identify it as any vortex domain wall. Could authors explain this difference? Perhaps, the polarization vector mapping on HAADF-STEM image will illustrate the issue more directly.  
+
+## Reviewer #3 (Remarks to the Author):  
+
+The manuscript by Susarla et al. presents a structural analysis of the 3D domain wall network in topological polar vortices formed in (SrTiO3)16/(PbTiO3)16/(SrTiO3)16 trilayers grown on SrRuO3- buffered DyScO3 substrates. The microstructure of the films is examined in detail by means 4D- STEM imaging. Thus, lateral and axial polarization maps are obtained by taking the normalized intensity difference be- tween opposite Friedel pair disks. This allows identifying three distinct types of domain wall configurations having different parallel/antiparallel axial/lateral components. Thus, two chiral and one achiral domain walls are identified. Finally, the authors observe that the domain walls meet at triple points, which typically appear in pairs. They hypothesize that these topological defects could lead to unique electrostatic and magnetic properties useful for quantum sensor applications.  
+
+The manuscript presents an original and very good experimental and analytical work on the microstructure of domain walls in topological polar vortices. Overall, the manuscript is clearly written and the figures are well elaborated. I enjoyed reading the manuscript, although I suggest introducing a couple of changes to make it a bit more comprehensible. In addition, I am listing also here some minor amendments.  
+
+1. In page 4 of the manuscript, "Supplementary Information" should be deleted, as there is no additional information in the SI referring to the trilayer stack.
+
+<--- Page Split --->
+
+2. At the end of page 5, it reads that in Figure 1b "a zig-zag type pattern, giving rise to a net in-plane polarization rotation along [001]o (lateral component) indicated as Px". I have difficulties seeing this net in-plane polarization. Could the authors plot the resulting in-plane polarization by averaging it along the horizontal direction and plotting it next to the image? This is not obvious from the figure. It seems to me that the Px at the top of the PTO film should cancel with the Px at the bottom of the PTO film.  
+
+3. Again, in Figure 1b, it seems there are vortex cores also in the bottom STO layer. Can the authors comment on this?  
+
+4. The scale is missing in Figure 1c.  
+
+5. It is a bit complicated to follow the discussion of the possible triple points depicted in Figure 3b. Could the authors correlate the triple points observed in Figure 3a (and also in the SI) to the triple point pairs represented in Figure 3b? This would make it easier to follow the explanation.  
+
+6. In the cation of Figure 4 it reads "The chirality for each type of domain is indicated by a sketched hand", but in the figure there are no sketched hands. Please, remove the sentence.  
+
+7. In the Materials and Methods the authors should indicate how was the sample for S/TEM prepared.  
+
+8. In page 14, please change "low-pass and high-pass Gaussian filters" for "band-pass Gaussian filters".  
+
+Based on my previous comments I recommend the publication of the manuscript of Susarla et al. in Nat. Commun. after minor revisions.
+
+<--- Page Split --->
+
+## REVIEWER COMMENTS  
+
+Reviewer #1 (Remarks to the Author):  
+
+This paper investigates the local polarization within tubular vortex topologies in STO/PTO/STO superlattice by 4D- STEM. Through quantifying the helicity based on polarization, they discussed the domain chirality separated by different domain walls. The authors discovered new pair of triple points with the opposite/same rotation at the junction of achiral and chiral domain walls. They further discussed that all possible configuration of three- point topologies with reliable analysis of the origin of the triple points. This article presents an interesting study of topologically driven chiral domain walls in oxide superlattices by using specialized electron microscopy. I have some comments:  
+
+We thank the reviewer for reading our manuscript. We have tried our best to answer the reviewer's questions.  
+
+Question 1. The polarization measurement based on 4D- STEM is an important basis of the article. The authors briefly mentioned in the article that 4D- STEM can precisely measure polarization in ferroelectrics due to the violation of Friedel's Law, I think it is necessary to further elaborate the principle and quantitative details, which is crucial for this article. For example, why choose the difference between 1, 2, 3, and 4 pairs to map the polarization, and can we get the same results by performing similar operations on other pairs?  
+
+Response: Friedel law states that the diffraction intensities from (hkl) and \(\overline{(hkl)}\) planes are similar. However, this rule breaks for non- centrosymmetric materials where the opposite Friedel pair disks have differential intensities due to multiple scattering effects. In this work, we have chosen to use the 1,2, 3 and 4 because these are the primary (hkl) directions along which the effect of Friedel's law breaking is the maximum. The intensity difference between a particular (hkl) Friedel pair is also coupled to the effective polarization along that direction. Since, we were concerned about purely lateral and axial polarization, we choose disks 1, 2, 3 and 4. The other pairs along different directions might have mixed lateral and axial polarization signals and hence it is difficult to interpret the resultant polarization maps.  
+
+We have now added a discussion about it in the main manuscript:  
+
+The polarization from the PTO layer can be determined qualitatively by subtracting the intensity of the opposite Friedel pair disks due to the violation of Friedel's law \(^{30 - 32}\) . This polarization mapping is slightly different from strain mapping where the accurate position of the diffracted disks is calculated \(^{35}\) . The method for subtracting the intensities of opposite Friedel's pair disks would also work for analogous \(\mathrm{Pb_xZr_{1 - x}TiO_3}\) (PZT) where polarization is suppressed under large tetragonality \(^{36}\) . The (hkl) Friedel pairs were chosen along \([001]_{0}\) and \([1\bar{1} 0]_{0}\) directions to determine pure lateral and axial polarization respectively. We note that the other (hkl) directions may also break Friedel's law, but the associated polarization will be a combination of lateral and axial component. The polarization maps corresponding to regions delimited by rectangles in Figure 2b are shown in Figure 2d- e.  
+
+Question 2. In fact, 4D- STEM only maps the tetragonality (strain). Although it is usually
+
+<--- Page Split --->
+
+positively correlated with the polarization, but sometimes it is not. For example, at the surface of PZT, the polarization is suppressed although the tetragonality (strain) is larger (Nature Communications | 7:11318 | DOI: 10.1038/ncomms11318). In the vortex system, I would expect this case is more complicated. I wonder how the author handle this. Related discussion is needed to avoid any misleading.  
+
+Response: We agree with the reviewer that the polarization and strain do not always go hand in hand. However, in 4D STEM, we determine the strain by the change in the position of the disks relative to the parent materials. The polarity is associated with the relative change in the intensity of the opposite Friedel pair disks, that measured in our current work. We have now added the discussion in main manuscript as shown in previous question. To help with the discussion, we have also cited the paper mentioned by the reviewer.  
+
+Question 3. Vortex is a three- dimensional polar structure. Obviously, in the STEM image the polarization along the viewing direction is not uniform for this sample, i.e., near the core and far away from the core should have different polarization. For the conventional HAADF- STEM method without the depth resolution, it is very difficult to precisely measure the in- plane polarization to extract the chirality information. However, I do not see how the 4D- STEM solve this problem. Did the authors simply ignore this and extract the averaged polarization information? Detailed discussion is needed.  
+
+Response: We thank the reviewer for pointing this aspect. Indeed, the polarization varies near and away from the vortex core. Hence, the HAADF- STEM fails in this regard. However, 4D- STEM allows us to image the lateral and axial polarization simultaneously which is not possible the HAADF- STEM. We cannot access the axial polarization directly with accuracy in conventional HAADF- STEM. However, in 4D- STEM we can access the pure axial polarization because it has a different diffraction condition (different Friedel's diffraction pairs) than lateral polarization. Additionally we primarily observe polarization from the top half of polar vortices in PbTiO₃ layer due to the limited depth resolution (http://arxiv.org/abs/2012.04134). Hence, the lateral polarization do not cancel out one another. We can't measure polarization quantitatively, but our qualitative measurements are good enough for the present purposes studying domain boundaries and triple points). We illustrate this better in the following diagram pasted paste in a figure from a previous paper (https://www.nature.com/articles/s41586-019-1092-8) where we can measure the topological polarization texture buried underneath SrTiO₃.
+
+<--- Page Split --->
+![](images/Figure_unknown_0.jpg)
+
+<center>Figure: a, b, Reversed Ti-displacement vector map (top) based on the atomically resolved plane-view HAADF-STEM image (bottom) of a single skyrmion bubble (marked by a white circle in Extended Data Fig. 6a), showing the hedgehog-like skyrmion structure. The sketch of the superlattice in b is overlaid with the planar-view dark-field TEM image and gives a top view of the superlattice. c, Ti-displacement vector map (front) based on the atomically resolved cross-sectional HAADF-STEM image (back), showing a cylindrical domain with anti-parallel (up–down) polarization. The sketch in b is overlaid with the cross-sectional dark-field TEM image and shows the cross sectional view of the superlattice. d, e, The 4D-STEM image of a [(PbTiO3)16/(SrTiO3)16]8 superlattice gives the ADF image (d) and maps of polar order using the probability current flow (e), which were reconstructed from the same 4D dataset. f, g, Multislice simulations of the beam propagation through the model structure from Fig. 2 show the ADF image (f) and the probability current flow (g), which were analyzed using the same process as the experimental data. The signals are not simple projections, but weighted by electron beam channelling towards the middle of the skyrmion bubble, where the polarization exhibits a Bloch-like character. </center>  
+
+We have also added this discussion in the main manuscript and modified the schematic in Figure 2.  
+
+. On the other hand, 4D- STEM allows us to collect a diffraction pattern at each probe position, which can then be used to create precise maps of physical quantities such as strain and polarization. The diffraction pattern in 4D- STEM offers a unique advantage over HAADF- STEM in polar vortices because direction of polarization can be accurately measured. For the present experiment, we performed 4D- STEM imaging on a trilayer STO/PTO/STO along the [110]o zone axis (Figure 2a). We used a probe size of \(\sim 7\) Å, larger than the STO/PTO unit cell dimensions ( \(\sim 4\) Å) to remove the atomic- resolution signal and to estimate the polarization at unit cell resolution.
+
+<--- Page Split --->
+![](images/Figure_unknown_1.jpg)
+  
+
+Question 4. I wonder how to determine the position of vortex cores from the BF- STEM image in Fig. 1a, I can only distinguish the dark contrast of the wave shape. How to correlate the bright/dark contrast with the core positions. Besides, it would be better to remove a few red circles to show more details.  
+
+Response: BF- STEM is more sensitive to the diffraction contrast. Vortex features are continuous rotation of polarization vectors and hence they are more visible at lower collection angles in BF- STEM mode. In the BF STEM mode, they usually appear as dark cross. For better clarity, we have removed some of the red circles as the reviewer was suggesting.  
+
+![](images/Figure_unknown_2.jpg)
+  
+
+Question 5. Both the virtual dark field image and the actual dark field image in Fig. 1c are constructed from diffraction information. Why are these domain walls invisible in virtual dark field images? In addition, the dark field image in Fig. 1c need to clarify the selected diffraction point.  
+
+Response: Some of the vortex domain walls may be missing in the virtual dark field images because they do not capture the entire information. Vortex domain walls originate from the change in lateral and axial polarization which can only be determined by difference in the intensity of the opposite Friedel pair disks.
+
+<--- Page Split --->
+
+Here is an example showing all the dark field images taken from disk 1, 2, 3 and 4. Even, if we combine everything, we will still find a few boundaries missing. It is only the normalized subtraction of (1- 2) and (3- 4) that reveals all of the vortex domain boundaries.  
+
+![](images/Figure_unknown_3.jpg)
+
+<center>Figure S4: Dark field images corresponding disks 1-4. </center>  
+
+Question 6. Can different domain walls be distinguished by dark field images? e.g., through branching or stripe periodicity.  
+
+Response: No, they cannot be distinguished by just dark field images. This is because the vortex domain walls are only identified by change lateral and axial polarization. The lateral and axial polarization cannot be identified by just one dark field image.  
+
+We have now also included the virtual dark field image created from four different disks. We cannot solely identify all of the polarization domain boundaries from just one dark field image.  
+
+Question 7. I found some problems in the cited references in this manuscript. For example, the authors mentioned the "The first experimental demonstration was in 25, where chirality switches...". In fact, at the same time another group published an article of switching chirality of polar vortex at the atomic scale STEM and dark- field TEM, which has been totally ignored in this manuscript. (Sci. China- Phys. Mech. Astron. 65, 237011 (2022), https://doi.org/10.1007/s11433- 021- 1820- 4). Another one is the authors mentioned that "novel polarization textures in ferroelectrics such as merons, polar flux- closure domains, vortices......in oxide superlattices", ignoring the representative polar antivortex (Nature Communications | (2021) 12:2054, https://doi.org/10.1038/s41467- 021- 22356- 0), and three- fold polar vertices (Nature Communications | (2022) 13:6340, https://doi.org/10.1038/s41467- 022- 33973- 8)  
+
+Response: We thank reviewer for the suggestions. We have included all the citations in the introduction of our manuscript.  
+
+Question 8. Some minor suggestions: Fig. 1c lacks the scale bar; Fig. 3a lacks the axis; "Once
+
+<--- Page Split --->
+
+the atoms were identified, the atomic planes were divided into different zone axis such as along [001]o and [001]o" seems to be a typo here.  
+
+Response: We have corrected the typo and added the scale bar in Figure 1c.  
+
+Reviewer #2 (Remarks to the Author):  
+
+S. Susarla et al. reported the chirality engineering of the topological polar vortex via atomic-scale symmetry-breaking operations. In this work, 4D-TEM results display the topology-driven three-dimensional domain walls, and the manuscript was well organized. As the author said, the chirality of the polar vortex is governed by the perpendicular and parallel polarization of the tubular vortex. In my opinion, polarization vector mapping of the vortex region in in-plane geometry will be a more intuitive way to demonstrate the vortex' chirality. Furthermore, the 4D-TEM data needs to be reanalyzed, otherwise it is difficult to support the existing conclusions. Two main concerns are as follows:  
+
+Response: We thank the reviewer for the comments. However the reviewer has not completely understood our motivation behind performing 4D- STEM. Polar vortices have both axial and lateral polarization components, out of which the former cannot be directly measured via HAADF- STEM due to the projection problem. Since 4D- STEM uses two different Friedel pair disks to map out the lateral and axial polarization, it is more intuitive to use 4D- STEM to quantify the polarization. A more detailed description is given in the latter responses.  
+
+Question 1. In Figure 1b, it is difficult for the reader to locate the vortex center, which is consistent with the position marked by the author.  
+
+Response: We have now reanalyzed Figure 1b which is shown below:  
+
+![](images/Figure_unknown_4.jpg)
+
+<center>Figure S1: Polarization vector maps overlaid on the drift-corrected HAADF-STEM images. The yellow vector indicates the direction of polarization. The underlying red/blue contrast is the curl of the displacement. </center>
+
+<--- Page Split --->
+
+We want to clarify that we are marking the vortex center at the maxima/ minima point of the polarization curl which is defined as below:  
+
+\[\theta = \frac{1}{2}\left(\frac{\partial u}{\partial y} -\frac{\partial v}{\partial x}\right)\]  
+
+This is how we have marked the center position of the vortices. We have put the zoomed- out version of the polarization maps, and their corresponding in- plane and out- of- plane strain maps in the supplementary information.  
+
+![](images/Figure_1b.jpg)
+
+<center>Figure S2: Strain maps (top two) and infinitesimal rotation (bottom) extracted by A-site fitting of atoms in Figure S1. </center>  
+
+Question 2. The combination of Figure 2 a, 2e and Figure 4e, there is a dislocation structure with respect to the tubular vortex across the \(\gamma\) - domain wall, in accordance with Figure 1b shows. However, in Fig. S3 and S4, the Virtual image has obviously the same boundary (in the middle of the image) as in Fig.2b, but the author does not identify it as any vortex domain wall. Could authors explain this difference? Perhaps, the polarization vector mapping on HAADF- STEM image will illustrate the issue more directly.  
+
+Response: We thank the reviewer for pointing out this aspect. We have now reanalyzed the 4D STEM datasets where we have included the missing vortex domain wall that reviewer pointed
+
+<--- Page Split --->
+
+out.  
+
+![](images/Figure_1c.jpg)
+
+<center>Figure S6: Virtual image, polarization, and helicity maps from different 4D STEM datasets showing the repeatability of different chiral/achiral boundaries in the PTO/STO trilayer. The presence of triple point topologies is evident whenever the chiral and achiral boundaries intersect one another. Scale bar: \(20 \mathrm{nm}\) for all panels. </center>  
+
+![PLACEHOLDER_11_1]
+
+<center>Figure S7: Virtual image, polarization, and helicity maps from different 4D STEM datasets showing the repeatability of different chiral/achiral boundaries in the PTO/STO trilayer. The presence of triple point topologies is evident whenever the chiral and achiral boundaries intersect one another. Scale bar: \(30 \mathrm{nm}\) </center>
+
+<--- Page Split --->
+
+The polarization maps on the in-plane HAADF- STEM image will not reveal all types of domain boundaries as the axial polarization cannot the measured directly in the HAADF- STEM.  
+
+Reviewer #3 (Remarks to the Author):  
+
+The manuscript by Susarla et al. presents a structural analysis of the 3D domain wall network in topological polar vortices formed in (SrTiO3)16/(PrTiO3)16/(SrTiO3)16 trilayers grown on SrRuO3- buffered DyScO3 substrates. The microstructure of the films is examined in detail by means 4D- STEM imaging. Thus, lateral, and axial polarization maps are obtained by taking the normalized intensity difference be- tween opposite Friedel pair disks. This allows identifying three distinct types of domain wall configurations having different parallel/antiparallel axial/lateral components. Thus, two chiral and one achiral domain walls are identified. Finally, the authors observe that the domain walls meet at triple points, which typically appear in pairs. They hypothesize that these topological defects could lead to unique electrostatic and magnetic properties useful for quantum sensor applications.  
+
+The manuscript presents an original and very good experimental and analytical work on the microstructure of domain walls in topological polar vortices. Overall, the manuscript is clearly written, and the figures are well elaborated. I enjoyed reading the manuscript, although I suggest introducing a couple of changes to make it a bit more comprehensible. In addition, I am listing also here some minor amendments.  
+
+Response: We thank the reviewer for appreciating our manuscript. We have answered almost all of the reviewer questions.  
+
+Question 1. In page 4 of the manuscript, "Supplementary Information" should be deleted, as there is no additional information in the SI referring to the trilayer stack.  
+
+Response: This has now been deleted.  
+
+Question 2. At the end of page 5, it reads that in Figure 1b "a zig- zag type pattern, giving rise to a net in- plane polarization rotation along [001]o (lateral component) indicated as Px". I have difficulties seeing this net in- plane polarization. Could the authors plot the resulting in- plane polarization by averaging it along the horizontal direction and plotting it next to the image? This is not obvious from the figure. It seems to me that the Px at the top of the PTO film should cancel with the Px at the bottom of the PTO film.  
+
+Response: We thank the reviewer for noticing this aspect. We have re- plotted our polarization vector maps in Figure 1b (i) where we observe that the top portion of lateral polarization is not equivalent with the bottom portion of the polarization. Additionally, we have a schematic in
+
+<--- Page Split --->
+![PLACEHOLDER_13_0]
+
+<center>Figure 1b (ii) explaining the origin of net lateral polarization. </center>  
+
+Question 3. Again, in Figure 1b, it seems there are vortex cores also in the bottom STO layer. Can the authors comment on this?  
+
+Response: We apologize to reviewers for this mistake. We had some errors in fitting the A sites. We re- did the A site gaussian fitting and replaced the earlier images better one as new Figure 1b. Refer to our response to reviewer 2 Question#1  
+
+![PLACEHOLDER_13_1]
+
+<center>Question 4. The scale is missing in Figure 1c. Response: The scale bar has been added now. </center>  
+
+Question 5. It is a bit complicated to follow the discussion of the possible triple points depicted in Figure 3b. Could the authors correlate the triple points observed in Figure 3a (and also in the SI) to the triple point pairs represented in Figure 3b? This would make it easier to follow the explanation.  
+
+Response: We thank the reviewers for the response. We have now included the labels in Figure 3b and also indicated their corresponding location in Figure 3a.
+
+<--- Page Split --->
+![PLACEHOLDER_14_0]
+  
+
+Question 6. In the cation of Figure 4 it reads "The chirality for each type of domain is indicated by a sketched hand", but in the figure there are no sketched hands. Please, remove the sentence. Response: We have removed that sentence.  
+
+Question 7. In the Materials and Methods the authors should indicate how was the sample for S/TEM prepared.  
+
+Response: We have now added the STEM sample preparation part in Materials and Methods.  
+
+STEM Sample preparation: In- plane [(PbTiO \(_3\) ) \(_{16}\) /(SrTiO \(_3\) ) \(_{16}\) ] trilayer grown on SrRuO \(_3\) /DyScO \(_3\) substrate were mechanically polished using a \(0.5^{\circ}\) wedge in Allied Multiprep. The samples were subsequently Ar ion milled in a Gatan Precision Ion Milling System, starting from \(3.5\mathrm{keV}\) at \(4^{\circ}\) down to \(1\mathrm{keV}\) at \(1^{\circ}\) for the final polish. The HAADF- STEM images were acquired using double aberration corrected TEAM I microscope operated at \(300\mathrm{kV}\) under nonmonochromated mode.  
+
+Question 8. In page 14, please change "low- pass and high- pass Gaussian filters" for "band- pass Gaussian filters".  
+
+Response: We have changed the typos  
+
+Based on my previous comments I recommend the publication of the manuscript of Susarla et al. in Nat. Commun. after minor revisions.
+
+<--- Page Split --->
+
+## REVIEWERS' COMMENTS  
+
+## Reviewer #1 (Remarks to the Author):  
+
+The revised manuscript addresses my concerns regarding the depth resolution problem in 4DSTEM and polarization information extraction, the authors state that the signal primarily originates from the top half of polar vortices in the PbTiO3 layer, as supported by simulation results (http://arxiv.org/abs/2012.04134). The virtual imaging ability and the ability to obtain pure axial polarization in 4DSTEM is indeed a valuable advantage over conventional STEM imaging. I understand that while quantitative measurements of polarization may not be feasible, the qualitative measurements in this work still provide valuable insights into studying domain boundaries and triple points. Overall, I'm happy with the response to all the questions and comments. Thus, I recommend the publication of this manuscript in NC.  
+
+## Reviewer #2 (Remarks to the Author):  
+
+The authors have addressed my concerns.  
+
+## Reviewer #3 (Remarks to the Author):  
+
+The authors have satisfactorily addressed most of my concerns and I therefore suggest publication of the manuscript by Susarla et al. in Nature Communications.
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__015d965c0cb0c74c387f9596e3f91c0197ff39a7333b1c0b5bef96993bcbbd18/supplementary_0_Peer Review File__015d965c0cb0c74c387f9596e3f91c0197ff39a7333b1c0b5bef96993bcbbd18_det.mmd

@@ -0,0 +1,350 @@
+<|ref|>title<|/ref|><|det|>[[99, 40, 506, 90]]<|/det|>
+# nature portfolio  
+
+<|ref|>title<|/ref|><|det|>[[102, 110, 373, 139]]<|/det|>
+# Peer Review File  
+
+<|ref|>text<|/ref|><|det|>[[125, 155, 870, 210]]<|/det|>
+The emergence of three- dimensional chiral domain walls in polar vortices  
+
+<|ref|>image<|/ref|><|det|>[[94, 732, 262, 780]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[270, 732, 880, 784]]<|/det|>
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[119, 83, 331, 100]]<|/det|>
+## REVIEWER COMMENTS  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 113, 449, 130]]<|/det|>
+## Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 143, 879, 265]]<|/det|>
+This paper investigates the local polarization within tubular vortex topologies in STO/PTO/STO superlattice by 4D- STEM. Through quantifying the helicity based on polarization, they discussed the domain chirality separated by different domain walls. The authors discovered new pair of triple points with the opposite/same rotation at the junction of achiral and chiral domain walls. They further discussed that all possible configuration of three point topologies with reliable analysis of the origin of the triple points. This article presents an interesting study of topologically driven chiral domain walls in oxide superlattices by using specialized electron microscopy. I have some comments:  
+
+<|ref|>text<|/ref|><|det|>[[118, 279, 847, 370]]<|/det|>
+1. The polarization measurement based on 4D-STEM is an important basis of the article. The authors briefly mentioned in the article that 4D-STEM can precisely measure polarization in ferroelectrics due to the violation of Friedel's Law, I think it is necessary to further elaborate the principle and quantitative details, which is crucial for this article. For example, why choose the difference between 1, 2, 3, and 4 pairs to map the polarization, and can we get the same results by performing similar operations on other pairs?  
+
+<|ref|>text<|/ref|><|det|>[[118, 384, 866, 476]]<|/det|>
+2. In fact, 4D-STEM only maps the tetragonality (strain). Although it is usually positively correlated with the polarization, but sometimes it is not. For example, at the surface of PZT, the polariton is suppressed although the tetragonality (strain) is larger (Nature Communications | 7:11318 | DOI: 10.1038/ncomms11318). In the vortex system, I would expect this case is more complicated. I wonder how the author handle this. Related discussion is needed to avoid any misleading.  
+
+<|ref|>text<|/ref|><|det|>[[117, 490, 870, 595]]<|/det|>
+3. Vortex is a three-dimensional polar structure. Obviously, in the STEM image the polarization along the viewing direction is not uniform for this sample, i.e., near the core and far away from the core should have different polarization. For the conventional HAADF-STEM method without the depth resolution, it is very difficult to precisely measure the in-plane polarization to extract the chirality information. However, I donot see how the 4D-STEM solve this problem. Did the authors simply ignore this and extract the averaged polarization information? Detailed discussion is needed.  
+
+<|ref|>text<|/ref|><|det|>[[118, 609, 857, 670]]<|/det|>
+4. I wonder how to determine the position of vortex cores from the BF-STEM image in Fig. 1a, I can only distinguish the dark contrast of the wave shape. How to correlate the bright/dark contrast with the core positions. Besides, it would be better to remove a few red circles to show more details.  
+
+<|ref|>text<|/ref|><|det|>[[118, 684, 870, 745]]<|/det|>
+5. Both the virtual dark field image and the actual dark field image in Fig. 1c are constructed from diffraction information. Why are these domain walls invisible in virtual dark field images? In addition, the dark field image in Fig. 1c need to clarify the selected diffraction point.  
+
+<|ref|>text<|/ref|><|det|>[[115, 759, 870, 790]]<|/det|>
+6. Can different domain walls be distinguished by dark field images? e.g., through branching or stripe periodicity.  
+
+<|ref|>text<|/ref|><|det|>[[118, 804, 870, 911]]<|/det|>
+7. I found some problems in the cited refereces in this manuscript. For example, the authors mentioned the "The first experimental demonstration was in 25, where chirality switches...". In fact, at the same time another group published an article of switching chirality of polar vortex at the atomic scale STEM and dark-field TEM, which has been totally ingored in this manuscript. (Sci. China-Phys.Mech. Astron. 65, 237011 (2022), https://doi.org/10.1007/s11433-021-1820-4). Another one is the authors mentioned that "novel polarization textures in ferroelectrics such as merons, polar flux-closure domains,
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 84, 870, 145]]<|/det|>
+vortices......in oxide superlattices", ignoring the representative polar antivortex (Nature Communications | (2021) 12:2054, https://doi.org/10.1038/s41467-021-22356-0), and threefold polar vertices (Nature Communications | (2022) 13:6340, https://doi.org/10.1038/s41467-022-33973-8)  
+
+<|ref|>text<|/ref|><|det|>[[118, 158, 870, 206]]<|/det|>
+8. Some minor suggestions: Fig. 1c lacks the scale bar; Fig. 3a lacks the axis; "Once the atoms were identified, the atomic planes were divided into different zone axis such as along [001]o and [001]o" seems to be a typo here.  
+
+<|ref|>sub_title<|/ref|><|det|>[[120, 263, 449, 280]]<|/det|>
+## Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 294, 873, 415]]<|/det|>
+S. Susarla et al. reported the chirality engineering of the topological polar vortex via atomic-scale symmetry-breaking operations. In this work, 4D-TEM results display the topology-driven three-dimensional domain walls, and the manuscript was well organized. As the author said, the chirality of the polar vortex is governed by the perpendicular and parallel polarization of the tubular vortex. In my opinion, polarization vector mapping of the vortex region in in-plane geometry will be a more intuitive way to demonstrate the vortex' chirality. Furthermore, the 4D-TEM data needs to be reanalysed, otherwise it is difficult to support the existing conclusions. Two main concerns are as follows:  
+
+<|ref|>text<|/ref|><|det|>[[118, 415, 875, 440]]<|/det|>
+1. In Figure 1b, it is difficult for the reader to locate the vortex center, which is consistent with the position marked by the author.  
+
+<|ref|>text<|/ref|><|det|>[[118, 441, 870, 536]]<|/det|>
+2. The combination of Figure 2 a, 2e and Figure 4e, there is a dislocation structure with respect to the tubular vortex across the \(\gamma\) -domain wall, in accordance with Figure 1b shows. However, in Fig. S3 and S4, the Virtual image has obviously the same boundary (in the middle of the image) as in Fig.2b, but the author does not identify it as any vortex domain wall. Could authors explain this difference? Perhaps, the polarization vector mapping on HAADF-STEM image will illustrate the issue more directly.  
+
+<|ref|>sub_title<|/ref|><|det|>[[120, 580, 449, 596]]<|/det|>
+## Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 609, 875, 761]]<|/det|>
+The manuscript by Susarla et al. presents a structural analysis of the 3D domain wall network in topological polar vortices formed in (SrTiO3)16/(PbTiO3)16/(SrTiO3)16 trilayers grown on SrRuO3- buffered DyScO3 substrates. The microstructure of the films is examined in detail by means 4D- STEM imaging. Thus, lateral and axial polarization maps are obtained by taking the normalized intensity difference be- tween opposite Friedel pair disks. This allows identifying three distinct types of domain wall configurations having different parallel/antiparallel axial/lateral components. Thus, two chiral and one achiral domain walls are identified. Finally, the authors observe that the domain walls meet at triple points, which typically appear in pairs. They hypothesize that these topological defects could lead to unique electrostatic and magnetic properties useful for quantum sensor applications.  
+
+<|ref|>text<|/ref|><|det|>[[118, 775, 875, 850]]<|/det|>
+The manuscript presents an original and very good experimental and analytical work on the microstructure of domain walls in topological polar vortices. Overall, the manuscript is clearly written and the figures are well elaborated. I enjoyed reading the manuscript, although I suggest introducing a couple of changes to make it a bit more comprehensible. In addition, I am listing also here some minor amendments.  
+
+<|ref|>text<|/ref|><|det|>[[118, 865, 860, 896]]<|/det|>
+1. In page 4 of the manuscript, "Supplementary Information" should be deleted, as there is no additional information in the SI referring to the trilayer stack.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 83, 870, 175]]<|/det|>
+2. At the end of page 5, it reads that in Figure 1b "a zig-zag type pattern, giving rise to a net in-plane polarization rotation along [001]o (lateral component) indicated as Px". I have difficulties seeing this net in-plane polarization. Could the authors plot the resulting in-plane polarization by averaging it along the horizontal direction and plotting it next to the image? This is not obvious from the figure. It seems to me that the Px at the top of the PTO film should cancel with the Px at the bottom of the PTO film.  
+
+<|ref|>text<|/ref|><|det|>[[118, 189, 875, 220]]<|/det|>
+3. Again, in Figure 1b, it seems there are vortex cores also in the bottom STO layer. Can the authors comment on this?  
+
+<|ref|>text<|/ref|><|det|>[[119, 234, 413, 250]]<|/det|>
+4. The scale is missing in Figure 1c.  
+
+<|ref|>text<|/ref|><|det|>[[118, 264, 878, 325]]<|/det|>
+5. It is a bit complicated to follow the discussion of the possible triple points depicted in Figure 3b. Could the authors correlate the triple points observed in Figure 3a (and also in the SI) to the triple point pairs represented in Figure 3b? This would make it easier to follow the explanation.  
+
+<|ref|>text<|/ref|><|det|>[[118, 339, 852, 385]]<|/det|>
+6. In the cation of Figure 4 it reads "The chirality for each type of domain is indicated by a sketched hand", but in the figure there are no sketched hands. Please, remove the sentence.  
+
+<|ref|>text<|/ref|><|det|>[[118, 399, 867, 430]]<|/det|>
+7. In the Materials and Methods the authors should indicate how was the sample for S/TEM prepared.  
+
+<|ref|>text<|/ref|><|det|>[[118, 444, 830, 475]]<|/det|>
+8. In page 14, please change "low-pass and high-pass Gaussian filters" for "band-pass Gaussian filters".  
+
+<|ref|>text<|/ref|><|det|>[[118, 489, 866, 520]]<|/det|>
+Based on my previous comments I recommend the publication of the manuscript of Susarla et al. in Nat. Commun. after minor revisions.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[116, 90, 350, 108]]<|/det|>
+## REVIEWER COMMENTS  
+
+<|ref|>text<|/ref|><|det|>[[116, 125, 422, 143]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 159, 883, 300]]<|/det|>
+This paper investigates the local polarization within tubular vortex topologies in STO/PTO/STO superlattice by 4D- STEM. Through quantifying the helicity based on polarization, they discussed the domain chirality separated by different domain walls. The authors discovered new pair of triple points with the opposite/same rotation at the junction of achiral and chiral domain walls. They further discussed that all possible configuration of three- point topologies with reliable analysis of the origin of the triple points. This article presents an interesting study of topologically driven chiral domain walls in oxide superlattices by using specialized electron microscopy. I have some comments:  
+
+<|ref|>text<|/ref|><|det|>[[116, 317, 808, 351]]<|/det|>
+We thank the reviewer for reading our manuscript. We have tried our best to answer the reviewer's questions.  
+
+<|ref|>text<|/ref|><|det|>[[115, 368, 879, 474]]<|/det|>
+Question 1. The polarization measurement based on 4D- STEM is an important basis of the article. The authors briefly mentioned in the article that 4D- STEM can precisely measure polarization in ferroelectrics due to the violation of Friedel's Law, I think it is necessary to further elaborate the principle and quantitative details, which is crucial for this article. For example, why choose the difference between 1, 2, 3, and 4 pairs to map the polarization, and can we get the same results by performing similar operations on other pairs?  
+
+<|ref|>text<|/ref|><|det|>[[115, 491, 881, 650]]<|/det|>
+Response: Friedel law states that the diffraction intensities from (hkl) and \(\overline{(hkl)}\) planes are similar. However, this rule breaks for non- centrosymmetric materials where the opposite Friedel pair disks have differential intensities due to multiple scattering effects. In this work, we have chosen to use the 1,2, 3 and 4 because these are the primary (hkl) directions along which the effect of Friedel's law breaking is the maximum. The intensity difference between a particular (hkl) Friedel pair is also coupled to the effective polarization along that direction. Since, we were concerned about purely lateral and axial polarization, we choose disks 1, 2, 3 and 4. The other pairs along different directions might have mixed lateral and axial polarization signals and hence it is difficult to interpret the resultant polarization maps.  
+
+<|ref|>text<|/ref|><|det|>[[116, 667, 632, 684]]<|/det|>
+We have now added a discussion about it in the main manuscript:  
+
+<|ref|>text<|/ref|><|det|>[[115, 684, 878, 860]]<|/det|>
+The polarization from the PTO layer can be determined qualitatively by subtracting the intensity of the opposite Friedel pair disks due to the violation of Friedel's law \(^{30 - 32}\) . This polarization mapping is slightly different from strain mapping where the accurate position of the diffracted disks is calculated \(^{35}\) . The method for subtracting the intensities of opposite Friedel's pair disks would also work for analogous \(\mathrm{Pb_xZr_{1 - x}TiO_3}\) (PZT) where polarization is suppressed under large tetragonality \(^{36}\) . The (hkl) Friedel pairs were chosen along \([001]_{0}\) and \([1\bar{1} 0]_{0}\) directions to determine pure lateral and axial polarization respectively. We note that the other (hkl) directions may also break Friedel's law, but the associated polarization will be a combination of lateral and axial component. The polarization maps corresponding to regions delimited by rectangles in Figure 2b are shown in Figure 2d- e.  
+
+<|ref|>text<|/ref|><|det|>[[115, 876, 820, 895]]<|/det|>
+Question 2. In fact, 4D- STEM only maps the tetragonality (strain). Although it is usually
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 879, 178]]<|/det|>
+positively correlated with the polarization, but sometimes it is not. For example, at the surface of PZT, the polarization is suppressed although the tetragonality (strain) is larger (Nature Communications | 7:11318 | DOI: 10.1038/ncomms11318). In the vortex system, I would expect this case is more complicated. I wonder how the author handle this. Related discussion is needed to avoid any misleading.  
+
+<|ref|>text<|/ref|><|det|>[[115, 195, 880, 300]]<|/det|>
+Response: We agree with the reviewer that the polarization and strain do not always go hand in hand. However, in 4D STEM, we determine the strain by the change in the position of the disks relative to the parent materials. The polarity is associated with the relative change in the intensity of the opposite Friedel pair disks, that measured in our current work. We have now added the discussion in main manuscript as shown in previous question. To help with the discussion, we have also cited the paper mentioned by the reviewer.  
+
+<|ref|>text<|/ref|><|det|>[[115, 316, 875, 440]]<|/det|>
+Question 3. Vortex is a three- dimensional polar structure. Obviously, in the STEM image the polarization along the viewing direction is not uniform for this sample, i.e., near the core and far away from the core should have different polarization. For the conventional HAADF- STEM method without the depth resolution, it is very difficult to precisely measure the in- plane polarization to extract the chirality information. However, I do not see how the 4D- STEM solve this problem. Did the authors simply ignore this and extract the averaged polarization information? Detailed discussion is needed.  
+
+<|ref|>text<|/ref|><|det|>[[114, 456, 881, 682]]<|/det|>
+Response: We thank the reviewer for pointing this aspect. Indeed, the polarization varies near and away from the vortex core. Hence, the HAADF- STEM fails in this regard. However, 4D- STEM allows us to image the lateral and axial polarization simultaneously which is not possible the HAADF- STEM. We cannot access the axial polarization directly with accuracy in conventional HAADF- STEM. However, in 4D- STEM we can access the pure axial polarization because it has a different diffraction condition (different Friedel's diffraction pairs) than lateral polarization. Additionally we primarily observe polarization from the top half of polar vortices in PbTiO₃ layer due to the limited depth resolution (http://arxiv.org/abs/2012.04134). Hence, the lateral polarization do not cancel out one another. We can't measure polarization quantitatively, but our qualitative measurements are good enough for the present purposes studying domain boundaries and triple points). We illustrate this better in the following diagram pasted paste in a figure from a previous paper (https://www.nature.com/articles/s41586-019-1092-8) where we can measure the topological polarization texture buried underneath SrTiO₃.
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[115, 87, 725, 344]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[114, 346, 877, 606]]<|/det|>
+<center>Figure: a, b, Reversed Ti-displacement vector map (top) based on the atomically resolved plane-view HAADF-STEM image (bottom) of a single skyrmion bubble (marked by a white circle in Extended Data Fig. 6a), showing the hedgehog-like skyrmion structure. The sketch of the superlattice in b is overlaid with the planar-view dark-field TEM image and gives a top view of the superlattice. c, Ti-displacement vector map (front) based on the atomically resolved cross-sectional HAADF-STEM image (back), showing a cylindrical domain with anti-parallel (up–down) polarization. The sketch in b is overlaid with the cross-sectional dark-field TEM image and shows the cross sectional view of the superlattice. d, e, The 4D-STEM image of a [(PbTiO3)16/(SrTiO3)16]8 superlattice gives the ADF image (d) and maps of polar order using the probability current flow (e), which were reconstructed from the same 4D dataset. f, g, Multislice simulations of the beam propagation through the model structure from Fig. 2 show the ADF image (f) and the probability current flow (g), which were analyzed using the same process as the experimental data. The signals are not simple projections, but weighted by electron beam channelling towards the middle of the skyrmion bubble, where the polarization exhibits a Bloch-like character. </center>  
+
+<|ref|>text<|/ref|><|det|>[[115, 623, 876, 658]]<|/det|>
+We have also added this discussion in the main manuscript and modified the schematic in Figure 2.  
+
+<|ref|>text<|/ref|><|det|>[[115, 675, 877, 817]]<|/det|>
+. On the other hand, 4D- STEM allows us to collect a diffraction pattern at each probe position, which can then be used to create precise maps of physical quantities such as strain and polarization. The diffraction pattern in 4D- STEM offers a unique advantage over HAADF- STEM in polar vortices because direction of polarization can be accurately measured. For the present experiment, we performed 4D- STEM imaging on a trilayer STO/PTO/STO along the [110]o zone axis (Figure 2a). We used a probe size of \(\sim 7\) Å, larger than the STO/PTO unit cell dimensions ( \(\sim 4\) Å) to remove the atomic- resolution signal and to estimate the polarization at unit cell resolution.
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[133, 93, 480, 277]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[115, 302, 882, 373]]<|/det|>
+Question 4. I wonder how to determine the position of vortex cores from the BF- STEM image in Fig. 1a, I can only distinguish the dark contrast of the wave shape. How to correlate the bright/dark contrast with the core positions. Besides, it would be better to remove a few red circles to show more details.  
+
+<|ref|>text<|/ref|><|det|>[[115, 389, 882, 460]]<|/det|>
+Response: BF- STEM is more sensitive to the diffraction contrast. Vortex features are continuous rotation of polarization vectors and hence they are more visible at lower collection angles in BF- STEM mode. In the BF STEM mode, they usually appear as dark cross. For better clarity, we have removed some of the red circles as the reviewer was suggesting.  
+
+<|ref|>image<|/ref|><|det|>[[123, 477, 763, 700]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[115, 718, 864, 790]]<|/det|>
+Question 5. Both the virtual dark field image and the actual dark field image in Fig. 1c are constructed from diffraction information. Why are these domain walls invisible in virtual dark field images? In addition, the dark field image in Fig. 1c need to clarify the selected diffraction point.  
+
+<|ref|>text<|/ref|><|det|>[[115, 805, 848, 877]]<|/det|>
+Response: Some of the vortex domain walls may be missing in the virtual dark field images because they do not capture the entire information. Vortex domain walls originate from the change in lateral and axial polarization which can only be determined by difference in the intensity of the opposite Friedel pair disks.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 866, 142]]<|/det|>
+Here is an example showing all the dark field images taken from disk 1, 2, 3 and 4. Even, if we combine everything, we will still find a few boundaries missing. It is only the normalized subtraction of (1- 2) and (3- 4) that reveals all of the vortex domain boundaries.  
+
+<|ref|>image<|/ref|><|det|>[[117, 143, 884, 417]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 422, 544, 441]]<|/det|>
+<center>Figure S4: Dark field images corresponding disks 1-4. </center>  
+
+<|ref|>text<|/ref|><|det|>[[115, 456, 844, 492]]<|/det|>
+Question 6. Can different domain walls be distinguished by dark field images? e.g., through branching or stripe periodicity.  
+
+<|ref|>text<|/ref|><|det|>[[115, 508, 877, 559]]<|/det|>
+Response: No, they cannot be distinguished by just dark field images. This is because the vortex domain walls are only identified by change lateral and axial polarization. The lateral and axial polarization cannot be identified by just one dark field image.  
+
+<|ref|>text<|/ref|><|det|>[[115, 559, 864, 595]]<|/det|>
+We have now also included the virtual dark field image created from four different disks. We cannot solely identify all of the polarization domain boundaries from just one dark field image.  
+
+<|ref|>text<|/ref|><|det|>[[115, 611, 879, 805]]<|/det|>
+Question 7. I found some problems in the cited references in this manuscript. For example, the authors mentioned the "The first experimental demonstration was in 25, where chirality switches...". In fact, at the same time another group published an article of switching chirality of polar vortex at the atomic scale STEM and dark- field TEM, which has been totally ignored in this manuscript. (Sci. China- Phys. Mech. Astron. 65, 237011 (2022), https://doi.org/10.1007/s11433- 021- 1820- 4). Another one is the authors mentioned that "novel polarization textures in ferroelectrics such as merons, polar flux- closure domains, vortices......in oxide superlattices", ignoring the representative polar antivortex (Nature Communications | (2021) 12:2054, https://doi.org/10.1038/s41467- 021- 22356- 0), and three- fold polar vertices (Nature Communications | (2022) 13:6340, https://doi.org/10.1038/s41467- 022- 33973- 8)  
+
+<|ref|>text<|/ref|><|det|>[[115, 823, 836, 858]]<|/det|>
+Response: We thank reviewer for the suggestions. We have included all the citations in the introduction of our manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[115, 874, 865, 893]]<|/det|>
+Question 8. Some minor suggestions: Fig. 1c lacks the scale bar; Fig. 3a lacks the axis; "Once
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 868, 126]]<|/det|>
+the atoms were identified, the atomic planes were divided into different zone axis such as along [001]o and [001]o" seems to be a typo here.  
+
+<|ref|>text<|/ref|><|det|>[[115, 142, 714, 161]]<|/det|>
+Response: We have corrected the typo and added the scale bar in Figure 1c.  
+
+<|ref|>text<|/ref|><|det|>[[116, 177, 420, 195]]<|/det|>
+Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 211, 881, 352]]<|/det|>
+S. Susarla et al. reported the chirality engineering of the topological polar vortex via atomic-scale symmetry-breaking operations. In this work, 4D-TEM results display the topology-driven three-dimensional domain walls, and the manuscript was well organized. As the author said, the chirality of the polar vortex is governed by the perpendicular and parallel polarization of the tubular vortex. In my opinion, polarization vector mapping of the vortex region in in-plane geometry will be a more intuitive way to demonstrate the vortex' chirality. Furthermore, the 4D-TEM data needs to be reanalyzed, otherwise it is difficult to support the existing conclusions. Two main concerns are as follows:  
+
+<|ref|>text<|/ref|><|det|>[[115, 369, 866, 474]]<|/det|>
+Response: We thank the reviewer for the comments. However the reviewer has not completely understood our motivation behind performing 4D- STEM. Polar vortices have both axial and lateral polarization components, out of which the former cannot be directly measured via HAADF- STEM due to the projection problem. Since 4D- STEM uses two different Friedel pair disks to map out the lateral and axial polarization, it is more intuitive to use 4D- STEM to quantify the polarization. A more detailed description is given in the latter responses.  
+
+<|ref|>text<|/ref|><|det|>[[115, 491, 816, 526]]<|/det|>
+Question 1. In Figure 1b, it is difficult for the reader to locate the vortex center, which is consistent with the position marked by the author.  
+
+<|ref|>text<|/ref|><|det|>[[115, 527, 671, 544]]<|/det|>
+Response: We have now reanalyzed Figure 1b which is shown below:  
+
+<|ref|>image<|/ref|><|det|>[[120, 551, 875, 811]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 813, 878, 866]]<|/det|>
+<center>Figure S1: Polarization vector maps overlaid on the drift-corrected HAADF-STEM images. The yellow vector indicates the direction of polarization. The underlying red/blue contrast is the curl of the displacement. </center>
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 845, 125]]<|/det|>
+We want to clarify that we are marking the vortex center at the maxima/ minima point of the polarization curl which is defined as below:  
+
+<|ref|>equation<|/ref|><|det|>[[423, 131, 568, 172]]<|/det|>
+\[\theta = \frac{1}{2}\left(\frac{\partial u}{\partial y} -\frac{\partial v}{\partial x}\right)\]  
+
+<|ref|>text<|/ref|><|det|>[[115, 188, 866, 241]]<|/det|>
+This is how we have marked the center position of the vortices. We have put the zoomed- out version of the polarization maps, and their corresponding in- plane and out- of- plane strain maps in the supplementary information.  
+
+<|ref|>image<|/ref|><|det|>[[152, 255, 491, 616]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 626, 866, 661]]<|/det|>
+<center>Figure S2: Strain maps (top two) and infinitesimal rotation (bottom) extracted by A-site fitting of atoms in Figure S1. </center>  
+
+<|ref|>text<|/ref|><|det|>[[115, 678, 881, 784]]<|/det|>
+Question 2. The combination of Figure 2 a, 2e and Figure 4e, there is a dislocation structure with respect to the tubular vortex across the \(\gamma\) - domain wall, in accordance with Figure 1b shows. However, in Fig. S3 and S4, the Virtual image has obviously the same boundary (in the middle of the image) as in Fig.2b, but the author does not identify it as any vortex domain wall. Could authors explain this difference? Perhaps, the polarization vector mapping on HAADF- STEM image will illustrate the issue more directly.  
+
+<|ref|>text<|/ref|><|det|>[[115, 800, 866, 836]]<|/det|>
+Response: We thank the reviewer for pointing out this aspect. We have now reanalyzed the 4D STEM datasets where we have included the missing vortex domain wall that reviewer pointed
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 94, 147, 106]]<|/det|>
+out.  
+
+<|ref|>image<|/ref|><|det|>[[118, 110, 685, 400]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 404, 881, 474]]<|/det|>
+<center>Figure S6: Virtual image, polarization, and helicity maps from different 4D STEM datasets showing the repeatability of different chiral/achiral boundaries in the PTO/STO trilayer. The presence of triple point topologies is evident whenever the chiral and achiral boundaries intersect one another. Scale bar: \(20 \mathrm{nm}\) for all panels. </center>  
+
+<|ref|>image<|/ref|><|det|>[[118, 495, 567, 809]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 814, 880, 883]]<|/det|>
+<center>Figure S7: Virtual image, polarization, and helicity maps from different 4D STEM datasets showing the repeatability of different chiral/achiral boundaries in the PTO/STO trilayer. The presence of triple point topologies is evident whenever the chiral and achiral boundaries intersect one another. Scale bar: \(30 \mathrm{nm}\) </center>
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 872, 125]]<|/det|>
+The polarization maps on the in-plane HAADF- STEM image will not reveal all types of domain boundaries as the axial polarization cannot the measured directly in the HAADF- STEM.  
+
+<|ref|>text<|/ref|><|det|>[[116, 142, 420, 160]]<|/det|>
+Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[115, 177, 872, 352]]<|/det|>
+The manuscript by Susarla et al. presents a structural analysis of the 3D domain wall network in topological polar vortices formed in (SrTiO3)16/(PrTiO3)16/(SrTiO3)16 trilayers grown on SrRuO3- buffered DyScO3 substrates. The microstructure of the films is examined in detail by means 4D- STEM imaging. Thus, lateral, and axial polarization maps are obtained by taking the normalized intensity difference be- tween opposite Friedel pair disks. This allows identifying three distinct types of domain wall configurations having different parallel/antiparallel axial/lateral components. Thus, two chiral and one achiral domain walls are identified. Finally, the authors observe that the domain walls meet at triple points, which typically appear in pairs. They hypothesize that these topological defects could lead to unique electrostatic and magnetic properties useful for quantum sensor applications.  
+
+<|ref|>text<|/ref|><|det|>[[115, 369, 877, 456]]<|/det|>
+The manuscript presents an original and very good experimental and analytical work on the microstructure of domain walls in topological polar vortices. Overall, the manuscript is clearly written, and the figures are well elaborated. I enjoyed reading the manuscript, although I suggest introducing a couple of changes to make it a bit more comprehensible. In addition, I am listing also here some minor amendments.  
+
+<|ref|>text<|/ref|><|det|>[[115, 473, 875, 508]]<|/det|>
+Response: We thank the reviewer for appreciating our manuscript. We have answered almost all of the reviewer questions.  
+
+<|ref|>text<|/ref|><|det|>[[115, 525, 847, 560]]<|/det|>
+Question 1. In page 4 of the manuscript, "Supplementary Information" should be deleted, as there is no additional information in the SI referring to the trilayer stack.  
+
+<|ref|>text<|/ref|><|det|>[[116, 560, 420, 577]]<|/det|>
+Response: This has now been deleted.  
+
+<|ref|>text<|/ref|><|det|>[[115, 594, 875, 700]]<|/det|>
+Question 2. At the end of page 5, it reads that in Figure 1b "a zig- zag type pattern, giving rise to a net in- plane polarization rotation along [001]o (lateral component) indicated as Px". I have difficulties seeing this net in- plane polarization. Could the authors plot the resulting in- plane polarization by averaging it along the horizontal direction and plotting it next to the image? This is not obvious from the figure. It seems to me that the Px at the top of the PTO film should cancel with the Px at the bottom of the PTO film.  
+
+<|ref|>text<|/ref|><|det|>[[115, 717, 861, 770]]<|/det|>
+Response: We thank the reviewer for noticing this aspect. We have re- plotted our polarization vector maps in Figure 1b (i) where we observe that the top portion of lateral polarization is not equivalent with the bottom portion of the polarization. Additionally, we have a schematic in
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[120, 115, 655, 243]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 90, 598, 110]]<|/det|>
+<center>Figure 1b (ii) explaining the origin of net lateral polarization. </center>  
+
+<|ref|>text<|/ref|><|det|>[[115, 260, 857, 296]]<|/det|>
+Question 3. Again, in Figure 1b, it seems there are vortex cores also in the bottom STO layer. Can the authors comment on this?  
+
+<|ref|>text<|/ref|><|det|>[[115, 296, 877, 350]]<|/det|>
+Response: We apologize to reviewers for this mistake. We had some errors in fitting the A sites. We re- did the A site gaussian fitting and replaced the earlier images better one as new Figure 1b. Refer to our response to reviewer 2 Question#1  
+
+<|ref|>image<|/ref|><|det|>[[120, 357, 875, 616]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[115, 635, 481, 671]]<|/det|>
+<center>Question 4. The scale is missing in Figure 1c. Response: The scale bar has been added now. </center>  
+
+<|ref|>text<|/ref|><|det|>[[115, 687, 870, 758]]<|/det|>
+Question 5. It is a bit complicated to follow the discussion of the possible triple points depicted in Figure 3b. Could the authors correlate the triple points observed in Figure 3a (and also in the SI) to the triple point pairs represented in Figure 3b? This would make it easier to follow the explanation.  
+
+<|ref|>text<|/ref|><|det|>[[115, 774, 870, 810]]<|/det|>
+Response: We thank the reviewers for the response. We have now included the labels in Figure 3b and also indicated their corresponding location in Figure 3a.
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[125, 90, 880, 288]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[115, 304, 872, 357]]<|/det|>
+Question 6. In the cation of Figure 4 it reads "The chirality for each type of domain is indicated by a sketched hand", but in the figure there are no sketched hands. Please, remove the sentence. Response: We have removed that sentence.  
+
+<|ref|>text<|/ref|><|det|>[[115, 373, 860, 408]]<|/det|>
+Question 7. In the Materials and Methods the authors should indicate how was the sample for S/TEM prepared.  
+
+<|ref|>text<|/ref|><|det|>[[115, 408, 856, 427]]<|/det|>
+Response: We have now added the STEM sample preparation part in Materials and Methods.  
+
+<|ref|>text<|/ref|><|det|>[[115, 450, 870, 556]]<|/det|>
+STEM Sample preparation: In- plane [(PbTiO \(_3\) ) \(_{16}\) /(SrTiO \(_3\) ) \(_{16}\) ] trilayer grown on SrRuO \(_3\) /DyScO \(_3\) substrate were mechanically polished using a \(0.5^{\circ}\) wedge in Allied Multiprep. The samples were subsequently Ar ion milled in a Gatan Precision Ion Milling System, starting from \(3.5\mathrm{keV}\) at \(4^{\circ}\) down to \(1\mathrm{keV}\) at \(1^{\circ}\) for the final polish. The HAADF- STEM images were acquired using double aberration corrected TEAM I microscope operated at \(300\mathrm{kV}\) under nonmonochromated mode.  
+
+<|ref|>text<|/ref|><|det|>[[115, 573, 870, 608]]<|/det|>
+Question 8. In page 14, please change "low- pass and high- pass Gaussian filters" for "band- pass Gaussian filters".  
+
+<|ref|>text<|/ref|><|det|>[[116, 609, 425, 626]]<|/det|>
+Response: We have changed the typos  
+
+<|ref|>text<|/ref|><|det|>[[115, 644, 877, 679]]<|/det|>
+Based on my previous comments I recommend the publication of the manuscript of Susarla et al. in Nat. Commun. after minor revisions.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[119, 83, 348, 100]]<|/det|>
+## REVIEWERS' COMMENTS  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 113, 450, 130]]<|/det|>
+## Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 144, 872, 279]]<|/det|>
+The revised manuscript addresses my concerns regarding the depth resolution problem in 4DSTEM and polarization information extraction, the authors state that the signal primarily originates from the top half of polar vortices in the PbTiO3 layer, as supported by simulation results (http://arxiv.org/abs/2012.04134). The virtual imaging ability and the ability to obtain pure axial polarization in 4DSTEM is indeed a valuable advantage over conventional STEM imaging. I understand that while quantitative measurements of polarization may not be feasible, the qualitative measurements in this work still provide valuable insights into studying domain boundaries and triple points. Overall, I'm happy with the response to all the questions and comments. Thus, I recommend the publication of this manuscript in NC.  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 308, 450, 325]]<|/det|>
+## Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[119, 339, 470, 355]]<|/det|>
+The authors have addressed my concerns.  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 384, 450, 401]]<|/det|>
+## Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 414, 835, 445]]<|/det|>
+The authors have satisfactorily addressed most of my concerns and I therefore suggest publication of the manuscript by Susarla et al. in Nature Communications.
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__0179e628a853118df9da75d50a227d9cf3c9b00dfa8825f8efe5e29d0d1c5f4c/images_list.json

@@ -0,0 +1,145 @@
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+  {
+    "type": "image",
+    "img_path": "images/Figure_1.jpg",
+    "caption": "Fig. 1 | Optical polarization property of TFBG: (a) Experimental polarized transmission spectra in electrolyte (red, S-pol input, and blue, P-pol input); (b) Radial (P-pol) and (c) azimuthal (S-pol) dependence of simulated E-field intensity of cut-off guided mode (the arrows show the E-field vector orientations in both cases).",
+    "footnote": [],
+    "bbox": [],
+    "page_idx": 0
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_2.jpg",
+    "caption": "Fig. 2 | The PEEK container.",
+    "footnote": [],
+    "bbox": [
+      [
+        162,
+        320,
+        835,
+        475
+      ]
+    ],
+    "page_idx": 5
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_3.jpg",
+    "caption": "Fig. 3 | Decoding sulfur concentration gradient of LSB by two sensors close to electrode.",
+    "footnote": [],
+    "bbox": [
+      [
+        150,
+        88,
+        846,
+        270
+      ]
+    ],
+    "page_idx": 6
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_4.jpg",
+    "caption": "Fig. 4 | Thermal calibration of electrolyte: (a) The thermal response in air and (b) The corresponding thermal sensitivity of core mode and target cladding mode (cutoff mode in electrolyte); (c) The thermal response in electrolyte and (d) sensitivity.",
+    "footnote": [],
+    "bbox": [
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+    "page_idx": 7
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_5.jpg",
+    "caption": "Fig 5. The adsorption test of polysulfide by SP or KB carbon.",
+    "footnote": [],
+    "bbox": [
+      [
+        297,
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+        690,
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+      ]
+    ],
+    "page_idx": 8
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_1.jpg",
+    "caption": "Fig. 1 | The morphology (SEM) of cathode (S: Super P=6: 4 wt%) at the beginning of discharging and end of charging",
+    "footnote": [],
+    "bbox": [
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+    "caption": "Fig. 2 | The PEEK container.",
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+      ]
+    ],
+    "page_idx": 11
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_3.jpg",
+    "caption": "Fig. 3 | Thermal calibration of electrolyte: (a) The thermal response in air and (b) the corresponding thermal sensitivity of core mode and target cladding mode (cutoff mode in electrolyte); (c) The thermal response in electrolyte and (d) sensitivity.",
+    "footnote": [],
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+    "caption": "Fig. 4 | The definition of consumption rate of sulfur under the galvanostatic condition.",
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+    "img_path": "images/Figure_5.jpg",
+    "caption": "Fig. 5 | The TFBG response to 100 mM \\(\\mathrm{Li}_2\\mathrm{S}_2\\) , \\(\\mathrm{Li}_2\\mathrm{S}_5\\) and \\(\\mathrm{Li}_2\\mathrm{S}_8\\) in electrolyte of 1 M LiTFSI, 0.5 M \\(\\mathrm{LiNO}_3\\) in DOL/DME (1:1, v/v)",
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peer_reviews/supplementary_0_Peer Review File__0179e628a853118df9da75d50a227d9cf3c9b00dfa8825f8efe5e29d0d1c5f4c/supplementary_0_Peer Review File__0179e628a853118df9da75d50a227d9cf3c9b00dfa8825f8efe5e29d0d1c5f4c.mmd

@@ -0,0 +1,294 @@
+
+# nature portfolio  
+
+Peer Review File  
+
+Detangling electrolyte chemical dynamics in lithium sulfur batteries by operando monitoring with optical resonance combs  
+
+![](images/Figure_1.jpg)
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to  
+
+the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+Reviewer #1 (Remarks to the Author):  
+
+In this work, TFBG were employed to operando track the chemical dynamics/states of the Li- S battery via electrolyte sulfur concentration, revealing the correlated relationship between the capacity fading and dynamic of dissolution/precipitation of polysulfides over cycling and at different cycling rates. Overall, I think is an interesting work, since the authors proposed a new application of optical fiber sensor for battery monitoring. Consequently, I will recommend its publication after a minor revision.  
+
+(1) In Fig1c, why did the author choose the \(^{**}n\) region as the sensing mode? Can other modes also be used as sensing modes, and if so, what are the differences in sensing performance?  
+
+(2) As we all know, the S cathodes exhibit around \(80\%\) volume changes during cycling. Therefore, in this manuscript, please explain whether the wavelength shift caused by the volume changes of the S cathode will affect the results.  
+
+(3) In manuscript, the author filled \(500\mu L\) of electrolyte into the cell, which is different from the common application scenario of optical fiber (the fiber is placed in a huge amount of liquid). Therefore, the author should consider whether the electrolyte was sufficient to completely infiltrate the fiber and can evenly wrap the optical fiber, which will affect the result.  
+
+(4) Ultimately, optical fiber is a linear sensor with a limited detection range. In manuscript, the author also mentioned that the "delay" due to position of the sensor in the cell. So can the author show the evolution of sulfur concentration in other locations, such as near the electrode or at the same level as the current fiber?  
+
+(5) In Fig. 4b, I observed a slight dissonance in 12th cycle where the drop in temperature caused the wavelength to decrease. However, the double effect of temperature dropping and Li2S nucleation will result in smaller wavelength. Why is the valley of sulfur concentration in 12th cycle greater than that in other cycles?  
+
+(6) In Fig. 4b, an increase of the background of sulfur concentration was attributed to the strong shuttle effect with less sulfur utilization caused by the high E/S ratio. However, other components in the electrolyte also undergo irreversible chemical reactions during cycling (> 400 h). So how can you confirm that the increase of the background of sulfur concentration is solely due to the strong shuttle effect with less sulfur utilization?  
+
+(7) KB was better than SP as physical nonpolar sulfur confinement host. Therefore, the sulfur concentration in KB/S should be smaller than SP/S. In manuscript, the sulfur concentration in KB/S was \(\sim 700 \text{mM}\) and the sulfur concentration in SP/S was \(\sim 500 \text{mM}\) . Please explain it.  
+
+(8) Typos or hard to understand. The authors need to rephrase the following parts.
+
+<--- Page Split --->
+
+-In abstract, "...the nucleation pathway and crystallization of Li2S and sulfur governs the cycling performance..." should be "...the nucleation pathway and crystallization of Li2S and sulfur govern the cycling performance..."  
+
+-On page 1, "...and shuttle effect caused by soluble polysulfide in electrolyte..." should be "...and the shuttle effect caused by soluble polysulfide in electrolyte..."  
+
+-On page 11, "...On the upper voltage plateau the solid sulfur dissolution or recrystallization..." should be "...On the upper voltage plateau, the solid sulfur dissolution or recrystallization..."  
+
+-On page 15, "...changing from progressive to an instantaneous pathway..." should be "...changing from a progressive to an instantaneous pathway ..."  
+
+Reviewer #2 (Remarks to the Author):  
+
+The paper entitled "Detangling electrolyte chemical dynamics and evolution in Li- S batteries by operando monitoring with optical resonance combs" reports about TFBG technique for studying electrolyte chemical dynamics and evolution in Li- S batteries by tracking sulfur concentration and demonstrate that the nucleation pathway and crystallization of Li2S and sulfur governs the cycling performance. Although the similar technique has already been used in their previous work to track electrolyte concentration through refractive index (DOI: 10.1039/d1ee02186a), this work brings a new insight on understanding the mechanism and electrolyte chemical dynamics of LSB. Thus, this paper could be considered for publication after a minor revision, detailed in the following comments:  
+
+1. Why the operando XRD in fig 2a doesn't show the recrystallization process of sulfur at the end of charging?  
+
+2. There is a sudden drop of sulfur concentration from A to B in the beginning of discharge in fig 3c, which also happens at the beginning of next discharge period. What leds to this? The sulfur concentration from A to B in fig 3c reacts different from in fig 3a and b. It doesn't seem to be attributed to "delay".  
+
+3. The soluble sulfur transport flux calculation should be based on equilibrium state, as the result is used for representing the whole process. So I think it is more reasonable to exclude region from A to B and C to D during transport flux calculation. Then the net transport flux should be VBC/(S × t).  
+
+4. The explanation of green triangle in fig 3e is "Transport flux of soluble sulfur based on current pulse (kinetics process)". Actually, the transport flux of soluble sulfur based on current pulse is (VBC-VDE) / (S × t), because the electrochemical process is accompanied by disproportionation process. Or the green triangle should be described as the net transport flux of soluble sulfur.
+
+<--- Page Split --->
+
+5. Adding a fig comparing cycling performance under three cycle mode in fig 4e would clearly show how the five stage affect battery performance and enhance persuasion of this part.  
+
+6. Please explain how you control internal temperature as well as strain stable during charging and discharging processes to eliminate their influence on the wavelength shift.  
+
+7. Could you please specify the definition of the ratio which indicates the consumption rate of sulfur under the galvanostatic condition on Page 7? Furthermore, the sulfur concentration variation rate corresponding to each plateau would better be given in Fig. S4a.  
+
+8. In "TFBG fabrication and sensing system", please add the specific structure of the TFBG sensor employed in the measurements should be introduced, including the core/cladding diameter, the coating material, the period of grating and so on.  
+
+9. In supplementary video, the amplitude and the wavelength have same trends. What is the difference between them? Can we draw the same conclusion from amplitude instead of wavelength?  
+
+10. How to achieve optical fiber inserted into the battery without leaking electrolyte and how to make sure there is no side effect or carryover effect due to inserted fibre?  
+
+11. How reproducible is the experiment?
+
+<--- Page Split --->
+
+## Reviewer #1:  
+
+In this work, TFBGs were employed to operando track the chemical dynamics/states of the Li- S battery via electrolyte sulfur concentration, revealing the correlated relationship between the capacity fading and dynamic of dissolution/precipitation of polysulfides over cycling and at different cycling rates. Overall, I think is an interesting work, since the authors proposed a new application of optical fiber sensor for battery monitoring. Consequently, I will recommend its publication after a minor revision.  
+
+Author response: We highly appreciate the positive comments from the reviewer, and they are all considered in corrected manuscript.  
+
+Question 1: In Fig1c, why did the author choose the "\\*\\* region as the sensing mode? Can other modes also be used as sensing modes, and if so, what are the differences in sensing performance?  
+
+Author response: The optical fiber sensor resonance marked by "\\*\\*" was chosen as the preferred sensing mode due to the response sensitivity and particular optical polarization property.  
+
+Regarding the sensing sensitivity, the primary advantage of the chosen resonance is that, for the accessible spectrum, it shows largest refractive index sensitivity (and hence response to sulfur concentration) compared to other guided modes at longer wavelengths that could also be considered [R1]. It is also referred to as the "cut- off" mode, where the surrounding refractive index (i.e. concentration of polysulfide dissolved in electrolyte) becomes equal to its mode effective index, and thereby it is observed that the mode resonance shifts more rapidly as its evanescent field penetrates more into the outer medium (electrolyte). With the goal of collecting valuable details regarding polysulfide chemical dynamics and evolution in electrolyte, the cut- off mode marked by "\\*\\*" should be the best choice.  
+
+Turning to the optical polarization of the relevant guided mode, it is well known that there are two group resonances in transmission spectra: P- polarization (blue line in Fig. 1(a)) corresponding to guide mode electric field azimuthally polarized; S- polarization (red line in Fig. 1(a)) corresponding to guided mode electric field radially polarized [R2]. Given the fact that the cutoff mode (marked by "\\*\\*") will be polarization insensitive (See Fig 1b, 1c insets) and there is no wavelength shift except some amplitude variation, it inspires confidence that it is possible to decouple the sulfur concentration in electrolyte by tracking the wavelength shift of cutoff mode with un- polarized input light and without a polarizer. By doing so, it allows for a simplified sensing system which still ensures that detection is both stable and repeatable.
+
+<--- Page Split --->
+![](images/Figure_2.jpg)
+
+<center>Fig. 1 | Optical polarization property of TFBG: (a) Experimental polarized transmission spectra in electrolyte (red, S-pol input, and blue, P-pol input); (b) Radial (P-pol) and (c) azimuthal (S-pol) dependence of simulated E-field intensity of cut-off guided mode (the arrows show the E-field vector orientations in both cases). </center>  
+
+[R1] Chan, C.- F., Chen, C., Jafari, A., Laronche, A., Thomson, D. J. & Albert, J. Optical fiber refractometer using narrowband cladding-mode resonance shifts. Appl. Opt. 46, 1142- 1149 (2007). [R2] Alam, M.- Z., Albert, J. Selective excitation of radially and azimuthally polarized optical fiber cladding modes. J. Lightw. Technol. 31, 3167- 3175 (2013).  
+
+Question 2: As we all know, the S cathodes exhibit around \(80\%\) volume changes during cycling. Therefore, in this manuscript, please explain whether the wavelength shift caused by the volume changes of the S cathode will affect the results.  
+
+Author response: The reviewer has rightfully noted a particular challenge with the Li- S system, but this is something where our sensor design has a particular advantage. The volume changes of sulfur during cycling (both expansive and contractive) don't affect the wavelength shift of our sensor due to the specific cell design, mentioned in Fig. S3 in supplementary information. Basically, we are first placing a \(2 \text{mm}\) thick, \(12.8 \text{mm}\) diameter polyether ether ketone (PEEK) spacer ring into the swagelok assembly. This ring is pierced in the middle such that we can inject the fiber (which has a \(1 \text{cm}\) long TFBG sensor inscribed segment) into the cross- sectional center of the cell. Within the Swagelok, the PEEK ring separates the cathode (sulfur and Super P carbon composite (60/40 wt.%) and lithium anode so that fiber sensor is perfectly immersed inside the electrolyte but not touching, nor at risk of touching, either electrode regardless of the respective volume change.  
+
+During our experiments, we ensure temperature and strain remain effectively constant because 1) the slow cycling rate gives minimal heat generation from overpotential and did not result in any temperature fluctuation of the sensor; additionally noted is that the cells are placed in a well- regulated thermostatic oven and 2) with the TFBG being effectively isolated and solely in the liquid electrolyte, it should not be sensitive to strain related to the electrodes. This is the particular case here, as we are using a high sulfur ratio (E/S ratio, \(\sim 100 \mu \text{L} /\text{mg}\) ).
+
+<--- Page Split --->
+
+Question 3: In manuscript, the author filled 500 μL of electrolyte into the cell, which is different from the common application scenario of optical fiber (the fiber is placed in a huge amount of liquid). Therefore, the author should consider whether the electrolyte was sufficient to completely infiltrate the fiber and can evenly wrap the optical fiber, which will affect the result.  
+
+Author response: We fully agree with the reviewer's consideration that the fiber must be fully immersed in the electrolyte, which is the key point for ensuring that the sensor works properly. As mentioned above, a PEEK ring is used in the Swagelok assembly stack, and it is through the middle of this ring where the TFBG sensor is inserted. The volume inside this PEEK ring serves as a container, which is filled electrolyte. As the ring thickness of \(2 \text{mm}\) is much greater than the fiber diameter of \(0.125 \text{mm}\) , the PEEK ring provides a nice pool of liquid to constantly immerse the fiber during cell operation. To help illustrate the geometric considerations, Fig. 2 is shared below.  
+
+![](images/Figure_3.jpg)
+
+<center>Fig. 2 | The PEEK container. </center>  
+
+Question 4: Ultimately, optical fiber is a linear sensor with a limited detection range. In manuscript, the author also mentioned that the "delay" due to position of the sensor in the cell. So can the author show the evolution of sulfur concentration in other locations, such as near the electrode or at the same level as the current fiber?  
+
+Author response: The question regarding the position of sensors is well spotted. This is an important aspect that we have also explored because any significant ion transport latency would have important implications for understanding dynamic reactions, as is the case here.  
+
+Regarding our terminology, "delay" is used to refer to the time needed by the new polysulfide species generated at the positive electrode to lead an equilibrium state within the electrolyte. To quantify the extent of "delay", a further experiment was carried out by placing two TFBGs sensors at different positions within the volume of electrolyte. This allows us to see the sulfur concentration dynamics close to Li metal and sulfur electrode surfaces, respectively. As shown in Fig. 3, the sulfur concentration detected by TFBG1, closer to the surface of Li (red line), typically "falls behind" that of the concentration detected by TFBG2, near to the surface of sulfur (blue line). A second and notable change is the lower amplitude of the S concentration near the surface of Li, as compared to the blue curve associated with the polysulfide of the positive electrode. While certainly the reviewer is aware of the value hidden within the details of the two sensor signals, given their positions, further assessment is beyond the scope of the work reported herein. Nevertheless, the position dependent differences observed here are small and do not impact our overall conclusions nor understanding of mechanisms.
+
+<--- Page Split --->
+![](images/Figure_4.jpg)
+
+<center>Fig. 3 | Decoding sulfur concentration gradient of LSB by two sensors close to electrode. </center>  
+
+Question 5: In Fig. 4b, I observed a slight dissonance in 12th cycle where the drop in temperature caused the wavelength to decrease. However, the double effect of temperature dropping and Li2S nucleation will result in smaller wavelength. Why is the valley of sulfur concentration in 12th cycle greater than that in other cycles?  
+
+Author response: We thank the reviewer for this keen observation and would firstly like to apologize for the confusion stemming from this anomaly. The "valley" mentioned by the referee is not a "real" wavelength shift induced by a temperature change or generated polysulfides. It is the result of an error in plotting the data which we should have spotted ourselves. This anomaly comes from a recording failure of the integrator software that lasted for about 10 hours due to Windows update of computer system, and during this time period there was no data recorded. We have replotted Fig. 4b in the main text with "dot" instead of "line", which we hope makes sense in terms of the wavelength shift.  
+
+Regarding the second remark by the referee about the double effect of temperature, thermal effects can be totally removed by a thorough thermal calibration process that enlists several steps as follows:  
+
+Step 1: By testing the temperature response of the fiber sensor in air (Fig. 4a,b), the thermal sensitivity of the core mode is determined to be \(10.2 \mathrm{pm / ^{\circ}C}\) and the target cladding mode sensitivity (cutoff mode in electrolyte) is \(9.7 \mathrm{pm / ^{\circ}C}\) (the cladding mode thermal sensitivity is always smaller than that of core mode) [R3].  
+
+Step 2: By testing temperature response of the fiber sensor immersed in electrolyte (Fig. 4c,d), the total wavelength shift of the cutoff mode comprises two parts: temperature (9.7 pm/°C obtained from step 1) and the temperature- modulated refractive index of the electrolyte (- 9.5 pm/°C in Fig. 4d).  
+
+When the cell is cycled with the fiber sensor, the observed wavelength shift will be composed of temperature, temperature- induced refractive index, and polysulfide- induced refractive index of electrolyte. By manually compensating for the thermal effects on the basis of steps 1 and 2, the wavelength shift will be linked solely to the polysulfide generated.  
+
+For the consideration of the reviewer and future readers, the above discussions have been added in the revised supplementary information as Fig. 4S.
+
+<--- Page Split --->
+![](images/Figure_5.jpg)
+
+<center>Fig. 4 | Thermal calibration of electrolyte: (a) The thermal response in air and (b) The corresponding thermal sensitivity of core mode and target cladding mode (cutoff mode in electrolyte); (c) The thermal response in electrolyte and (d) sensitivity. </center>  
+
+[R3] Imas, J. J., Bai, X., Zamarreño, C. R., Matías, I. R. & Albert, J. Accurate compensation and prediction of the temperature cross- sensitivity of tilted FBG cladding mode resonance. Appl. Opt. 62, E8- E15 (2023).  
+
+Question 6: In Fig. 4b, an increase of the background of sulfur concentration was attributed to the strong shuttle effect with less sulfur utilization caused by the high E/S ratio. However, other components in the electrolyte also undergo irreversible chemical reactions during cycling (> 400 h). So how can you confirm that the increase of the background of sulfur concentration is solely due to the strong shuttle effect with less sulfur utilization?  
+
+Author response: The reviewer has made another interesting suggestion concerning the increase in background noise, in addition to the lower sulfur usage caused by the high E/S ratio. It is certainly true that other irreversible reactions processes such as the formation of a solid electrolyte interphase (SEI) by the consumption of the additive \(\mathrm{LiNO_3}\) , and the decomposition of the solvents DOL and DME [R4] could affect the sensing results by altering the interlinking refractive index of the electrolyte. However, this is exactly the reason why we have pursued a high E/S ratio so that the electrolyte consumption due to SEI formation is quite limited by using small amount of active material comparing to electrolyte (5- 6 mg sulfur and 500 μL electrolyte). To us, this is well- confirmed by the experiment (Fig. 5d in the main text) involving the cathode composite based on Ketjen black carbon (KB) or MOF- 801(Zr), which shows that the sulfur concentration background is fairly stable during cycling due to the higher efficiency of the nucleation pathway and crystallization of \(\mathrm{Li_2S}\) .
+
+<--- Page Split --->
+
+[R4] Xiong, S., Xie, K., Diao, Y & Hong, X. Characterization of solid electrolyte interphase on lithium anode for preventing the shuttle mechanism in lithium- sulfur batteries. J. Power Sources 246, 840- 845 (2014).  
+
+Question 7: KB was better than SP as physical nonpolar sulfur confinement host. Therefore, the sulfur concentration in KB/S should be smaller than SP/S. In manuscript, the sulfur concentration in KB/S was \(\sim 700 \text{mM}\) and the sulfur concentration in SP/S was \(\sim 500 \text{mM}\) . Please explain it.  
+
+Author response: We totally agree with the reviewer's argument that theoretically higher surface area carbon (KB) should lead to trapping more sulfur. There could be several explanations for these discrepancies:  
+
+To start with, both carbons are non- polar and the adsorption ability is very small compared to, for example, oxygenated porous architectures [R5]. Inspired by the reviewer's comment, we tested the adsorption ability of SP and KB carbons by mimicking the similar sulfur/carbon ratio we have used in our cells. Both carbons showed almost no adsorption ability, which was visually detected after resting for 22 hours (Fig. 5).  
+
+![](images/Figure_1.jpg)
+
+<center>Fig 5. The adsorption test of polysulfide by SP or KB carbon. </center>  
+
+In addition to that, as was pointed in Fig. S10 in supplementary information with the XRD patterns before and after heat treatment, sulfur only partially penetrated into the nanostructure of KB, contrary to our own expectation, as well as literature reports. Moreover, physical nonpolar sulfur confinement by Ketjen black (KB) is very limited in our experiment, supported by Fig. S11 in supplementary information there is no sulfur left tested by energy- dispersive X- ray spectroscopy (EDX) inside the KB after the first plateau of discharge. We must therefore infer that all sulfur is converted to polysulfide and dissolved in electrolyte where it is detected by the fiber sensor.  
+
+Another reason may rise from our experimental condition in which we deliberately used a much higher E/S ratio ( \(\sim 100\) ) than usual ( \(\leq 8\) ) so that the solubility of polysulfides would be feasible even for the highly porous hosts. To help with detection, a relatively small current density (C/20) gives time for dissolved polysulfide to equilibrate in the electrolyte, regardless of how much sulfur is trapped into the pores.  
+
+Finally, the slightly higher sulfur concentration of KB/S than SP/S could be due to the fact that the weight of the active material is slightly larger than that of the Super P/S composite disk, which
+
+<--- Page Split --->
+
+can be attributed to the manual process of mixing the electrode composite with PTFE, forming it into a film and punching it into disks. As a result, there will be some deviation in the weight of the active material, but this is generally within a controllable range, and doesn't materially impact any of the results presented.  
+
+[R5]. Demir- Cakan, R., Morcrette, M., Nouar, F., Davoisne, C., Devic, T., Gonbeau, D., Dominko, R., Serre, C., Ferey, G & Tarascon, J.- M. Cathode composites for Li- S batteries via the use of oxygenated porous architectures. J. Am. Chem. Soc. 133, 40, 16154- 16160 (2011).  
+
+Question 8: Typos or hard to understand. The authors need to rephrase the following parts. - In abstract, "...the nucleation pathway and crystallization of Li2S and sulfur governs the cycling performance..." should be "...the nucleation pathway and crystallization of Li2S and sulfur govern the cycling performance..." - On page 1, "...and shuttle effect caused by soluble polysulfide in electrolyte..." should be "...and the shuttle effect caused by soluble polysulfide in electrolyte..." - On page 11, "...On the upper voltage plateau the solid sulfur dissolution or recrystallization..." should be "...On the upper voltage plateau, the solid sulfur dissolution or recrystallization..." - On page 15, "...changing from progressive to an instantaneous pathway..." should be "...changing from a progressive to an instantaneous pathway..."  
+
+Author response: We really appreciate the time spent by the referee in identifying and editing inconsistencies found in our submitted manuscript. We have accepted all suggestions and reworded the passages in the text accordingly.
+
+<--- Page Split --->
+
+## Reviewer #2:  
+
+The paper entitled "Detangling electrolyte chemical dynamics and evolution in Li- S batteries by operando monitoring with optical resonance combs" reports about TFBG technique for studying electrolyte chemical dynamics and evolution in Li- S batteries by tracking sulfur concentration and demonstrate that the nucleation pathway and crystallization of \(\mathsf{L i}_{2}\mathsf{S}\) and sulfur governs the cycling performance. Although the similar technique has already been used in their previous work to track electrolyte concentration through refractive index (DOI: 10.1039/d1ee02186a), this work brings a new insight on understanding the mechanism and electrolyte chemical dynamics of LSB. Thus, this paper could be considered for publication after a minor revision, detailed in the following comments:  
+
+Author response: We highly appreciate the positive comments from the reviewer, and they are all considered in corrected manuscript.  
+
+Question 1: Why the operando XRD in fig 2a doesn't show the recrystallization process of sulfur at the end of charging?  
+
+Author response: The reviewer has indeed made a careful observation. We believe it is a question of quantity and state of sulfur (amorphous vs crystallize). From our TFBG decoupling experiment we can deduce that only \(\sim 10\%\) solid sulfur will be reformed without providing clues on its state (crystallize or amorphous). Ten percent of crystallized sulfur should be easily detected by XRDs. To check this point that the re-formed sulfur is amorphous, a fully charged sulfur- loaded carbon electrode was recovered by washing and drying to remove any soluble polysulfide as well as remaining electrolyte salts and investigated by SEM. Fig. 1 compares two SEM taken shots of the pristine and fully charge samples suggesting the presence of amorphous sulfur, hence confirming the XRDs. We may also note that sulfur is readily amorphized in the presence of organic compounds under mild conditions, which has been well- established in various sulfur industries going back 70 years, at least [R1]  
+
+[R1] Bartlett, P.- D., Meguerian, G. Reactions of elemental sulfur. I. The uncatalyzed reaction of sulfur with triarylphosphines. J. Am. Chem. Soc. 78, 15, 3710- 3715 (1956).  
+
+![](images/Figure_2.jpg)
+
+<center>Fig. 1 | The morphology (SEM) of cathode (S: Super P=6: 4 wt%) at the beginning of discharging and end of charging </center>
+
+<--- Page Split --->
+
+Question 2: There is a sudden drop of sulfur concentration from A to B in the beginning of discharge in fig 3c, which also happens at the beginning of next discharge period. What leads to this? The sulfur concentration from A to B in fig 3c reacts different from in fig 3a and b. It doesn't seem to be attributed to "delay".  
+
+Author response: Regarding the reason leading to the "tiny" sudden drop of sulfur concentration from A to B in Fig.3c during charging (transition from rest mode to cycle mode), this can be attributed to the instantaneous response of electrolyte to a current pulse, arising from the redistribution of polysulfide (concentration gradient) because of sudden electric field. In this way, it is not exactly a "delay", as noted by the referee. While it is quite different for the case in Fig. 3a and b that concentration variation from A to B is opposite to that in Fig. 3c, this should be attributed to the change of the concentration gradient direction during discharge. We also note that the amplitude of concentration variation from A to B is smaller than that in Fig. 3c, resulting from the fact that the fiber sensor is physically/geometrically closer to cathode during assembly process, and therefore it leads to an asymmetric concentration variation.  
+
+Question 3: The soluble sulfur transport flux calculation should be based on equilibrium state, as the result is used for representing the whole process. So I think it is more reasonable to exclude region from A to B and C to D during transport flux calculation. Then the net transport flux should be \(VBC / (S \times t)\) .  
+
+Author response: We sincerely appreciate that the referee makes the definition of net transport flux clearer \((V_{BC} / (S \times t))\) and we fully agree with the change. It has been corrected in main text of Fig. 3.  
+
+Question 4: The explanation of green triangle in fig 3e is "Transport flux of soluble sulfur based on current pulse (kinetics process)". Actually, the transport flux of soluble sulfur based on current pulse is (VBC- VDE)/(S x t), because the electrochemical process is accompanied by disproportionation process. Or the green triangle should be described as the net transport flux of soluble sulfur.  
+
+Author response: The referee is perfectly right to assert that the net transport flux of soluble sulfur is based on electrochemical process accompanied by disproportionation process in Fig. 3e. It has been corrected in main text.  
+
+Question 5: Adding a fig comparing cycling performance under three cycle mode in fig 4e would clearly show how the five stages affect battery performance and enhance persuasion of this part.  
+
+Author response: The referee's suggestion is a very good one and accordingly a figure has been prepared regarding cycling performance. It can now be found added to Fig. 4 in main text.  
+
+Question 6: Please explain how you control internal temperature as well as strain stable during charging and discharging processes to eliminate their influence on the wavelength shift.  
+
+Author response: Interestingly, the same question was asked by referee 1 (questions 3 and 5) and we have provided effectively the same answers. To eliminate any deformation induced by the large volume changes (around \(80\%\) ) of the active material, the cell is specifically designed as mentioned in Fig. S3 in supplementary information, as well as Fig 2 shared again below. Basically, we are first placing a \(2 \text{mm}\) thick, \(12.8 \text{mm}\) diameter polyether ether ketone (PEEK)
+
+<--- Page Split --->
+
+spacer ring into the swagelok assembly. This ring is pierced in the middle such that we can inject the fiber (which has a \(1 \text{cm}\) long TFBG sensor inscribed segment) into the cross- sectional center of the cell. Within the Swagelok, the PEEK ring separates the cathode (sulfur and Super P carbon composite (60/40 wt.%) and lithium anode so that fiber sensor is perfectly immersed inside the electrolyte but not touching, nor at risk of touching, either electrode regardless of the respective volume change.  
+
+![](images/Figure_3.jpg)
+
+<center>Fig. 2 | The PEEK container. </center>  
+
+During our experiments, we ensure temperature and strain remain effectively constant because 1) the slow cycling rate gives minimal heat generation from overpotential and did not result in any temperature fluctuation of the sensor; additionally noted is that the cells are place in a well- regulated thermostatic oven and 2) with the TFBG being effectively isolated and solely in the liquid electrolyte, it should not be sensitive to strain related to the electrodes. This is the particular case here, as we are using a high sulfur ratio (E/S ratio, \(\sim 100 \mu \text{L} /\text{mg}\) ).  
+
+Any thermal effect during cycling can be totally removed by a thorough thermal calibration process that enlists several steps as follows:  
+
+Step 1: By testing the temperature response of the fiber sensor in air (Fig. 3a,b), the thermal sensitivity of the core mode is determined to be \(10.2 \text{pm} /\text{oC}\) and the target cladding mode sensitivity (cutoff mode in electrolyte) is \(9.7 \text{pm} /\text{oC}\) (the cladding mode thermal sensitivity is always smaller than that of core mode) [R2].  
+
+Step 2: By testing temperature response of the fiber sensor immersed in electrolyte (Fig. 3c,d), the total wavelength shift of the cutoff mode comprises two parts: temperature (9.7 pm/°C obtained from step 1) and the temperature- modulated refractive index of the electrolyte (- 9.5 pm/°C in Fig. 3d).  
+
+When the cell is cycled with the fiber sensor, the observed wavelength shift will be composed of temperature, temperature- induced refractive index, and polysulfide- induced refractive index of electrolyte. By manually compensating for the thermal effects on the basis of steps 1 and 2, the wavelength shift will be linked solely to the polysulfide generated.  
+
+For the consideration of the reviewer and future readers, the above discussions have been added in the revised supplementary information as Fig. 4S.
+
+<--- Page Split --->
+![](images/Figure_4.jpg)
+
+<center>Fig. 3 | Thermal calibration of electrolyte: (a) The thermal response in air and (b) the corresponding thermal sensitivity of core mode and target cladding mode (cutoff mode in electrolyte); (c) The thermal response in electrolyte and (d) sensitivity. </center>  
+
+[R2] Imas, J. J., Bai, X., Zamarreño, C. R., Matías, I. R. & Albert, J. Accurate compensation and prediction of the temperature cross- sensitivity of tilted FBG cladding mode resonance. Appl. Opt. 62, E8- E15 (2023).  
+
+Question 7: Could you please specify the definition of the ratio, which indicates the consumption rate of sulfur under the galvanostatic condition on Page 7? Furthermore, the sulfur concentration variation rate corresponding to each plateau would better be given in Fig. S4a.  
+
+Author response: We thank the referee for noting that this was not clear in our initial submission. The ratio indicating the consumption rate of sulfur under the galvanostatic condition is defined by: \(Ratio = \left| \frac{\text{first plateau concentration slope}}{\text{second plateau concentration slope}} \right|\) . Therefore, the \(Ratio_{discharge} = \left| \frac{S_1}{S_2} \right| = 3.88\) , \(Ratio_{charge} = \left| \frac{S_4}{S_3} \right| = 0.84\) . A figure regarding to sulfur concentration variation rate has been added to Fig. S5 in the supplementary information, which should help visually clarify the origin of these ratios.
+
+<--- Page Split --->
+![](images/Figure_5.jpg)
+
+<center>Fig. 4 | The definition of consumption rate of sulfur under the galvanostatic condition. </center>  
+
+Question 8: In "TFBG fabrication and sensing system", please add the specific structure of the TFBG sensor employed in the measurements should be introduced, including the core/cladding diameter, the coating material, the period of grating and so on.  
+
+Author response: We have added more details about the specific structure of TFBG to "TFBG fabrication and sensing system" in main text in accordance with the referee's request.  
+
+Question 9: In supplementary video, the amplitude and the wavelength have same trends. What is the difference between them? Can we draw the same conclusion from amplitude instead of wavelength?  
+
+Author response: Indeed, the amplitude and wavelength of TFBG cutoff resonance are strongly correlated by refractive index (sulfur concentration) variation. The cutoff resonance decreases sharply in amplitude together with the wavelength shift (Fig. 5), indicating loss of total internal reflection at the point where the cladding mode effective index becomes equal to the surrounding refractive index of polysulfide solution. Amplitude and wavelength will follow the same trend when the surrounding solution is a "pure" liquid, except in the special case of high turbidity, which leads to the irreversible disappearance of all cladding mode resonances [R3]. Thus, the observation of similar amplitude and wavelength trends in our video means that the effects of turbidity in our system, if any, are negligible.  
+
+![PLACEHOLDER_15_1]
+
+<center>Fig. 5 | The TFBG response to 100 mM \(\mathrm{Li}_2\mathrm{S}_2\) , \(\mathrm{Li}_2\mathrm{S}_5\) and \(\mathrm{Li}_2\mathrm{S}_8\) in electrolyte of 1 M LiTFSI, 0.5 M \(\mathrm{LiNO}_3\) in DOL/DME (1:1, v/v) </center>
+
+<--- Page Split --->
+
+[R3] Huang, J., Han, X., Liu, F., Gervillie, C., Blanquer, L. A., Guo, T. & Tarascon, J.- M. Monitoring battery electrolyte chemistry via in- operando tilted fiber Bragg grating sensors. Energy Environ. Sci. 14, 6464- 6475 (2021).  
+
+Question 10: How to achieve optical fiber inserted into the battery without leaking electrolyte and how to make sure there is no side effect or carryover effect due to inserted fibre?  
+
+Author response: As shown in Fig. 2 we have used a Swagelok cell design for our experiments, which is nearly a worldwide standard in battery research labs because of its relative ease of assembly and air/moisture tightness. Swagelok ferrules typically rely on plastic deformation to ensure excellent sealing properties. The cell is then diametrically drilled through the PEEK spacer to accommodate the fiber that supports the TFBG and is hermetically sealed with epoxy at the fiber entry and exit positions in the Swagelok assembly. With this method we have never experienced electrolyte leakage.  
+
+As far as secondary or carry- over effects due to the inserted fiber are concerned, they are minimal here because the fiber is immersed in an electrolyte bath (PEEK ring) and is largely separated from the positive and negative electrode. Of course, the story would have been very different if we had placed the fiber in the sulfur electrode, due to the limitations of ion transport and induced current inhomogeneity!  
+
+## Question 11: How reproducible is the experiment?  
+
+Author response: These experiments enlist several key steps out of which three, namely i) the preparation of consistent C- S electrodes ii) the proper positioning of the fiber and iii) the feasibility of having similar TFBG sensors, were found to be the most critical for overall reproducibility. Nevertheless, we could well- master the first two steps in- house and the third one by working with our TFBG's producer, such that highly reproducible and dependable data could be obtained. Overall, both sensors and cells needed to be developed to a point that we can ensure our data is repeatable and reproducible (with respect to sulfur concentration evolution during cell operation). As per the question of the referee we should have addressed this point by adding the sentence "all the data has been at least be duplicated 2 or 3 times prior to being reported", which has now been included in the main text.
+
+<--- Page Split --->
+
+REVIEWERS' COMMENTS  
+
+Reviewer #1 (Remarks to the Author):  
+
+The authors have well addressed the previous concerns. I think this paper can be accepted.  
+
+Reviewer #2 (Remarks to the Author):  
+
+The manuscript can be accepted.
+
+<--- Page Split --->

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+Detangling electrolyte chemical dynamics in lithium sulfur batteries by operando monitoring with optical resonance combs  
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+<|ref|>image<|/ref|><|det|>[[56, 732, 240, 780]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[250, 732, 912, 784]]<|/det|>
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to  
+
+<|ref|>text<|/ref|><|det|>[[56, 785, 934, 924]]<|/det|>
+the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 145, 405, 161]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 204, 875, 313]]<|/det|>
+In this work, TFBG were employed to operando track the chemical dynamics/states of the Li- S battery via electrolyte sulfur concentration, revealing the correlated relationship between the capacity fading and dynamic of dissolution/precipitation of polysulfides over cycling and at different cycling rates. Overall, I think is an interesting work, since the authors proposed a new application of optical fiber sensor for battery monitoring. Consequently, I will recommend its publication after a minor revision.  
+
+<|ref|>text<|/ref|><|det|>[[118, 327, 877, 363]]<|/det|>
+(1) In Fig1c, why did the author choose the \(^{**}n\) region as the sensing mode? Can other modes also be used as sensing modes, and if so, what are the differences in sensing performance?  
+
+<|ref|>text<|/ref|><|det|>[[118, 377, 880, 430]]<|/det|>
+(2) As we all know, the S cathodes exhibit around \(80\%\) volume changes during cycling. Therefore, in this manuscript, please explain whether the wavelength shift caused by the volume changes of the S cathode will affect the results.  
+
+<|ref|>text<|/ref|><|det|>[[118, 444, 875, 517]]<|/det|>
+(3) In manuscript, the author filled \(500\mu L\) of electrolyte into the cell, which is different from the common application scenario of optical fiber (the fiber is placed in a huge amount of liquid). Therefore, the author should consider whether the electrolyte was sufficient to completely infiltrate the fiber and can evenly wrap the optical fiber, which will affect the result.  
+
+<|ref|>text<|/ref|><|det|>[[118, 531, 860, 602]]<|/det|>
+(4) Ultimately, optical fiber is a linear sensor with a limited detection range. In manuscript, the author also mentioned that the "delay" due to position of the sensor in the cell. So can the author show the evolution of sulfur concentration in other locations, such as near the electrode or at the same level as the current fiber?  
+
+<|ref|>text<|/ref|><|det|>[[118, 617, 864, 689]]<|/det|>
+(5) In Fig. 4b, I observed a slight dissonance in 12th cycle where the drop in temperature caused the wavelength to decrease. However, the double effect of temperature dropping and Li2S nucleation will result in smaller wavelength. Why is the valley of sulfur concentration in 12th cycle greater than that in other cycles?  
+
+<|ref|>text<|/ref|><|det|>[[118, 704, 875, 793]]<|/det|>
+(6) In Fig. 4b, an increase of the background of sulfur concentration was attributed to the strong shuttle effect with less sulfur utilization caused by the high E/S ratio. However, other components in the electrolyte also undergo irreversible chemical reactions during cycling (> 400 h). So how can you confirm that the increase of the background of sulfur concentration is solely due to the strong shuttle effect with less sulfur utilization?  
+
+<|ref|>text<|/ref|><|det|>[[118, 808, 860, 861]]<|/det|>
+(7) KB was better than SP as physical nonpolar sulfur confinement host. Therefore, the sulfur concentration in KB/S should be smaller than SP/S. In manuscript, the sulfur concentration in KB/S was \(\sim 700 \text{mM}\) and the sulfur concentration in SP/S was \(\sim 500 \text{mM}\) . Please explain it.  
+
+<|ref|>text<|/ref|><|det|>[[125, 876, 761, 893]]<|/det|>
+(8) Typos or hard to understand. The authors need to rephrase the following parts.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 84, 879, 138]]<|/det|>
+-In abstract, "...the nucleation pathway and crystallization of Li2S and sulfur governs the cycling performance..." should be "...the nucleation pathway and crystallization of Li2S and sulfur govern the cycling performance..."  
+
+<|ref|>text<|/ref|><|det|>[[118, 150, 872, 186]]<|/det|>
+-On page 1, "...and shuttle effect caused by soluble polysulfide in electrolyte..." should be "...and the shuttle effect caused by soluble polysulfide in electrolyte..."  
+
+<|ref|>text<|/ref|><|det|>[[118, 199, 830, 234]]<|/det|>
+-On page 11, "...On the upper voltage plateau the solid sulfur dissolution or recrystallization..." should be "...On the upper voltage plateau, the solid sulfur dissolution or recrystallization..."  
+
+<|ref|>text<|/ref|><|det|>[[118, 248, 860, 283]]<|/det|>
+-On page 15, "...changing from progressive to an instantaneous pathway..." should be "...changing from a progressive to an instantaneous pathway ..."  
+
+<|ref|>text<|/ref|><|det|>[[119, 357, 404, 373]]<|/det|>
+Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[117, 416, 864, 581]]<|/det|>
+The paper entitled "Detangling electrolyte chemical dynamics and evolution in Li- S batteries by operando monitoring with optical resonance combs" reports about TFBG technique for studying electrolyte chemical dynamics and evolution in Li- S batteries by tracking sulfur concentration and demonstrate that the nucleation pathway and crystallization of Li2S and sulfur governs the cycling performance. Although the similar technique has already been used in their previous work to track electrolyte concentration through refractive index (DOI: 10.1039/d1ee02186a), this work brings a new insight on understanding the mechanism and electrolyte chemical dynamics of LSB. Thus, this paper could be considered for publication after a minor revision, detailed in the following comments:  
+
+<|ref|>text<|/ref|><|det|>[[118, 624, 863, 659]]<|/det|>
+1. Why the operando XRD in fig 2a doesn't show the recrystallization process of sulfur at the end of charging?  
+
+<|ref|>text<|/ref|><|det|>[[118, 672, 863, 744]]<|/det|>
+2. There is a sudden drop of sulfur concentration from A to B in the beginning of discharge in fig 3c, which also happens at the beginning of next discharge period. What leds to this? The sulfur concentration from A to B in fig 3c reacts different from in fig 3a and b. It doesn't seem to be attributed to "delay".  
+
+<|ref|>text<|/ref|><|det|>[[118, 758, 875, 811]]<|/det|>
+3. The soluble sulfur transport flux calculation should be based on equilibrium state, as the result is used for representing the whole process. So I think it is more reasonable to exclude region from A to B and C to D during transport flux calculation. Then the net transport flux should be VBC/(S × t).  
+
+<|ref|>text<|/ref|><|det|>[[118, 824, 878, 896]]<|/det|>
+4. The explanation of green triangle in fig 3e is "Transport flux of soluble sulfur based on current pulse (kinetics process)". Actually, the transport flux of soluble sulfur based on current pulse is (VBC-VDE) / (S × t), because the electrochemical process is accompanied by disproportionation process. Or the green triangle should be described as the net transport flux of soluble sulfur.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 84, 864, 119]]<|/det|>
+5. Adding a fig comparing cycling performance under three cycle mode in fig 4e would clearly show how the five stage affect battery performance and enhance persuasion of this part.  
+
+<|ref|>text<|/ref|><|det|>[[115, 133, 860, 168]]<|/det|>
+6. Please explain how you control internal temperature as well as strain stable during charging and discharging processes to eliminate their influence on the wavelength shift.  
+
+<|ref|>text<|/ref|><|det|>[[118, 181, 867, 235]]<|/det|>
+7. Could you please specify the definition of the ratio which indicates the consumption rate of sulfur under the galvanostatic condition on Page 7? Furthermore, the sulfur concentration variation rate corresponding to each plateau would better be given in Fig. S4a.  
+
+<|ref|>text<|/ref|><|det|>[[118, 248, 848, 301]]<|/det|>
+8. In "TFBG fabrication and sensing system", please add the specific structure of the TFBG sensor employed in the measurements should be introduced, including the core/cladding diameter, the coating material, the period of grating and so on.  
+
+<|ref|>text<|/ref|><|det|>[[118, 315, 816, 368]]<|/det|>
+9. In supplementary video, the amplitude and the wavelength have same trends. What is the difference between them? Can we draw the same conclusion from amplitude instead of wavelength?  
+
+<|ref|>text<|/ref|><|det|>[[118, 382, 840, 416]]<|/det|>
+10. How to achieve optical fiber inserted into the battery without leaking electrolyte and how to make sure there is no side effect or carryover effect due to inserted fibre?  
+
+<|ref|>text<|/ref|><|det|>[[120, 432, 430, 448]]<|/det|>
+11. How reproducible is the experiment?
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[149, 93, 267, 111]]<|/det|>
+## Reviewer #1:  
+
+<|ref|>text<|/ref|><|det|>[[148, 131, 851, 241]]<|/det|>
+In this work, TFBGs were employed to operando track the chemical dynamics/states of the Li- S battery via electrolyte sulfur concentration, revealing the correlated relationship between the capacity fading and dynamic of dissolution/precipitation of polysulfides over cycling and at different cycling rates. Overall, I think is an interesting work, since the authors proposed a new application of optical fiber sensor for battery monitoring. Consequently, I will recommend its publication after a minor revision.  
+
+<|ref|>text<|/ref|><|det|>[[149, 251, 850, 287]]<|/det|>
+Author response: We highly appreciate the positive comments from the reviewer, and they are all considered in corrected manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[149, 297, 857, 334]]<|/det|>
+Question 1: In Fig1c, why did the author choose the "\\*\\* region as the sensing mode? Can other modes also be used as sensing modes, and if so, what are the differences in sensing performance?  
+
+<|ref|>text<|/ref|><|det|>[[149, 344, 850, 380]]<|/det|>
+Author response: The optical fiber sensor resonance marked by "\\*\\*" was chosen as the preferred sensing mode due to the response sensitivity and particular optical polarization property.  
+
+<|ref|>text<|/ref|><|det|>[[148, 381, 851, 546]]<|/det|>
+Regarding the sensing sensitivity, the primary advantage of the chosen resonance is that, for the accessible spectrum, it shows largest refractive index sensitivity (and hence response to sulfur concentration) compared to other guided modes at longer wavelengths that could also be considered [R1]. It is also referred to as the "cut- off" mode, where the surrounding refractive index (i.e. concentration of polysulfide dissolved in electrolyte) becomes equal to its mode effective index, and thereby it is observed that the mode resonance shifts more rapidly as its evanescent field penetrates more into the outer medium (electrolyte). With the goal of collecting valuable details regarding polysulfide chemical dynamics and evolution in electrolyte, the cut- off mode marked by "\\*\\*" should be the best choice.  
+
+<|ref|>text<|/ref|><|det|>[[148, 548, 851, 712]]<|/det|>
+Turning to the optical polarization of the relevant guided mode, it is well known that there are two group resonances in transmission spectra: P- polarization (blue line in Fig. 1(a)) corresponding to guide mode electric field azimuthally polarized; S- polarization (red line in Fig. 1(a)) corresponding to guided mode electric field radially polarized [R2]. Given the fact that the cutoff mode (marked by "\\*\\*") will be polarization insensitive (See Fig 1b, 1c insets) and there is no wavelength shift except some amplitude variation, it inspires confidence that it is possible to decouple the sulfur concentration in electrolyte by tracking the wavelength shift of cutoff mode with un- polarized input light and without a polarizer. By doing so, it allows for a simplified sensing system which still ensures that detection is both stable and repeatable.
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[200, 88, 800, 359]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[152, 372, 844, 426]]<|/det|>
+<center>Fig. 1 | Optical polarization property of TFBG: (a) Experimental polarized transmission spectra in electrolyte (red, S-pol input, and blue, P-pol input); (b) Radial (P-pol) and (c) azimuthal (S-pol) dependence of simulated E-field intensity of cut-off guided mode (the arrows show the E-field vector orientations in both cases). </center>  
+
+<|ref|>text<|/ref|><|det|>[[147, 437, 850, 510]]<|/det|>
+[R1] Chan, C.- F., Chen, C., Jafari, A., Laronche, A., Thomson, D. J. & Albert, J. Optical fiber refractometer using narrowband cladding-mode resonance shifts. Appl. Opt. 46, 1142- 1149 (2007). [R2] Alam, M.- Z., Albert, J. Selective excitation of radially and azimuthally polarized optical fiber cladding modes. J. Lightw. Technol. 31, 3167- 3175 (2013).  
+
+<|ref|>text<|/ref|><|det|>[[149, 520, 850, 575]]<|/det|>
+Question 2: As we all know, the S cathodes exhibit around \(80\%\) volume changes during cycling. Therefore, in this manuscript, please explain whether the wavelength shift caused by the volume changes of the S cathode will affect the results.  
+
+<|ref|>text<|/ref|><|det|>[[148, 584, 851, 787]]<|/det|>
+Author response: The reviewer has rightfully noted a particular challenge with the Li- S system, but this is something where our sensor design has a particular advantage. The volume changes of sulfur during cycling (both expansive and contractive) don't affect the wavelength shift of our sensor due to the specific cell design, mentioned in Fig. S3 in supplementary information. Basically, we are first placing a \(2 \text{mm}\) thick, \(12.8 \text{mm}\) diameter polyether ether ketone (PEEK) spacer ring into the swagelok assembly. This ring is pierced in the middle such that we can inject the fiber (which has a \(1 \text{cm}\) long TFBG sensor inscribed segment) into the cross- sectional center of the cell. Within the Swagelok, the PEEK ring separates the cathode (sulfur and Super P carbon composite (60/40 wt.%) and lithium anode so that fiber sensor is perfectly immersed inside the electrolyte but not touching, nor at risk of touching, either electrode regardless of the respective volume change.  
+
+<|ref|>text<|/ref|><|det|>[[148, 788, 851, 900]]<|/det|>
+During our experiments, we ensure temperature and strain remain effectively constant because 1) the slow cycling rate gives minimal heat generation from overpotential and did not result in any temperature fluctuation of the sensor; additionally noted is that the cells are placed in a well- regulated thermostatic oven and 2) with the TFBG being effectively isolated and solely in the liquid electrolyte, it should not be sensitive to strain related to the electrodes. This is the particular case here, as we are using a high sulfur ratio (E/S ratio, \(\sim 100 \mu \text{L} /\text{mg}\) ).
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[148, 85, 850, 157]]<|/det|>
+Question 3: In manuscript, the author filled 500 μL of electrolyte into the cell, which is different from the common application scenario of optical fiber (the fiber is placed in a huge amount of liquid). Therefore, the author should consider whether the electrolyte was sufficient to completely infiltrate the fiber and can evenly wrap the optical fiber, which will affect the result.  
+
+<|ref|>text<|/ref|><|det|>[[147, 167, 850, 315]]<|/det|>
+Author response: We fully agree with the reviewer's consideration that the fiber must be fully immersed in the electrolyte, which is the key point for ensuring that the sensor works properly. As mentioned above, a PEEK ring is used in the Swagelok assembly stack, and it is through the middle of this ring where the TFBG sensor is inserted. The volume inside this PEEK ring serves as a container, which is filled electrolyte. As the ring thickness of \(2 \text{mm}\) is much greater than the fiber diameter of \(0.125 \text{mm}\) , the PEEK ring provides a nice pool of liquid to constantly immerse the fiber during cell operation. To help illustrate the geometric considerations, Fig. 2 is shared below.  
+
+<|ref|>image<|/ref|><|det|>[[162, 320, 835, 475]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[391, 492, 604, 510]]<|/det|>
+<center>Fig. 2 | The PEEK container. </center>  
+
+<|ref|>text<|/ref|><|det|>[[148, 519, 850, 592]]<|/det|>
+Question 4: Ultimately, optical fiber is a linear sensor with a limited detection range. In manuscript, the author also mentioned that the "delay" due to position of the sensor in the cell. So can the author show the evolution of sulfur concentration in other locations, such as near the electrode or at the same level as the current fiber?  
+
+<|ref|>text<|/ref|><|det|>[[148, 602, 850, 657]]<|/det|>
+Author response: The question regarding the position of sensors is well spotted. This is an important aspect that we have also explored because any significant ion transport latency would have important implications for understanding dynamic reactions, as is the case here.  
+
+<|ref|>text<|/ref|><|det|>[[147, 659, 850, 900]]<|/det|>
+Regarding our terminology, "delay" is used to refer to the time needed by the new polysulfide species generated at the positive electrode to lead an equilibrium state within the electrolyte. To quantify the extent of "delay", a further experiment was carried out by placing two TFBGs sensors at different positions within the volume of electrolyte. This allows us to see the sulfur concentration dynamics close to Li metal and sulfur electrode surfaces, respectively. As shown in Fig. 3, the sulfur concentration detected by TFBG1, closer to the surface of Li (red line), typically "falls behind" that of the concentration detected by TFBG2, near to the surface of sulfur (blue line). A second and notable change is the lower amplitude of the S concentration near the surface of Li, as compared to the blue curve associated with the polysulfide of the positive electrode. While certainly the reviewer is aware of the value hidden within the details of the two sensor signals, given their positions, further assessment is beyond the scope of the work reported herein. Nevertheless, the position dependent differences observed here are small and do not impact our overall conclusions nor understanding of mechanisms.
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[150, 88, 846, 270]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[168, 280, 825, 298]]<|/det|>
+<center>Fig. 3 | Decoding sulfur concentration gradient of LSB by two sensors close to electrode. </center>  
+
+<|ref|>text<|/ref|><|det|>[[149, 307, 850, 380]]<|/det|>
+Question 5: In Fig. 4b, I observed a slight dissonance in 12th cycle where the drop in temperature caused the wavelength to decrease. However, the double effect of temperature dropping and Li2S nucleation will result in smaller wavelength. Why is the valley of sulfur concentration in 12th cycle greater than that in other cycles?  
+
+<|ref|>text<|/ref|><|det|>[[148, 389, 851, 538]]<|/det|>
+Author response: We thank the reviewer for this keen observation and would firstly like to apologize for the confusion stemming from this anomaly. The "valley" mentioned by the referee is not a "real" wavelength shift induced by a temperature change or generated polysulfides. It is the result of an error in plotting the data which we should have spotted ourselves. This anomaly comes from a recording failure of the integrator software that lasted for about 10 hours due to Windows update of computer system, and during this time period there was no data recorded. We have replotted Fig. 4b in the main text with "dot" instead of "line", which we hope makes sense in terms of the wavelength shift.  
+
+<|ref|>text<|/ref|><|det|>[[149, 539, 850, 592]]<|/det|>
+Regarding the second remark by the referee about the double effect of temperature, thermal effects can be totally removed by a thorough thermal calibration process that enlists several steps as follows:  
+
+<|ref|>text<|/ref|><|det|>[[149, 594, 850, 667]]<|/det|>
+Step 1: By testing the temperature response of the fiber sensor in air (Fig. 4a,b), the thermal sensitivity of the core mode is determined to be \(10.2 \mathrm{pm / ^{\circ}C}\) and the target cladding mode sensitivity (cutoff mode in electrolyte) is \(9.7 \mathrm{pm / ^{\circ}C}\) (the cladding mode thermal sensitivity is always smaller than that of core mode) [R3].  
+
+<|ref|>text<|/ref|><|det|>[[149, 669, 850, 741]]<|/det|>
+Step 2: By testing temperature response of the fiber sensor immersed in electrolyte (Fig. 4c,d), the total wavelength shift of the cutoff mode comprises two parts: temperature (9.7 pm/°C obtained from step 1) and the temperature- modulated refractive index of the electrolyte (- 9.5 pm/°C in Fig. 4d).  
+
+<|ref|>text<|/ref|><|det|>[[149, 743, 850, 815]]<|/det|>
+When the cell is cycled with the fiber sensor, the observed wavelength shift will be composed of temperature, temperature- induced refractive index, and polysulfide- induced refractive index of electrolyte. By manually compensating for the thermal effects on the basis of steps 1 and 2, the wavelength shift will be linked solely to the polysulfide generated.  
+
+<|ref|>text<|/ref|><|det|>[[149, 817, 849, 852]]<|/det|>
+For the consideration of the reviewer and future readers, the above discussions have been added in the revised supplementary information as Fig. 4S.
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[147, 85, 850, 437]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[172, 446, 825, 502]]<|/det|>
+<center>Fig. 4 | Thermal calibration of electrolyte: (a) The thermal response in air and (b) The corresponding thermal sensitivity of core mode and target cladding mode (cutoff mode in electrolyte); (c) The thermal response in electrolyte and (d) sensitivity. </center>  
+
+<|ref|>text<|/ref|><|det|>[[148, 510, 850, 565]]<|/det|>
+[R3] Imas, J. J., Bai, X., Zamarreño, C. R., Matías, I. R. & Albert, J. Accurate compensation and prediction of the temperature cross- sensitivity of tilted FBG cladding mode resonance. Appl. Opt. 62, E8- E15 (2023).  
+
+<|ref|>text<|/ref|><|det|>[[148, 575, 851, 666]]<|/det|>
+Question 6: In Fig. 4b, an increase of the background of sulfur concentration was attributed to the strong shuttle effect with less sulfur utilization caused by the high E/S ratio. However, other components in the electrolyte also undergo irreversible chemical reactions during cycling (> 400 h). So how can you confirm that the increase of the background of sulfur concentration is solely due to the strong shuttle effect with less sulfur utilization?  
+
+<|ref|>text<|/ref|><|det|>[[147, 677, 851, 899]]<|/det|>
+Author response: The reviewer has made another interesting suggestion concerning the increase in background noise, in addition to the lower sulfur usage caused by the high E/S ratio. It is certainly true that other irreversible reactions processes such as the formation of a solid electrolyte interphase (SEI) by the consumption of the additive \(\mathrm{LiNO_3}\) , and the decomposition of the solvents DOL and DME [R4] could affect the sensing results by altering the interlinking refractive index of the electrolyte. However, this is exactly the reason why we have pursued a high E/S ratio so that the electrolyte consumption due to SEI formation is quite limited by using small amount of active material comparing to electrolyte (5- 6 mg sulfur and 500 μL electrolyte). To us, this is well- confirmed by the experiment (Fig. 5d in the main text) involving the cathode composite based on Ketjen black carbon (KB) or MOF- 801(Zr), which shows that the sulfur concentration background is fairly stable during cycling due to the higher efficiency of the nucleation pathway and crystallization of \(\mathrm{Li_2S}\) .
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[148, 84, 850, 139]]<|/det|>
+[R4] Xiong, S., Xie, K., Diao, Y & Hong, X. Characterization of solid electrolyte interphase on lithium anode for preventing the shuttle mechanism in lithium- sulfur batteries. J. Power Sources 246, 840- 845 (2014).  
+
+<|ref|>text<|/ref|><|det|>[[148, 150, 850, 204]]<|/det|>
+Question 7: KB was better than SP as physical nonpolar sulfur confinement host. Therefore, the sulfur concentration in KB/S should be smaller than SP/S. In manuscript, the sulfur concentration in KB/S was \(\sim 700 \text{mM}\) and the sulfur concentration in SP/S was \(\sim 500 \text{mM}\) . Please explain it.  
+
+<|ref|>text<|/ref|><|det|>[[148, 215, 850, 269]]<|/det|>
+Author response: We totally agree with the reviewer's argument that theoretically higher surface area carbon (KB) should lead to trapping more sulfur. There could be several explanations for these discrepancies:  
+
+<|ref|>text<|/ref|><|det|>[[148, 270, 850, 362]]<|/det|>
+To start with, both carbons are non- polar and the adsorption ability is very small compared to, for example, oxygenated porous architectures [R5]. Inspired by the reviewer's comment, we tested the adsorption ability of SP and KB carbons by mimicking the similar sulfur/carbon ratio we have used in our cells. Both carbons showed almost no adsorption ability, which was visually detected after resting for 22 hours (Fig. 5).  
+
+<|ref|>image<|/ref|><|det|>[[297, 374, 690, 572]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[275, 585, 720, 603]]<|/det|>
+<center>Fig 5. The adsorption test of polysulfide by SP or KB carbon. </center>  
+
+<|ref|>text<|/ref|><|det|>[[147, 612, 850, 760]]<|/det|>
+In addition to that, as was pointed in Fig. S10 in supplementary information with the XRD patterns before and after heat treatment, sulfur only partially penetrated into the nanostructure of KB, contrary to our own expectation, as well as literature reports. Moreover, physical nonpolar sulfur confinement by Ketjen black (KB) is very limited in our experiment, supported by Fig. S11 in supplementary information there is no sulfur left tested by energy- dispersive X- ray spectroscopy (EDX) inside the KB after the first plateau of discharge. We must therefore infer that all sulfur is converted to polysulfide and dissolved in electrolyte where it is detected by the fiber sensor.  
+
+<|ref|>text<|/ref|><|det|>[[148, 761, 850, 852]]<|/det|>
+Another reason may rise from our experimental condition in which we deliberately used a much higher E/S ratio ( \(\sim 100\) ) than usual ( \(\leq 8\) ) so that the solubility of polysulfides would be feasible even for the highly porous hosts. To help with detection, a relatively small current density (C/20) gives time for dissolved polysulfide to equilibrate in the electrolyte, regardless of how much sulfur is trapped into the pores.  
+
+<|ref|>text<|/ref|><|det|>[[148, 853, 850, 889]]<|/det|>
+Finally, the slightly higher sulfur concentration of KB/S than SP/S could be due to the fact that the weight of the active material is slightly larger than that of the Super P/S composite disk, which
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[148, 84, 851, 158]]<|/det|>
+can be attributed to the manual process of mixing the electrode composite with PTFE, forming it into a film and punching it into disks. As a result, there will be some deviation in the weight of the active material, but this is generally within a controllable range, and doesn't materially impact any of the results presented.  
+
+<|ref|>text<|/ref|><|det|>[[148, 167, 851, 223]]<|/det|>
+[R5]. Demir- Cakan, R., Morcrette, M., Nouar, F., Davoisne, C., Devic, T., Gonbeau, D., Dominko, R., Serre, C., Ferey, G & Tarascon, J.- M. Cathode composites for Li- S batteries via the use of oxygenated porous architectures. J. Am. Chem. Soc. 133, 40, 16154- 16160 (2011).  
+
+<|ref|>text<|/ref|><|det|>[[148, 233, 852, 419]]<|/det|>
+Question 8: Typos or hard to understand. The authors need to rephrase the following parts. - In abstract, "...the nucleation pathway and crystallization of Li2S and sulfur governs the cycling performance..." should be "...the nucleation pathway and crystallization of Li2S and sulfur govern the cycling performance..." - On page 1, "...and shuttle effect caused by soluble polysulfide in electrolyte..." should be "...and the shuttle effect caused by soluble polysulfide in electrolyte..." - On page 11, "...On the upper voltage plateau the solid sulfur dissolution or recrystallization..." should be "...On the upper voltage plateau, the solid sulfur dissolution or recrystallization..." - On page 15, "...changing from progressive to an instantaneous pathway..." should be "...changing from a progressive to an instantaneous pathway..."  
+
+<|ref|>text<|/ref|><|det|>[[149, 427, 850, 483]]<|/det|>
+Author response: We really appreciate the time spent by the referee in identifying and editing inconsistencies found in our submitted manuscript. We have accepted all suggestions and reworded the passages in the text accordingly.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[149, 85, 252, 101]]<|/det|>
+## Reviewer #2:  
+
+<|ref|>text<|/ref|><|det|>[[148, 111, 852, 277]]<|/det|>
+The paper entitled "Detangling electrolyte chemical dynamics and evolution in Li- S batteries by operando monitoring with optical resonance combs" reports about TFBG technique for studying electrolyte chemical dynamics and evolution in Li- S batteries by tracking sulfur concentration and demonstrate that the nucleation pathway and crystallization of \(\mathsf{L i}_{2}\mathsf{S}\) and sulfur governs the cycling performance. Although the similar technique has already been used in their previous work to track electrolyte concentration through refractive index (DOI: 10.1039/d1ee02186a), this work brings a new insight on understanding the mechanism and electrolyte chemical dynamics of LSB. Thus, this paper could be considered for publication after a minor revision, detailed in the following comments:  
+
+<|ref|>text<|/ref|><|det|>[[149, 288, 849, 325]]<|/det|>
+Author response: We highly appreciate the positive comments from the reviewer, and they are all considered in corrected manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[149, 335, 849, 371]]<|/det|>
+Question 1: Why the operando XRD in fig 2a doesn't show the recrystallization process of sulfur at the end of charging?  
+
+<|ref|>text<|/ref|><|det|>[[148, 380, 852, 586]]<|/det|>
+Author response: The reviewer has indeed made a careful observation. We believe it is a question of quantity and state of sulfur (amorphous vs crystallize). From our TFBG decoupling experiment we can deduce that only \(\sim 10\%\) solid sulfur will be reformed without providing clues on its state (crystallize or amorphous). Ten percent of crystallized sulfur should be easily detected by XRDs. To check this point that the re-formed sulfur is amorphous, a fully charged sulfur- loaded carbon electrode was recovered by washing and drying to remove any soluble polysulfide as well as remaining electrolyte salts and investigated by SEM. Fig. 1 compares two SEM taken shots of the pristine and fully charge samples suggesting the presence of amorphous sulfur, hence confirming the XRDs. We may also note that sulfur is readily amorphized in the presence of organic compounds under mild conditions, which has been well- established in various sulfur industries going back 70 years, at least [R1]  
+
+<|ref|>text<|/ref|><|det|>[[147, 594, 850, 631]]<|/det|>
+[R1] Bartlett, P.- D., Meguerian, G. Reactions of elemental sulfur. I. The uncatalyzed reaction of sulfur with triarylphosphines. J. Am. Chem. Soc. 78, 15, 3710- 3715 (1956).  
+
+<|ref|>image<|/ref|><|det|>[[201, 639, 800, 824]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[186, 834, 810, 871]]<|/det|>
+<center>Fig. 1 | The morphology (SEM) of cathode (S: Super P=6: 4 wt%) at the beginning of discharging and end of charging </center>
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[148, 85, 851, 157]]<|/det|>
+Question 2: There is a sudden drop of sulfur concentration from A to B in the beginning of discharge in fig 3c, which also happens at the beginning of next discharge period. What leads to this? The sulfur concentration from A to B in fig 3c reacts different from in fig 3a and b. It doesn't seem to be attributed to "delay".  
+
+<|ref|>text<|/ref|><|det|>[[147, 167, 851, 353]]<|/det|>
+Author response: Regarding the reason leading to the "tiny" sudden drop of sulfur concentration from A to B in Fig.3c during charging (transition from rest mode to cycle mode), this can be attributed to the instantaneous response of electrolyte to a current pulse, arising from the redistribution of polysulfide (concentration gradient) because of sudden electric field. In this way, it is not exactly a "delay", as noted by the referee. While it is quite different for the case in Fig. 3a and b that concentration variation from A to B is opposite to that in Fig. 3c, this should be attributed to the change of the concentration gradient direction during discharge. We also note that the amplitude of concentration variation from A to B is smaller than that in Fig. 3c, resulting from the fact that the fiber sensor is physically/geometrically closer to cathode during assembly process, and therefore it leads to an asymmetric concentration variation.  
+
+<|ref|>text<|/ref|><|det|>[[148, 363, 850, 435]]<|/det|>
+Question 3: The soluble sulfur transport flux calculation should be based on equilibrium state, as the result is used for representing the whole process. So I think it is more reasonable to exclude region from A to B and C to D during transport flux calculation. Then the net transport flux should be \(VBC / (S \times t)\) .  
+
+<|ref|>text<|/ref|><|det|>[[148, 445, 850, 500]]<|/det|>
+Author response: We sincerely appreciate that the referee makes the definition of net transport flux clearer \((V_{BC} / (S \times t))\) and we fully agree with the change. It has been corrected in main text of Fig. 3.  
+
+<|ref|>text<|/ref|><|det|>[[148, 510, 850, 601]]<|/det|>
+Question 4: The explanation of green triangle in fig 3e is "Transport flux of soluble sulfur based on current pulse (kinetics process)". Actually, the transport flux of soluble sulfur based on current pulse is (VBC- VDE)/(S x t), because the electrochemical process is accompanied by disproportionation process. Or the green triangle should be described as the net transport flux of soluble sulfur.  
+
+<|ref|>text<|/ref|><|det|>[[148, 612, 850, 666]]<|/det|>
+Author response: The referee is perfectly right to assert that the net transport flux of soluble sulfur is based on electrochemical process accompanied by disproportionation process in Fig. 3e. It has been corrected in main text.  
+
+<|ref|>text<|/ref|><|det|>[[148, 677, 850, 713]]<|/det|>
+Question 5: Adding a fig comparing cycling performance under three cycle mode in fig 4e would clearly show how the five stages affect battery performance and enhance persuasion of this part.  
+
+<|ref|>text<|/ref|><|det|>[[148, 723, 850, 760]]<|/det|>
+Author response: The referee's suggestion is a very good one and accordingly a figure has been prepared regarding cycling performance. It can now be found added to Fig. 4 in main text.  
+
+<|ref|>text<|/ref|><|det|>[[148, 770, 850, 805]]<|/det|>
+Question 6: Please explain how you control internal temperature as well as strain stable during charging and discharging processes to eliminate their influence on the wavelength shift.  
+
+<|ref|>text<|/ref|><|det|>[[148, 807, 850, 899]]<|/det|>
+Author response: Interestingly, the same question was asked by referee 1 (questions 3 and 5) and we have provided effectively the same answers. To eliminate any deformation induced by the large volume changes (around \(80\%\) ) of the active material, the cell is specifically designed as mentioned in Fig. S3 in supplementary information, as well as Fig 2 shared again below. Basically, we are first placing a \(2 \text{mm}\) thick, \(12.8 \text{mm}\) diameter polyether ether ketone (PEEK)
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[147, 84, 851, 195]]<|/det|>
+spacer ring into the swagelok assembly. This ring is pierced in the middle such that we can inject the fiber (which has a \(1 \text{cm}\) long TFBG sensor inscribed segment) into the cross- sectional center of the cell. Within the Swagelok, the PEEK ring separates the cathode (sulfur and Super P carbon composite (60/40 wt.%) and lithium anode so that fiber sensor is perfectly immersed inside the electrolyte but not touching, nor at risk of touching, either electrode regardless of the respective volume change.  
+
+<|ref|>image<|/ref|><|det|>[[160, 209, 825, 362]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[391, 381, 604, 399]]<|/det|>
+<center>Fig. 2 | The PEEK container. </center>  
+
+<|ref|>text<|/ref|><|det|>[[147, 408, 851, 520]]<|/det|>
+During our experiments, we ensure temperature and strain remain effectively constant because 1) the slow cycling rate gives minimal heat generation from overpotential and did not result in any temperature fluctuation of the sensor; additionally noted is that the cells are place in a well- regulated thermostatic oven and 2) with the TFBG being effectively isolated and solely in the liquid electrolyte, it should not be sensitive to strain related to the electrodes. This is the particular case here, as we are using a high sulfur ratio (E/S ratio, \(\sim 100 \mu \text{L} /\text{mg}\) ).  
+
+<|ref|>text<|/ref|><|det|>[[148, 520, 848, 556]]<|/det|>
+Any thermal effect during cycling can be totally removed by a thorough thermal calibration process that enlists several steps as follows:  
+
+<|ref|>text<|/ref|><|det|>[[148, 557, 850, 630]]<|/det|>
+Step 1: By testing the temperature response of the fiber sensor in air (Fig. 3a,b), the thermal sensitivity of the core mode is determined to be \(10.2 \text{pm} /\text{oC}\) and the target cladding mode sensitivity (cutoff mode in electrolyte) is \(9.7 \text{pm} /\text{oC}\) (the cladding mode thermal sensitivity is always smaller than that of core mode) [R2].  
+
+<|ref|>text<|/ref|><|det|>[[148, 632, 850, 704]]<|/det|>
+Step 2: By testing temperature response of the fiber sensor immersed in electrolyte (Fig. 3c,d), the total wavelength shift of the cutoff mode comprises two parts: temperature (9.7 pm/°C obtained from step 1) and the temperature- modulated refractive index of the electrolyte (- 9.5 pm/°C in Fig. 3d).  
+
+<|ref|>text<|/ref|><|det|>[[148, 706, 850, 778]]<|/det|>
+When the cell is cycled with the fiber sensor, the observed wavelength shift will be composed of temperature, temperature- induced refractive index, and polysulfide- induced refractive index of electrolyte. By manually compensating for the thermal effects on the basis of steps 1 and 2, the wavelength shift will be linked solely to the polysulfide generated.  
+
+<|ref|>text<|/ref|><|det|>[[148, 780, 848, 815]]<|/det|>
+For the consideration of the reviewer and future readers, the above discussions have been added in the revised supplementary information as Fig. 4S.
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[147, 85, 848, 435]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[172, 446, 826, 501]]<|/det|>
+<center>Fig. 3 | Thermal calibration of electrolyte: (a) The thermal response in air and (b) the corresponding thermal sensitivity of core mode and target cladding mode (cutoff mode in electrolyte); (c) The thermal response in electrolyte and (d) sensitivity. </center>  
+
+<|ref|>text<|/ref|><|det|>[[147, 510, 850, 565]]<|/det|>
+[R2] Imas, J. J., Bai, X., Zamarreño, C. R., Matías, I. R. & Albert, J. Accurate compensation and prediction of the temperature cross- sensitivity of tilted FBG cladding mode resonance. Appl. Opt. 62, E8- E15 (2023).  
+
+<|ref|>text<|/ref|><|det|>[[147, 576, 850, 630]]<|/det|>
+Question 7: Could you please specify the definition of the ratio, which indicates the consumption rate of sulfur under the galvanostatic condition on Page 7? Furthermore, the sulfur concentration variation rate corresponding to each plateau would better be given in Fig. S4a.  
+
+<|ref|>text<|/ref|><|det|>[[147, 640, 850, 789]]<|/det|>
+Author response: We thank the referee for noting that this was not clear in our initial submission. The ratio indicating the consumption rate of sulfur under the galvanostatic condition is defined by: \(Ratio = \left| \frac{\text{first plateau concentration slope}}{\text{second plateau concentration slope}} \right|\) . Therefore, the \(Ratio_{discharge} = \left| \frac{S_1}{S_2} \right| = 3.88\) , \(Ratio_{charge} = \left| \frac{S_4}{S_3} \right| = 0.84\) . A figure regarding to sulfur concentration variation rate has been added to Fig. S5 in the supplementary information, which should help visually clarify the origin of these ratios.
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[193, 85, 802, 266]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[185, 280, 810, 298]]<|/det|>
+<center>Fig. 4 | The definition of consumption rate of sulfur under the galvanostatic condition. </center>  
+
+<|ref|>text<|/ref|><|det|>[[149, 307, 850, 361]]<|/det|>
+Question 8: In "TFBG fabrication and sensing system", please add the specific structure of the TFBG sensor employed in the measurements should be introduced, including the core/cladding diameter, the coating material, the period of grating and so on.  
+
+<|ref|>text<|/ref|><|det|>[[149, 371, 850, 408]]<|/det|>
+Author response: We have added more details about the specific structure of TFBG to "TFBG fabrication and sensing system" in main text in accordance with the referee's request.  
+
+<|ref|>text<|/ref|><|det|>[[149, 417, 851, 472]]<|/det|>
+Question 9: In supplementary video, the amplitude and the wavelength have same trends. What is the difference between them? Can we draw the same conclusion from amplitude instead of wavelength?  
+
+<|ref|>text<|/ref|><|det|>[[148, 482, 851, 650]]<|/det|>
+Author response: Indeed, the amplitude and wavelength of TFBG cutoff resonance are strongly correlated by refractive index (sulfur concentration) variation. The cutoff resonance decreases sharply in amplitude together with the wavelength shift (Fig. 5), indicating loss of total internal reflection at the point where the cladding mode effective index becomes equal to the surrounding refractive index of polysulfide solution. Amplitude and wavelength will follow the same trend when the surrounding solution is a "pure" liquid, except in the special case of high turbidity, which leads to the irreversible disappearance of all cladding mode resonances [R3]. Thus, the observation of similar amplitude and wavelength trends in our video means that the effects of turbidity in our system, if any, are negligible.  
+
+<|ref|>image<|/ref|><|det|>[[245, 660, 750, 843]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[155, 853, 842, 890]]<|/det|>
+<center>Fig. 5 | The TFBG response to 100 mM \(\mathrm{Li}_2\mathrm{S}_2\) , \(\mathrm{Li}_2\mathrm{S}_5\) and \(\mathrm{Li}_2\mathrm{S}_8\) in electrolyte of 1 M LiTFSI, 0.5 M \(\mathrm{LiNO}_3\) in DOL/DME (1:1, v/v) </center>
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[149, 85, 850, 140]]<|/det|>
+[R3] Huang, J., Han, X., Liu, F., Gervillie, C., Blanquer, L. A., Guo, T. & Tarascon, J.- M. Monitoring battery electrolyte chemistry via in- operando tilted fiber Bragg grating sensors. Energy Environ. Sci. 14, 6464- 6475 (2021).  
+
+<|ref|>text<|/ref|><|det|>[[149, 150, 850, 186]]<|/det|>
+Question 10: How to achieve optical fiber inserted into the battery without leaking electrolyte and how to make sure there is no side effect or carryover effect due to inserted fibre?  
+
+<|ref|>text<|/ref|><|det|>[[148, 196, 851, 325]]<|/det|>
+Author response: As shown in Fig. 2 we have used a Swagelok cell design for our experiments, which is nearly a worldwide standard in battery research labs because of its relative ease of assembly and air/moisture tightness. Swagelok ferrules typically rely on plastic deformation to ensure excellent sealing properties. The cell is then diametrically drilled through the PEEK spacer to accommodate the fiber that supports the TFBG and is hermetically sealed with epoxy at the fiber entry and exit positions in the Swagelok assembly. With this method we have never experienced electrolyte leakage.  
+
+<|ref|>text<|/ref|><|det|>[[149, 326, 850, 417]]<|/det|>
+As far as secondary or carry- over effects due to the inserted fiber are concerned, they are minimal here because the fiber is immersed in an electrolyte bath (PEEK ring) and is largely separated from the positive and negative electrode. Of course, the story would have been very different if we had placed the fiber in the sulfur electrode, due to the limitations of ion transport and induced current inhomogeneity!  
+
+<|ref|>sub_title<|/ref|><|det|>[[150, 428, 512, 445]]<|/det|>
+## Question 11: How reproducible is the experiment?  
+
+<|ref|>text<|/ref|><|det|>[[148, 455, 851, 640]]<|/det|>
+Author response: These experiments enlist several key steps out of which three, namely i) the preparation of consistent C- S electrodes ii) the proper positioning of the fiber and iii) the feasibility of having similar TFBG sensors, were found to be the most critical for overall reproducibility. Nevertheless, we could well- master the first two steps in- house and the third one by working with our TFBG's producer, such that highly reproducible and dependable data could be obtained. Overall, both sensors and cells needed to be developed to a point that we can ensure our data is repeatable and reproducible (with respect to sulfur concentration evolution during cell operation). As per the question of the referee we should have addressed this point by adding the sentence "all the data has been at least be duplicated 2 or 3 times prior to being reported", which has now been included in the main text.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 85, 309, 100]]<|/det|>
+REVIEWERS' COMMENTS  
+
+<|ref|>text<|/ref|><|det|>[[118, 144, 405, 160]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[116, 204, 803, 220]]<|/det|>
+The authors have well addressed the previous concerns. I think this paper can be accepted.  
+
+<|ref|>text<|/ref|><|det|>[[118, 296, 405, 311]]<|/det|>
+Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 356, 368, 371]]<|/det|>
+The manuscript can be accepted.
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__018ea56f56282342b1a9e7b0cea3a8dc2890bbe74f3ff129238a91951bc7905d/images_list.json

@@ -0,0 +1,153 @@
+[
+  {
+    "type": "image",
+    "img_path": "images/Supplementary_Figure_6.jpg",
+    "caption": "Supplementary Figure 6. (a) Temperature dependence of Hall coefficient \\((R_{\\mathrm{H}})\\) of \\(\\mathrm{Pb_{0.98}Na_{0.02}Se - 2.05\\% AgInSe_2}\\) . (b) Effective mass as a function of temperature for \\(\\mathrm{Pb_{0.98}Na_{0.02}Se - 2.05\\% AgInSe_2}\\) and \\(\\mathrm{Pb_{1 - x}Na_xSe}\\) . (c) Temperature-dependent Hall mobility of \\(\\mathrm{Pb_{0.98}Na_{0.02}Se - 2.05\\% AgInSe_2}\\) , which displays a \\(\\mathrm{T}^{-3 / 2}\\) behavior when \\(\\mathrm{T}< 600 \\mathrm{K}\\) , demonstrating that acoustic-phonon scattering dominates. The deviation from \\(\\mathrm{T}^{-3 / 2}\\) relation at high temperature is due to the increase of effective mass.",
+    "footnote": [],
+    "bbox": [],
+    "page_idx": 0
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_1.jpg",
+    "caption": "Figure 1. (a) Comparison of thermal diffusivity \\((D)\\) between measurement and reference for standard sample (Inconel). (b) The uncertainty of \\(D\\) .",
+    "footnote": [],
+    "bbox": [
+      [
+        228,
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+      ]
+    ],
+    "page_idx": 6
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_2.jpg",
+    "caption": "Figure 2. Comparison of (a) electrical resistivity \\((\\rho)\\) and (c) Seebeck coefficient \\((S)\\) between measurement and standard sample (Constantan), respectively. The corresponding uncertainties are shown in (b) and (d).",
+    "footnote": [],
+    "bbox": [
+      [
+        227,
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+        413
+      ]
+    ],
+    "page_idx": 6
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_1.jpg",
+    "caption": "Fig. 1 Multiple valence bands enable high \\(ZT\\) values in p-type PbSe. a Schmatic diagram of multi-bands \\((\\mathrm{L}, \\Sigma , \\Lambda)\\) involvement in transport. The Brillouin zone shows that the degeneracies at the \\(\\mathrm{L}, \\Sigma\\) , and \\(\\Lambda\\) points are 4, 12, and 8, respectively. b The activated third band \\(\\Lambda\\) enables higher \\(ZT\\) values compared with the single-band and two-band PbSe-based materials.",
+    "footnote": [],
+    "bbox": [],
+    "page_idx": 7
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_6.jpg",
+    "caption": "Fig. 6 Thermal transport properties and dimensionless figure-of-merit \\(ZT\\) as a function of temperature for \\(\\mathrm{Pb_{0.98}Na_{0.02}Se - x\\% AgInSe_2}\\) (LISS) compounds. a Total thermal conductivity. b Lattice thermal conductivity. Inset shows the room-temperature lattice thermal conductivities departure from the theoretical line calculated by the Callaway model. c The average sound velocity \\((v_{\\mathrm{avg}})\\) versus lattice thermal conductivity \\((\\kappa_{\\mathrm{L}})\\) for LISS compounds at room temperature. d Temperature-dependent \\(ZT\\) for LISS samples.",
+    "footnote": [],
+    "bbox": [
+      [
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+        816,
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+      ]
+    ],
+    "page_idx": 9
+  },
+  {
+    "type": "image",
+    "img_path": "images/Supplementary_Figure_8.jpg",
+    "caption": "Supplementary Figure 8. Temperature-dependent (a) electrical conductivity, (b) Seebeck coefficient, (c) total thermal conductivity, and (d) dimensionless figure-of-merit \\(ZT\\) for several \\(x = 2.05\\) and \\(x = 2.1\\) samples, respectively.",
+    "footnote": [],
+    "bbox": [
+      [
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+      ]
+    ],
+    "page_idx": 10
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_2.jpg",
+    "caption": "Figure 2 in reference [Luo, Z. Z. et al. Angew. Chem. 2021, 133, 272 - 277]. Electronic band structures and density-of-states (DOS) for Ag-doped PbSe (a, b), Ag-doped and SrSe-alloyed PbSe (c, d), and Ag-doped and BaSe-alloyed PbSe (e, f).",
+    "footnote": [],
+    "bbox": [
+      [
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+      ]
+    ],
+    "page_idx": 11
+  },
+  {
+    "type": "image",
+    "img_path": "images/Supplementary_Figure_2.jpg",
+    "caption": "Supplementary Figure 2. Electronic band structures of (a) \\(\\mathrm{Pb_{26}AgSe_{27}}\\) and (b) \\(\\mathrm{Pb_{26}InSe_{27}}\\) . (c) The energy offset \\((\\Delta E_{1:2})\\) between L and \\(\\Sigma\\) valence band. (d) Theoretical bandgaps \\((E_{\\mathrm{g}})\\) for pristine, Ag-doped, In-doped and Ag-In co-doped PbSe.",
+    "footnote": [],
+    "bbox": [
+      [
+        185,
+        458,
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+        800
+      ]
+    ],
+    "page_idx": 12
+  },
+  {
+    "type": "image",
+    "img_path": "images/Supplementary_Figure_3.jpg",
+    "caption": "Supplementary Figure 3. The atomic orbital projected band structure of \\(\\mathrm{Pb_{25}AgInSe_{27}}\\) . (a) The conduction band is dominated by Pb-p orbitals, while the valence band contain considerable Pb-s character. (b) The Se-p orbital primarily contributes to the valence band. (c) The Ag-d orbitals have a considerable contribution to the valence band. (d) There is distinct In-s character at the conduction band.",
+    "footnote": [],
+    "bbox": [
+      [
+        174,
+        88,
+        820,
+        450
+      ]
+    ],
+    "page_idx": 15
+  },
+  {
+    "type": "image",
+    "img_path": "images/Figure_5.jpg",
+    "caption": "Fig. 5 Electronic band structure. a Electronic band structure of Pb27Se27 (black) and Pb25AgInSe27 (red). b Electronic density of states (DOS) near the Fermi level for Pb27Se27 (black), Pb26AgSe27 (green), Pb26InSe27 (blue) and Pb25AgInSe27 (red), respectively.",
+    "footnote": [],
+    "bbox": [
+      [
+        150,
+        309,
+        843,
+        523
+      ]
+    ],
+    "page_idx": 16
+  },
+  {
+    "type": "image",
+    "img_path": "images/Supplementary_Figure_2.jpg",
+    "caption": "Supplementary Figure 2. Electronic band structures of (a) \\(\\mathrm{Pb_{26}AgSe_{27}}\\) and (b) \\(\\mathrm{Pb_{26}InSe_{27}}\\) . (c) The energy offset \\((\\Delta E_{1:2})\\) between L and \\(\\Sigma\\) valence band. (d) Theoretical bandgaps \\((E_{\\mathrm{g}})\\) for pristine, Ag-doped, In-doped and Ag-In co-doped PbSe.",
+    "footnote": [],
+    "bbox": [
+      [
+        200,
+        85,
+        792,
+        411
+      ]
+    ],
+    "page_idx": 17
+  }
+]

peer_reviews/supplementary_0_Peer Review File__018ea56f56282342b1a9e7b0cea3a8dc2890bbe74f3ff129238a91951bc7905d/supplementary_0_Peer Review File__018ea56f56282342b1a9e7b0cea3a8dc2890bbe74f3ff129238a91951bc7905d.mmd

@@ -0,0 +1,325 @@
+
+# nature portfolio  
+
+Peer Review File  
+
+Multiple valence bands convergence and strong phonon scattering lead to high thermoelectric performance in p- type PbSe  
+
+![](images/Supplementary_Figure_6.jpg)
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+## REVIEWER COMMENTS  
+
+## Reviewer #1 (Remarks to the Author):  
+
+Band convergence is an effective way to improve the electrical properties of thermoelectric materials. Here, the authors reported that multiple valence bands could be activated in p- type PbSe- AgInSe2 system, which is a novel point compared with the traditional two- band convergence. These results will motivate researchers to find more strategies on band structure engineering. AgInSe2 has an intrinsic low lattice thermal conductivity. The introduction of AgInSe2 leads to strong phonon scattering verified by the nano- scale precipitates and dislocations. As a result, a large ZT ( \(\sim 2.1\) ) was achieved in this work and it shows a good reproducible thermoelectric performance. The paper reported an extensive study on p- type PbSe- AgInSe2 and it is well written. Therefore, I recommend the paper to be published in Nature communications. Suggestions or comments are given below.  
+
+1. The microstructure study didn't give a quantitative results on the occupation of Ag and In atoms in this system. So, the sentence -"Local structure and microstructure analysis reveal that about 80 percent of Ag and In atoms form AgInSe2 as nano-scale precipitates" -in the Abstract may make misunderstanding, which should be modified.  
+
+2. The temperature-dependent of band-gap may be another evidence for the band convergence. The increase tendency of band gap become unobvious above \(\sim 500K\) , which indicate that the heavy valence bands may dominates above this temperature. This phenomenon is also consistent with the temperature-dependent Hall measurement that RH peak appears around 500K (Figure S5).  
+
+3. Mechanical property is important for the transport performance of materials. What is the effect on the mechanical property of PbSe with the introduction of AgInSe2?  
+
+4. The Lorenz number was calculated assuming acoustic-phonon scattering dominates. How to prove this?  
+
+5. What is the uncertainty for the thermoelectric properties measurements?  
+
+## Reviewer #2 (Remarks to the Author):  
+
+In this manuscript, the authors investigated the thermoelectric properties of Pb0.98Na0.02Se- \(\times \%\) AgInSe2 (LISS). An exceptional figure- of- merit ZT of \(\sim 2.1\) at 873K was achieved. They performed a systematic study on the electrical/thermal transport properties and microstructures for this system. The introduction of AgInSe2 enlarges the band gap, suppressing the bipolar effect. It is very important to achieve high thermoelectric performance at high temperature regime, but has been challenging to obtain. Interestingly, the incorporation of AgInSe2 facilitates the convergence of multiple valence bands, resulting in high weighted mobility and large power factor. The presence of nano- scale AgInSe2 precipitates and dislocations results in strong phonon scattering. As a result, a combination of band convergence and strong phonon scattering gives record- high thermoelectric performance in PbSe. The local structure analysis by XAFS is very interesting, providing a microscopic perspective to understand the role of doped elements. This is a very solid work and it is suitable for the scope of Nature Communications. This work shows that PbSe thermoelectrics can compete with the much expensive PbTe. It is highly recommended to be published in Nature Communications after addressing minor points as given below:  
+
+1. In the "Introduction" section, please cite related works when you mentioned the quality factor B (The lattice thermal conductivity is another important parameter for the thermoelectric performance indicated by the quality factor B).  
+
+2. It is mentioned that "the tetragonal AgInSe2 is perfectly inserted to the PbSe matrix as nano-scale
+
+<--- Page Split --->
+
+precipitates revealed by the transmission electron microscopy". This sentence can make readers confused because a part of Ag and In atoms occupies Pb sites.  
+
+3. The effective masses increase with the introduction of AgInSe2 as indicated by the Pisarenko plot. Please show the effective masses for each sample, which is more straightforward for the readers.  
+
+4. The measurement of sound velocities is not mentioned in the experimental section.  
+
+5. The heat capacity (Cp) calculated by the Dulong-Petit law will underestimate the thermal conductivity at high temperature. Estimating the Cp by the empirical equation is more accurate. It is not necessary to use the Cp value estimated by the Dulong-Petit law.  
+
+## Reviewer #3 (Remarks to the Author):  
+
+see separate file
+
+<--- Page Split --->
+
+The present manuscript provides interesting data describing the thermoelectric properties of PbSe doped with AgInSe2. This doping / alloying leads to a reduction in the thermal conductivity and an improvement of band convergence enabling a zT value slightly above 2. Thermoelectric materials are considered a viable option to improve energy conversion since they convert waste heat into electrical energy or enable efficient cooling. Hence identifying promising thermoelectric materials is a timely topic, which could be suitable for Nature Communications. Nevertheless, there are several reasons why the present manuscript does not meet my expectations for a manuscript to be published in Nature Communications.  
+
+One of the main claims is that there is multi- band convergence. Yet, the nature of these band and how they could possibly be described and explained is missing. One of the main claims of the present manuscript is the idea that more than 3 bands can contribute, yet the proof for this claim and the explanation of the nature of these bands appears rather incomplete. I would like to see very strong and convincing evidence that indeed more than two bands contribute and what their nature is. To mention one option to provide such evidence: several groups have recently employed tight- binding methods to explain the band structure of related chalcogenides [1,2]. Such calculations could be performed to explore the potential nature and more importantly origin of the bands involved. I am not aware of any study that has claimed and proven so far that three bands can provide a contribution to the thermoelectric performance of PbSe.  
+
+Then, I am also concerned about the apparent disagreement between theory and experiment. The authors claim that their experiment shows an increase of band gap upon alloying with AgInSe2. Yet, the DFT calculations presented in fig. 5b do not seem to support this conclusion.  
+
+Finally, in recent years the thermoelectric properties in lead chalcogenides have been discussed in terms of the underlying bonding mechanism, which must be related to the corresponding band structure [1,3]. A discussion of the fundamental bonding mechanism relevant here is missing. Such a discussion is important since it can help to predict and explain which materials and changes of bonding can improve the performance of a given thermoelectric material.  
+
+[1] Chemistry of Materials 32 (22), 9771- 9779 (2020)  [2] Advanced Materials 30, 1801787 (2018)  [3] Advanced Materials 32, 202005533 (2020)
+
+<--- Page Split --->
+
+## Reviewer #1 (Remarks to the Author):  
+
+## General comment:  
+
+Band convergence is an effective way to improve the electrical properties of thermoelectric materials. Here, the authors reported that multiple valence bands could be activated in p- type PbSe- AgInSe \(_2\) system, which is a novel point compared with the traditional two- band convergence. These results will motivate researchers to find more strategies on band structure engineering. AgInSe \(_2\) has an intrinsic low lattice thermal conductivity. The introduction of AgInSe \(_2\) leads to strong phonon scattering verified by the nano- scale precipitates and dislocations. As a result, a large ZT ( \(\sim 2.1\) ) was achieved in this work and it shows a good reproducible thermoelectric performance. The paper reported an extensive study on p- type PbSe- AgInSe \(_2\) and it is well written. Therefore, I recommend the paper to be published in Nature Communications. Suggestions or comments are given below.  
+
+Response: We appreciate the reviewer 1 for his/her solid summary and affirmation for our work. Your insightful comments will strengthen our work.  
+
+Comment 1: The microstructure study didn't give a quantitative results on the occupation of Ag and In atoms in this system. So, the sentence - "Local structure and microstructure analysis reveal that about 80 percent of Ag and In atoms form AgInSe \(_2\) as nano- scale precipitates" - in the Abstract may make misunderstanding, which should be modified.  
+
+Response: Thanks for your good suggestions. We have made a revision for this sentence. In addition, we made a change to the "abstract" to meet the requirement of Nature Communications (150 words or fewer).  
+
+Revision: Abundant nano- scale precipitates and dislocations result in strong phonon scattering and thus ultralow lattice thermal conductivity. Consequently, we achieve an exceptional ZT of \(\sim 1.9\) at 873 K in p- type PbSe. This work demonstrates that a combination of band manipulation and microstructure engineering can be realized by tuning the composition, which is expected to be a general strategy for improving the thermoelectric performance in bulk materials.  
+
+Comment 2: The temperature- dependent of band- gap may be another evidence for the band convergence. The increase tendency of band gap become unobvious above \(\sim 500\mathrm{K}\) , which indicate that the heavy valence bands may dominates above this temperature. This phenomenon is also consistent with the temperature- dependent Hall measurement that \(\mathrm{R_H}\) peak appears around 500K (Figure S5).  
+
+Response: It is a good point. A related discussion has been added in our manuscript.  
+
+Revision: The unobvious increase tendency of bandgap above \(\sim 500\mathrm{K}\) may be attributed to the band convergence, where the heavy valence bands dominate and the
+
+<--- Page Split --->
+
+position of heavy valence bands are almost temperature independent<sup>1</sup>.  
+
+1. Pei, Y. et al. Convergence of electronic bands for high performance bulk thermoelectrics. Nature 473, 66-69 (2011).  
+
+Comment 3: Mechanical property is important for the transport performance of materials. What is the effect on the mechanical property of PbSe with the introduction of \(\mathrm{AgInSe}_2\) ?  
+
+Response: Thanks for your comments. We calculated the bulk modulus \((K)\) as shown in Table S1. There is no significant effect on the mechanical property of PbSe with introducing \(\mathrm{AgInSe}_2\) because the bulk modulus has no obvious change.  
+
+Revision: The deduced Grüneisen parameters \((\gamma)\) and bulk modulus \((K)\) of LISS have no obvious change (Table S1).  
+
+Supplementary Table 1. Various parameters (longitudinal sound velocity \((v_{1})\) , transverse sound velocity \((v_{t})\) , average sound velocity \((v_{\mathrm{avg}})\) , Poisson ration \((v_{\mathrm{p}})\) , Grüneisen parameter \((\gamma)\) , and bulk modulus \((K)\) ) of \(\mathrm{Pb_{0.98}Na_{0.02}Se - x\% AgInSe_2}\) . The Poisson ration \((v_{\mathrm{p}})\) is calculated by \(v_{\mathrm{p}} = \frac{1 - 2(v_{t} / v_{t})^{2}}{2 - 2(v_{t} / v_{t})^{2}}\) , the Grüneisen parameter \((\gamma)\) is obtained using \(\gamma = \frac{3}{2}\left(\frac{1 + v_{p}}{2 - 3v_{p}}\right)\) and the bulk modulus \((K)\) is given by \(K = \rho \left(v_{t}^{2} - \frac{4}{3} v_{t}^{2}\right)\) \((\rho\) is the density of sample).  
+
+<table><tr><td>Sample</td><td>v1 (m/s)</td><td>v1 (m/s)</td><td>vavg (m/s)</td><td>vp</td><td>γ</td><td>K (GPa)</td></tr><tr><td>x=0</td><td>3165.6</td><td>1708.6</td><td>1907.1</td><td>0.294</td><td>1.74</td><td>48.9</td></tr><tr><td>x=0.5</td><td>3192.9</td><td>1726.9</td><td>1927.3</td><td>0.293</td><td>1.73</td><td>50.2</td></tr><tr><td>x=1</td><td>3214.7</td><td>1726.1</td><td>1927.4</td><td>0.297</td><td>1.75</td><td>51.2</td></tr><tr><td>x=1.5</td><td>3151.7</td><td>1715.4</td><td>1913.5</td><td>0.289</td><td>1.71</td><td>48.0</td></tr><tr><td>x=2</td><td>3217.4</td><td>1720.9</td><td>1922.1</td><td>0.299</td><td>1.77</td><td>50.9</td></tr><tr><td>x=2.05</td><td>3148.7</td><td>1718.3</td><td>1916.4</td><td>0.288</td><td>1.70</td><td>47.6</td></tr><tr><td>x=2.1</td><td>3179.8</td><td>1720.3</td><td>1919.9</td><td>0.293</td><td>1.73</td><td>48.3</td></tr><tr><td>x=2.15</td><td>3149.7</td><td>1718.8</td><td>1916.9</td><td>0.288</td><td>1.70</td><td>47.4</td></tr></table>  
+
+Comment 4: The Lorenz number was calculated assuming acoustic- phonon scattering dominates. How to prove this?  
+
+Response: Thanks for your comments. The carrier scattering mechanism can be revealed in the log \((\mu_{\mathrm{H}})\) - log (T) relation. A log \((\mu_{\mathrm{H}})\) - log (T) plot displays \(\mathrm{T}^{- 3 / 2}\) behavior for \(x = 2.05\) sample when \(\mathrm{T} < 600 \mathrm{K}\) (see below Figure), demonstrating that acoustic- phonon scattering dominates. The deviation from \(\mathrm{T}^{- 3 / 2}\) relation at high temperature is due to the increase of effective mass.
+
+<--- Page Split --->
+
+Revision: The \(\kappa_{\mathrm{e}}\) was calculated by the Wiedemann- Franz relation, \(\kappa_{\mathrm{e}} = L\sigma T\) , where \(L\) (Figure S7a) is estimated by SPB model assuming acoustic phonon scattering dominates (Figure S6c).  
+
+![](images/Figure_1.jpg)
+
+<center>Supplementary Figure 6. (a) Temperature dependence of Hall coefficient \((R_{\mathrm{H}})\) of \(\mathrm{Pb_{0.98}Na_{0.02}Se - 2.05\% AgInSe_2}\) . (b) Effective mass as a function of temperature for \(\mathrm{Pb_{0.98}Na_{0.02}Se - 2.05\% AgInSe_2}\) and \(\mathrm{Pb_{1 - x}Na_xSe}\) . (c) Temperature-dependent Hall mobility of \(\mathrm{Pb_{0.98}Na_{0.02}Se - 2.05\% AgInSe_2}\) , which displays a \(\mathrm{T}^{-3 / 2}\) behavior when \(\mathrm{T}< 600 \mathrm{K}\) , demonstrating that acoustic-phonon scattering dominates. The deviation from \(\mathrm{T}^{-3 / 2}\) relation at high temperature is due to the increase of effective mass. </center>  
+
+Comment 5: What is the uncertainty for the thermoelectric properties measurements?  
+
+Response: Thanks for your comments. The standard samples were measured and these measurement data are compared with the reference value. The uncertainties of thermoelectric parameters are given below. In Figure 1 and 2, the measurements of thermal diffusivity \((D)\) , electrical resistivity \((\rho)\) and Seebeck coefficient \((S)\) are well consistent with their reference values. The error of thermal diffusivity is less than \(2\%\) (Figure 1). The uncertainties of electrical resistivity \((\rho)\) and Seebeck coefficient \((S)\) are less than \(2\%\) and \(5\%\) , respectively (Figure 2). The combined uncertainty of all measurements for determining the \(ZT\) is less than \(20\%\) .  
+
+![](images/Figure_2.jpg)
+
+<center>Figure 1. (a) Comparison of thermal diffusivity \((D)\) between measurement and reference for standard sample (Inconel). (b) The uncertainty of \(D\) . </center>
+
+<--- Page Split --->
+![](images/Figure_1.jpg)
+
+<center>Figure 2. Comparison of (a) electrical resistivity \((\rho)\) and (c) Seebeck coefficient \((S)\) between measurement and standard sample (Constantan), respectively. The corresponding uncertainties are shown in (b) and (d). </center>
+
+<--- Page Split --->
+
+## Reviewer #2 (Remarks to the Author):  
+
+## General comment:  
+
+In this manuscript, the authors investigated the thermoelectric properties of \(\mathrm{Pb_{0.98}Na_{0.02}Se - x\%AgInSe_2}\) (LISS). An exceptional figure- of- merit ZT of \(\sim 2.1\) at \(873\mathrm{K}\) was achieved. They performed a systematic study on the electrical/thermal transport properties and microstructures for this system. The introduction of \(\mathrm{AgInSe_2}\) enlarges the band gap, suppressing the bipolar effect. It is very important to achieve high thermoelectric performance at high temperature regime, but has been challenging to obtain. Interestingly, the incorporation of \(\mathrm{AgInSe_2}\) facilitates the convergence of multiple valence bands, resulting in high weighted mobility and large power factor. The presence of nano- scale \(\mathrm{AgInSe_2}\) precipitates and dislocations results in strong phonon scattering. As a result, a combination of band convergence and strong phonon scattering gives record- high thermoelectric performance in \(\mathrm{PbSe}\) . The local structure analysis by XAFS is very interesting, providing a microscopic perspective to understand the role of doped elements. This is a very solid work and it is suitable for the scope of Nature Communications. This work shows that \(\mathrm{PbSe}\) thermoelectrics can compete with the much expensive \(\mathrm{PbTe}\) . It is highly recommended to be published in Nature Communications after addressing minor points as given below:  
+
+Response: We thank the reviewer 2 for his/her positive comments and valuable suggestions, which is a publishable justification for our submission.  
+
+Comment 1: In the "Introduction" section, please cite related works when you mentioned the quality factor B (The lattice thermal conductivity is another important parameter for the thermoelectric performance indicated by the quality factor B).  
+
+Response: Thanks for your comments. References about quality factor B are cited in the sentence.  
+
+Revision: The lattice thermal conductivity is another important parameter for the thermoelectric performance indicated by the quality factor \(B\) ( \(B \propto \mu \nu / \kappa_{\mathrm{L}}\) ) \(^{30,31}\) .  
+
+30. Kang, S. D., Snyder G. J. Transport property analysis method for thermoelectric materials material: quality factor and the effective mass model. arXiv:1710.06896 [cond-mat.mtrl-sci] (2017).  
+
+31. Tan, G., Zhao, L.-D. & Kanatzidis, M. G. Rationally Designing High-Performance Bulk Thermoelectric Materials. Chem. Rev. 116, 12123-12149 (2016).  
+
+Comment 2: It is mentioned that "the tetragonal \(\mathrm{AgInSe_2}\) is perfectly inserted to the \(\mathrm{PbSe}\) matrix as nano- scale precipitates revealed by the transmission electron microscopy". This sentence can make readers confused because a part of Ag and In atoms occupies Pb sites.  
+
+Response: We appreciate your comments. This sentence has been modified.
+
+<--- Page Split --->
+
+Revision: The sentence mentioned above is changed to "Nano- scale AgInSe₂ precipitates are revealed by the transmission electron microscopy (TEM)."  
+
+Comment 3: The effective masses increase with the introduction of AgInSe₂ as indicated by the Pisarenko plot. Please show the effective masses for each sample, which is more straightforward for the readers.  
+
+Response: Thanks for your good suggestions. The effective masses as a function of AgInSe₂ content are given in our manuscript.  
+
+Revision: the effective mass \((m^{*})\) of LISS is largely increased from \(0.44 m_{e}\) to 0.81 \(m_{e}\) with the introduction of AgInSe₂ (Figure 4a, Figure S1b).  
+
+![](images/Figure_6.jpg)
+  
+
+Supplementary Figure 1. (a) Hall carrier concentrations and (b) density- of- states effective mass of \(\mathrm{Pb_{0.98}Na_{0.02}Se}\) - \(x\%\) AgInSe₂ (LISS) with increasing AgInSe₂ content at 303K.  
+
+Comment 4: The measurement of sound velocities is not mentioned in the experimental section.  
+
+Response: Thanks for your comments. The measurements of sound velocity have added in the experimental section.  
+
+Revision: Pulse- echo method was used to measure the speed of sound and the waveforms were recorded using a Tektronix TBS 1102 oscilloscope.
+
+<--- Page Split --->
+
+Comment 5: The heat capacity \(\mathrm{(C_p)}\) calculated by the Dulong- Petit law will underestimate the thermal conductivity at high temperature. Estimating the \(\mathrm{C_p}\) by the empirical equation is more accurate. It is not necessary to use the \(\mathrm{C_p}\) value estimated by the Dulong- Petit law.  
+
+Response: We appreciate your good suggestions. We estimated the heat capacity using the empirical equation for all samples. The total thermal conductivity \((\kappa_{\mathrm{tot}})\) , lattice thermal conductivity \((\kappa_{\mathrm{L}})\) and the figure- of- merit \(ZT\) have been recalculated.  
+
+Revision: we recalculated the total thermal conductivity \((\kappa_{\mathrm{tot}})\) , lattice thermal conductivity \((\kappa_{\mathrm{L}})\) and the dimensionless figure- of- merit \(ZT\) . Accordingly, Figure 1, Figure 6 and Figure S8 have been revised.  
+
+![](images/Supplementary_Figure_8.jpg)
+
+<center>Fig. 1 Multiple valence bands enable high \(ZT\) values in p-type PbSe. a Schmatic diagram of multi-bands \((\mathrm{L}, \Sigma , \Lambda)\) involvement in transport. The Brillouin zone shows that the degeneracies at the \(\mathrm{L}, \Sigma\) , and \(\Lambda\) points are 4, 12, and 8, respectively. b The activated third band \(\Lambda\) enables higher \(ZT\) values compared with the single-band and two-band PbSe-based materials. </center>
+
+<--- Page Split --->
+![](images/Figure_2.jpg)
+
+<center>Fig. 6 Thermal transport properties and dimensionless figure-of-merit \(ZT\) as a function of temperature for \(\mathrm{Pb_{0.98}Na_{0.02}Se - x\% AgInSe_2}\) (LISS) compounds. a Total thermal conductivity. b Lattice thermal conductivity. Inset shows the room-temperature lattice thermal conductivities departure from the theoretical line calculated by the Callaway model. c The average sound velocity \((v_{\mathrm{avg}})\) versus lattice thermal conductivity \((\kappa_{\mathrm{L}})\) for LISS compounds at room temperature. d Temperature-dependent \(ZT\) for LISS samples. </center>
+
+<--- Page Split --->
+![](images/Supplementary_Figure_2.jpg)
+
+<center>Supplementary Figure 8. Temperature-dependent (a) electrical conductivity, (b) Seebeck coefficient, (c) total thermal conductivity, and (d) dimensionless figure-of-merit \(ZT\) for several \(x = 2.05\) and \(x = 2.1\) samples, respectively. </center>
+
+<--- Page Split --->
+
+## Reviewer #3 (Remarks to the Author):  
+
+## General comment:  
+
+The present manuscript provides interesting data describing the thermoelectric properties of PbSe doped with AgInSe2. This doping / alloying leads to a reduction in the thermal conductivity and an improvement of band convergence enabling a zT value slightly above 2. Thermoelectric materials are considered a viable option to improve energy conversion since they convert waste heat into electrical energy or enable efficient cooling. Hence identifying promising thermoelectric materials is a timely topic, which could be suitable for Nature Communications. Nevertheless, there are several reasons why the present manuscript does not meet my expectations for a manuscript to be published in Nature Communications.  
+
+Response: We appreciate your valuable comments and suggestions, which will strengthen our work. Hopefully, our revised manuscript could meet your expectations.  
+
+Comment 1: One of the main claims is that there is multi- band convergence. Yet, the nature of these band and how they could possibly be described and explained is missing. One of the main claims of the present manuscript is the idea that more than 3 bands can contribute, yet the proof for this claim and the explanation of the nature of these bands appears rather incomplete. I would like to see very strong and convincing evidence that indeed more than two bands contribute and what their nature is. To mention one option to provide such evidence: several groups have recently employed tight- binding methods to explain the band structure of related chalcogenides [1,2]. Such calculations could be performed to explore the potential nature and more importantly origin of the bands involved. I am not aware of any study that has claimed and proven so far that three bands can provide a contribution to the thermoelectric performance of PbSe.  
+
+[1] Chemistry of Materials 32 (22), 9771- 9779 (2020)  [2] Advanced Materials 30, 1801787 (2018)  
+
+Response: Thanks for your insightful comments. Our DFT calculations reveal that a third valence band \(\Lambda\) along \(\Gamma\) - L is activated. The large weighted mobility and effective mass also reflect the multi- band convergence indirectly. A comparison of the density- of- states effective mass for various p- type PbSe- based materials are shown in the Table 1 below.  
+
+Indeed, the tight- binding methods are powerful tool to understand the nature of electronic band structure. However, it is hard to employ tight- binding calculations for Ag- In co- doped PbSe since the supercells contain too many atoms. Instead, we calculated the atomic orbital projected band structure by DFT to understand their nature. The conduction and valence bands of PbSe system are dominated by the Pb- p and Se- p states, respectively, which is in line with the tight- binding calculations for PbTe [Brod, M. K., et al. Chem. Mater. 32, 9771- 9779 (2020)]. Owing to the rock- salt structure of PbSe, these p- bands are half- filled forming a \(\sigma\) - bond, which is
+
+<--- Page Split --->
+
+characteristic of metavalent bonding [Wuttig, M., et al. Adv. Mater. 30, e1803777 (2018)]. Similar to PbTe, the valence band maximum (L band) of PbSe is contributed by the p- states. The projected electronic band structure also implies that the third valence band \(\Lambda\) show a large contribution by the Ag 4d state and Se 4p state. This may explain the promoted band convergence in PbSe by alloying with AgInSe₂.  
+
+A similar electronic band structure can also be found in Ag- Sr co- doped PbSe system [Luo, Z. Z. et al. Angew. Chem. 2021, 133, 272 - 277]. As shown in Figure 2 below, the valence band 2 (Σ) and valence band 3 (Λ) are almost at the same energy level. The energy offset between valence band 1 (L) and the other two valence bands (Σ and Λ) is \(\sim 0.17 \mathrm{eV}\) . They found a strong band convergence behavior in this system. However, they didn't mention the underlying multi- band convergence behavior.  
+
+Table 1. Density-of-states effective masses \((m^{*})\) for various p-type PbSe-based materials.   
+
+<table><tr><td>sample</td><td>m*(me) at 300K</td><td>m*(me) at 773K</td><td>reference</td></tr><tr><td>PbSe-Na-Ag-In</td><td>0.81</td><td>2.16</td><td>This work</td></tr><tr><td>PbSe-Na</td><td>0.28</td><td>0.7</td><td>1</td></tr><tr><td>PbSe-Ag-Sr</td><td>0.4</td><td>1.1</td><td>2</td></tr><tr><td>PbSe-Ag-Ba</td><td>0.4</td><td>1.0</td><td>2</td></tr><tr><td>PbSe-Na-Hg</td><td>0.45</td><td>1.3</td><td>3</td></tr><tr><td>PbSe-Cd-Na-Te</td><td>0.57</td><td></td><td>4</td></tr><tr><td>PbSe-Ag</td><td>0.35</td><td></td><td>5</td></tr><tr><td>PbSe-Na-Ca</td><td>0.56</td><td></td><td>6</td></tr><tr><td>PbSe-Na-Ba</td><td>0.56</td><td></td><td>6</td></tr><tr><td>PbSe-Na-Sr</td><td>0.48</td><td></td><td>6</td></tr></table>  
+
+1 Wang, H., Pei, Y., LaLonde, A. D. & Snyder, G. J. Heavily doped p- type PbSe with high thermoelectric performance: an alternative for PbTe. Adv. Mater. 23, 1366- 1370 (2011). 2 Luo, Z. Z. et al. Strong Valence Band Convergence to Enhance Thermoelectric Performance in PbSe with Two Chemically Independent Controls. Angew. Chem. Int. Ed. 60, 268- 273 (2021). 3 Hodges, J. M. et al. Chemical Insights into PbSe- x%HgSe: High Power Factor and Improved Thermoelectric Performance by Alloying with Discordant Atoms. J. Am. Chem. Soc. 140, 18115- 18123 (2018). 4 Tan, G., Zhao, L.- D. & Kanatzidis, M. G. Rationally Designing High- Performance Bulk Thermoelectric Materials. Chem. Rev. 116, 12123- 12149 (2016). 5 Wang, S. et al. Exploring the doping effects of Ag in p- type PbSe compounds with enhanced thermoelectric performance. J. Phys. D: Appl. Phys. 44, 475304 (2011). 6 Lee, Y. et al. High- performance tellurium- free thermoelectrics: all- scale
+
+<--- Page Split --->
+
+hierarchical structuring of p-type PbSe- MSe systems (M = Ca, Sr, Ba). J. Am. Chem. Soc. 135, 5152- 5160 (2013).  
+
+![](images/Supplementary_Figure_3.jpg)
+
+<center>Figure 2 in reference [Luo, Z. Z. et al. Angew. Chem. 2021, 133, 272 - 277]. Electronic band structures and density-of-states (DOS) for Ag-doped PbSe (a, b), Ag-doped and SrSe-alloyed PbSe (c, d), and Ag-doped and BaSe-alloyed PbSe (e, f). </center>  
+
+Revision: The electronic band structures of Ag and In doped PbSe were calculated (Figure S2a, S2b) to understand their role in band manipulation. The Ag- doping and In- doping reflect p- type and n- type doping effect, respectively, which are consistent with previous experimental results<sup>42,43</sup>. Additionally, In- doping has a more important effect on decreasing energy offset ( \(\Delta E_{1 - 2}\) ) compared with the Ag- doping (Figure S2c), while Ag- doping plays a major role in enlarging the bandgap (Figure S2d). The orbital projected band structures reveal that the interaction between Pb- p and Se- p orbitals dominate the band structure (Figure S3a, S3b), which is consistent with previous study<sup>44</sup>. This is a typical feature of the metal- valent bonding system<sup>13,45,46</sup>. The tight binding calculations reveal that the cation states have important effect on the shape of valence band although their orbital projections are not obvious<sup>44</sup>. Indeed, the Ag- d orbitals play an important role in modulating the third valence band \(\Lambda\) along \(\Gamma\) - L (Figure S3c). A similar phenomenon was also observed in Ag- Sr co- dope PbSe system<sup>47</sup>. In addition, the cation- site doping can also contribute to the conduction band (Figure S3d) depending on the nature of cation states. Our results indicate that Ag- In co- doping enable multiple valence band convergence, verifying that the cation- site doping is an effective way to modulate the valence band in PbSe. Similar effects can be expected in other materials, such as PbTe and GeTe, by employing the same chemical bonding mechanism as PbSe.
+
+<--- Page Split --->
+
+13. Wuttig, M., Deringer, V. L., Gonze, X., Bichara, C. & Raty, J. Y. Incipient metals: functional materials with a unique bonding mechanism. Adv. Mater. 30, e1803777 (2018).  
+
+42. Wang, S. et al. Exploring the doping effects of Ag in p-type PbSe compounds with enhanced thermoelectric performance. J. Phys. D: Appl. Phys. 44, 475304 (2011).  
+
+43. Androulakis, J., Lee, Y., Todorov, I., Chung, D.-Y. & Kanatzidis, M. High-temperature thermoelectric properties of n-type PbSe doped with Ga, In, and Pb. Phys. Rev. B 83 (2011).  
+
+44. Brod, M. K., Toriyama, M. Y. & Snyder, G. J. Orbital chemistry that leads to high valley degeneracy in PbTe. Chem. Mater. 32, 9771-9779 (2020).  
+
+45. Maier, S. et al. Discovering electron-transfer-driven changes in chemical bonding in lead chalcogenides (PbX, where X = Te, Se, S, O). Adv. Mater. 32, e2005533 (2020).  
+
+46. Raty, J. Y. et al. A quantum-mechanical map for bonding and properties in solids. Adv. Mater. 31, e1806280 (2019).  
+
+47. Luo, Z. Z. et al. Strong valence band convergence to enhance thermoelectric performance in PbSe with two chemically independent controls. Angew. Chem. Int. Ed. 60, 268-273 (2021).  
+
+![](images/Figure_5.jpg)
+
+<center>Supplementary Figure 2. Electronic band structures of (a) \(\mathrm{Pb_{26}AgSe_{27}}\) and (b) \(\mathrm{Pb_{26}InSe_{27}}\) . (c) The energy offset \((\Delta E_{1:2})\) between L and \(\Sigma\) valence band. (d) Theoretical bandgaps \((E_{\mathrm{g}})\) for pristine, Ag-doped, In-doped and Ag-In co-doped PbSe. </center>
+
+<--- Page Split --->
+![](images/Supplementary_Figure_2.jpg)
+
+<center>Supplementary Figure 3. The atomic orbital projected band structure of \(\mathrm{Pb_{25}AgInSe_{27}}\) . (a) The conduction band is dominated by Pb-p orbitals, while the valence band contain considerable Pb-s character. (b) The Se-p orbital primarily contributes to the valence band. (c) The Ag-d orbitals have a considerable contribution to the valence band. (d) There is distinct In-s character at the conduction band. </center>
+
+<--- Page Split --->
+
+Comment 2: Then, I am also concerned about the apparent disagreement between theory and experiment. The authors claim that their experiment shows an increase of band gap upon alloying with AgInSe₂. Yet, the DFT calculations presented in fig. 5b do not seem to support this conclusion.  
+
+Response: We are sorry for this confusion. It is not obvious to distinguish the bandgap from the electronic density- of- states (Figure 5b). Actually, our calculation is consistent with the experimental result that the bandgap increases with introducing AgInSe₂ in PbSe matrix (Figure 5a). The theoretical bandgap for pure PbSe, Ag- doped PbSe, In- doped PbSe and Ag- In co- doped PbSe are compared in Figure S2d shown below. A possible reason for this phenomenon is given in our manuscript.  
+
+![PLACEHOLDER_18_0]
+
+<center>Fig. 5 Electronic band structure. a Electronic band structure of Pb27Se27 (black) and Pb25AgInSe27 (red). b Electronic density of states (DOS) near the Fermi level for Pb27Se27 (black), Pb26AgSe27 (green), Pb26InSe27 (blue) and Pb25AgInSe27 (red), respectively. </center>
+
+<--- Page Split --->
+![PLACEHOLDER_19_0]
+
+<center>Supplementary Figure 2. Electronic band structures of (a) \(\mathrm{Pb_{26}AgSe_{27}}\) and (b) \(\mathrm{Pb_{26}InSe_{27}}\) . (c) The energy offset \((\Delta E_{1:2})\) between L and \(\Sigma\) valence band. (d) Theoretical bandgaps \((E_{\mathrm{g}})\) for pristine, Ag-doped, In-doped and Ag-In co-doped PbSe. </center>  
+
+Revision: The experimental bandgap is \(\sim 0.24 \mathrm{eV}\) for the pristine PbSe, while the bandgap increases obviously with the incorporation of \(\mathrm{AgInSe_2}\) and a large bandgap \(\sim 0.33 \mathrm{eV}\) is achieved for the PbSe - \(2\%\) \(\mathrm{AgInSe_2}\) sample (Figure 2d). The small bandgap of PbSe results from its unconventional chemical bonding mechanism (metavalent bonding). For a perfect half- filled p- band, the energy band structures resemble a metallic system. Yet, the bandgap opens due to a small Peierls distortion or charge transfer<sup>41</sup>. It is the charge transfer between Pb and Se that opens a small bandgap in PbSe given its perfect octahedral arrangements. DFT results show that the enlarged bandgap is mainly attributed to the incorporation of Ag. The eletronegativity difference between Ag and Te \((\sim 0.62)\) is larger than that between Pb and Te \((\sim 0.22)\) . Therefore, the substitution of Ag at Pb sites will strengthen the charge transfer between cation and anion, leading to an enlarged bandgap.  
+
+41. Yu, Y., Cagnoni, M., Cojocaru- Miré din, O. & Wuttig, M. Chalcogenide Thermoelectrics Empowered by an Unconventional Bonding Mechanism. Adv. Funct. Mater. 30, 1904862 (2019).
+
+<--- Page Split --->
+
+Comment 3: Finally, in recent years the thermoelectric properties in lead chalcogenides have been discussed in terms of the underlying bonding mechanism, which must be related to the corresponding band structure [1,3]. A discussion of the fundamental bonding mechanism relevant here is missing. Such a discussion is important since it can help to predict and explain which materials and changes of bonding can improve the performance of a given thermoelectric material.  
+
+[1] Chemistry of Materials 32 (22), 9771- 9779 (2020)  [3] Advanced Materials 32, 202005533 (2020)  
+
+Response: Thanks for your valuable suggestions. We have made discussions to understand the nature of electronic band structure and the bandgap behavior in chemical bonding perspective.  
+
+## Revision:  
+
+Introduction section: Materials with disordered or complex crystal structure \(^{9,10}\) , giant anharmonicity \(^{11,12}\) , metavalent bonding \(^{13}\) , and lone pair electrons \(^{14}\) often exhibit intrinsic low lattice thermal conductivity, which are promising candidates for thermoelectric applications.  
+
+Bandgap: The small bandgap of PbSe results from its unconventional chemical bonding mechanism (metavalent bonding). For a perfect half- filled p- band, the energy band structures resemble a metallic system. Yet, the bandgap opens due to a small Peierls distortion or charge transfer \(^{41}\) . It is the charge transfer between Pb and Se that opens a small bandgap in PbSe given its perfect octahedral arrangements. DFT results show that the enlarged bandgap is mainly attributed to the incorporation of Ag. The eletronegativity difference between Ag and Te ( \(\sim 0.62\) ) is larger than that between Pb and Te ( \(\sim 0.22\) ). Therefore, the substitution of Ag at Pb sites will strengthen the charge transfer between cation and anion, leading to an enlarged bandgap.  
+
+Electronic band structure: We calculated the orbital projected band structures to understand the nature of electronic band structure in chemical bonding perspective. The orbital projected band structures reveal that the interaction between Pb- p and Te- p orbitals dominate the band structure (Figure S3a, S3b), which is consistent with previous study \(^{46}\) . This is a typical feature of the metavalent bonding system \(^{13,45,46}\) . The Ag- d orbitals play an important role in modulating the third valence band \(\Lambda\) along \(\Gamma\) - L (Figure S3c). Our results indicate that Ag- In co- doping enable multiple valence band convergence, verifying that the cation- site doping is an effective way to modulate the valence band in PbSe. Similar effects can be expected in other materials, such as PbTe and GeTe, by employing the same chemical bonding mechanism as PbSe. A more detailed discussion is shown above when we answering the comment 1.  
+
+Temperature- dependent bandgap: Clearly, the bandgap increases with rising temperature, which is also verified experimentally (Figure 5d). As revealed by Brod et al. \(^{44}\) , there is sufficient interaction between Pb- p and Te- p (Se- p in our case) to
+
+<--- Page Split --->
+
+provide the molecular orbitals with the proper s- type symmetry to place the VBM at L point. The weak s- p hybridization is a small addition to this effect. The thermal expansion will lead to a reduction of orbital overlap between p- orbitals \(^{41}\) as well as a weakened s- p hybridization \(^{48}\) . As a result, the energy of VBM (L point) in the electronic band structure decreases \(^{49}\) , resulting in an enlarged bandgap \((E_{\mathrm{g}})\) .  
+
+13. Wuttig, M., Deringer, V. L., Gonze, X., Bichara, C. & Raty, J. Y. Incipient metals: functional materials with a unique bonding mechanism. Adv. Mater. 30, e1803777 (2018).  
+41. Yu, Y., Cagnoni, M., Cojocaru-Miré din, O. & Wuttig, M. Chalcogenide Thermoelectrics Empowered by an Unconventional Bonding Mechanism. Adv. Funct. Mater. 30, 1904862 (2019).  
+44. Brod, M. K., Toriyama, M. Y. & Snyder, G. J. Orbital chemistry that leads to high valley degeneracy in PbTe. Chem. Mater. 32, 9771-9779 (2020).  
+45. Maier, S. et al. Discovering electron-transfer-driven changes in chemical bonding in lead chalcogenides (PbX, where X = Te, Se, S, O). Adv. Mater. 32, e2005533 (2020).  
+46. Raty, J. Y. et al. A quantum-mechanical map for bonding and properties in solids. Adv. Mater. 31, e1806280 (2019).  
+48. Zeier, W. G. et al. Thinking like a chemist: intuition in thermoelectric materials. Angew. Chem. Int. Ed. 55, 6826-6841 (2016).  
+49. Cagnoni, M., Fuhren, D. & Wuttig, M. Thermoelectric performance of IV-VI compounds with octahedral-like coordination: a chemical-bonding perspective. Adv. Mater., e1801787 (2018).
+
+<--- Page Split --->
+
+## REVIEWERS' COMMENTS  
+
+## Reviewer #1 (Remarks to the Author):  
+
+The authors have answered all the raised questions from these three reviewers. I am satisfied with the response. So, I suggest to accept this paper to publish in Nature Communications.  
+
+## Reviewer #2 (Remarks to the Author):  
+
+The manuscript has been properly revised to address all the comments by the reviewers. Now it can be published as it is. This work is a milestone for PbSe thermoelectrics, which possibly can outperform PbTe thermoelectrics in the near future.  
+
+## Reviewer #3 (Remarks to the Author):  
+
+In the response to the questions and comments from the different reviewers, the authors have addressed all questions and concerns adequately. Hence, the manuscript is acceptable in its present form.
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__018ea56f56282342b1a9e7b0cea3a8dc2890bbe74f3ff129238a91951bc7905d/supplementary_0_Peer Review File__018ea56f56282342b1a9e7b0cea3a8dc2890bbe74f3ff129238a91951bc7905d_det.mmd

@@ -0,0 +1,420 @@
+<|ref|>title<|/ref|><|det|>[[99, 40, 508, 90]]<|/det|>
+# nature portfolio  
+
+<|ref|>text<|/ref|><|det|>[[102, 110, 373, 139]]<|/det|>
+Peer Review File  
+
+<|ref|>text<|/ref|><|det|>[[117, 155, 879, 239]]<|/det|>
+Multiple valence bands convergence and strong phonon scattering lead to high thermoelectric performance in p- type PbSe  
+
+<|ref|>image<|/ref|><|det|>[[93, 732, 262, 780]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[270, 732, 880, 784]]<|/det|>
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[119, 83, 312, 98]]<|/det|>
+## REVIEWER COMMENTS  
+
+<|ref|>sub_title<|/ref|><|det|>[[120, 112, 419, 127]]<|/det|>
+## Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 142, 877, 275]]<|/det|>
+Band convergence is an effective way to improve the electrical properties of thermoelectric materials. Here, the authors reported that multiple valence bands could be activated in p- type PbSe- AgInSe2 system, which is a novel point compared with the traditional two- band convergence. These results will motivate researchers to find more strategies on band structure engineering. AgInSe2 has an intrinsic low lattice thermal conductivity. The introduction of AgInSe2 leads to strong phonon scattering verified by the nano- scale precipitates and dislocations. As a result, a large ZT ( \(\sim 2.1\) ) was achieved in this work and it shows a good reproducible thermoelectric performance. The paper reported an extensive study on p- type PbSe- AgInSe2 and it is well written. Therefore, I recommend the paper to be published in Nature communications. Suggestions or comments are given below.  
+
+<|ref|>text<|/ref|><|det|>[[118, 289, 866, 349]]<|/det|>
+1. The microstructure study didn't give a quantitative results on the occupation of Ag and In atoms in this system. So, the sentence -"Local structure and microstructure analysis reveal that about 80 percent of Ag and In atoms form AgInSe2 as nano-scale precipitates" -in the Abstract may make misunderstanding, which should be modified.  
+
+<|ref|>text<|/ref|><|det|>[[118, 363, 870, 422]]<|/det|>
+2. The temperature-dependent of band-gap may be another evidence for the band convergence. The increase tendency of band gap become unobvious above \(\sim 500K\) , which indicate that the heavy valence bands may dominates above this temperature. This phenomenon is also consistent with the temperature-dependent Hall measurement that RH peak appears around 500K (Figure S5).  
+
+<|ref|>text<|/ref|><|det|>[[116, 436, 864, 467]]<|/det|>
+3. Mechanical property is important for the transport performance of materials. What is the effect on the mechanical property of PbSe with the introduction of AgInSe2?  
+
+<|ref|>text<|/ref|><|det|>[[115, 481, 872, 511]]<|/det|>
+4. The Lorenz number was calculated assuming acoustic-phonon scattering dominates. How to prove this?  
+
+<|ref|>text<|/ref|><|det|>[[118, 525, 677, 541]]<|/det|>
+5. What is the uncertainty for the thermoelectric properties measurements?  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 584, 419, 599]]<|/det|>
+## Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[117, 613, 877, 820]]<|/det|>
+In this manuscript, the authors investigated the thermoelectric properties of Pb0.98Na0.02Se- \(\times \%\) AgInSe2 (LISS). An exceptional figure- of- merit ZT of \(\sim 2.1\) at 873K was achieved. They performed a systematic study on the electrical/thermal transport properties and microstructures for this system. The introduction of AgInSe2 enlarges the band gap, suppressing the bipolar effect. It is very important to achieve high thermoelectric performance at high temperature regime, but has been challenging to obtain. Interestingly, the incorporation of AgInSe2 facilitates the convergence of multiple valence bands, resulting in high weighted mobility and large power factor. The presence of nano- scale AgInSe2 precipitates and dislocations results in strong phonon scattering. As a result, a combination of band convergence and strong phonon scattering gives record- high thermoelectric performance in PbSe. The local structure analysis by XAFS is very interesting, providing a microscopic perspective to understand the role of doped elements. This is a very solid work and it is suitable for the scope of Nature Communications. This work shows that PbSe thermoelectrics can compete with the much expensive PbTe. It is highly recommended to be published in Nature Communications after addressing minor points as given below:  
+
+<|ref|>text<|/ref|><|det|>[[118, 834, 855, 879]]<|/det|>
+1. In the "Introduction" section, please cite related works when you mentioned the quality factor B (The lattice thermal conductivity is another important parameter for the thermoelectric performance indicated by the quality factor B).  
+
+<|ref|>text<|/ref|><|det|>[[115, 893, 864, 909]]<|/det|>
+2. It is mentioned that "the tetragonal AgInSe2 is perfectly inserted to the PbSe matrix as nano-scale
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[118, 83, 839, 113]]<|/det|>
+precipitates revealed by the transmission electron microscopy". This sentence can make readers confused because a part of Ag and In atoms occupies Pb sites.  
+
+<|ref|>text<|/ref|><|det|>[[115, 127, 868, 157]]<|/det|>
+3. The effective masses increase with the introduction of AgInSe2 as indicated by the Pisarenko plot. Please show the effective masses for each sample, which is more straightforward for the readers.  
+
+<|ref|>text<|/ref|><|det|>[[118, 171, 755, 187]]<|/det|>
+4. The measurement of sound velocities is not mentioned in the experimental section.  
+
+<|ref|>text<|/ref|><|det|>[[118, 201, 858, 246]]<|/det|>
+5. The heat capacity (Cp) calculated by the Dulong-Petit law will underestimate the thermal conductivity at high temperature. Estimating the Cp by the empirical equation is more accurate. It is not necessary to use the Cp value estimated by the Dulong-Petit law.  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 275, 420, 290]]<|/det|>
+## Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 306, 244, 320]]<|/det|>
+see separate file
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[148, 82, 849, 220]]<|/det|>
+The present manuscript provides interesting data describing the thermoelectric properties of PbSe doped with AgInSe2. This doping / alloying leads to a reduction in the thermal conductivity and an improvement of band convergence enabling a zT value slightly above 2. Thermoelectric materials are considered a viable option to improve energy conversion since they convert waste heat into electrical energy or enable efficient cooling. Hence identifying promising thermoelectric materials is a timely topic, which could be suitable for Nature Communications. Nevertheless, there are several reasons why the present manuscript does not meet my expectations for a manuscript to be published in Nature Communications.  
+
+<|ref|>text<|/ref|><|det|>[[148, 230, 846, 401]]<|/det|>
+One of the main claims is that there is multi- band convergence. Yet, the nature of these band and how they could possibly be described and explained is missing. One of the main claims of the present manuscript is the idea that more than 3 bands can contribute, yet the proof for this claim and the explanation of the nature of these bands appears rather incomplete. I would like to see very strong and convincing evidence that indeed more than two bands contribute and what their nature is. To mention one option to provide such evidence: several groups have recently employed tight- binding methods to explain the band structure of related chalcogenides [1,2]. Such calculations could be performed to explore the potential nature and more importantly origin of the bands involved. I am not aware of any study that has claimed and proven so far that three bands can provide a contribution to the thermoelectric performance of PbSe.  
+
+<|ref|>text<|/ref|><|det|>[[149, 412, 838, 462]]<|/det|>
+Then, I am also concerned about the apparent disagreement between theory and experiment. The authors claim that their experiment shows an increase of band gap upon alloying with AgInSe2. Yet, the DFT calculations presented in fig. 5b do not seem to support this conclusion.  
+
+<|ref|>text<|/ref|><|det|>[[149, 473, 841, 558]]<|/det|>
+Finally, in recent years the thermoelectric properties in lead chalcogenides have been discussed in terms of the underlying bonding mechanism, which must be related to the corresponding band structure [1,3]. A discussion of the fundamental bonding mechanism relevant here is missing. Such a discussion is important since it can help to predict and explain which materials and changes of bonding can improve the performance of a given thermoelectric material.  
+
+<|ref|>text<|/ref|><|det|>[[149, 568, 512, 640]]<|/det|>
+[1] Chemistry of Materials 32 (22), 9771- 9779 (2020)  [2] Advanced Materials 30, 1801787 (2018)  [3] Advanced Materials 32, 202005533 (2020)
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[149, 85, 463, 101]]<|/det|>
+## Reviewer #1 (Remarks to the Author):  
+
+<|ref|>sub_title<|/ref|><|det|>[[149, 105, 310, 120]]<|/det|>
+## General comment:  
+
+<|ref|>text<|/ref|><|det|>[[148, 123, 850, 325]]<|/det|>
+Band convergence is an effective way to improve the electrical properties of thermoelectric materials. Here, the authors reported that multiple valence bands could be activated in p- type PbSe- AgInSe \(_2\) system, which is a novel point compared with the traditional two- band convergence. These results will motivate researchers to find more strategies on band structure engineering. AgInSe \(_2\) has an intrinsic low lattice thermal conductivity. The introduction of AgInSe \(_2\) leads to strong phonon scattering verified by the nano- scale precipitates and dislocations. As a result, a large ZT ( \(\sim 2.1\) ) was achieved in this work and it shows a good reproducible thermoelectric performance. The paper reported an extensive study on p- type PbSe- AgInSe \(_2\) and it is well written. Therefore, I recommend the paper to be published in Nature Communications. Suggestions or comments are given below.  
+
+<|ref|>text<|/ref|><|det|>[[149, 344, 848, 379]]<|/det|>
+Response: We appreciate the reviewer 1 for his/her solid summary and affirmation for our work. Your insightful comments will strengthen our work.  
+
+<|ref|>text<|/ref|><|det|>[[148, 399, 850, 490]]<|/det|>
+Comment 1: The microstructure study didn't give a quantitative results on the occupation of Ag and In atoms in this system. So, the sentence - "Local structure and microstructure analysis reveal that about 80 percent of Ag and In atoms form AgInSe \(_2\) as nano- scale precipitates" - in the Abstract may make misunderstanding, which should be modified.  
+
+<|ref|>text<|/ref|><|det|>[[148, 510, 850, 564]]<|/det|>
+Response: Thanks for your good suggestions. We have made a revision for this sentence. In addition, we made a change to the "abstract" to meet the requirement of Nature Communications (150 words or fewer).  
+
+<|ref|>text<|/ref|><|det|>[[148, 584, 850, 695]]<|/det|>
+Revision: Abundant nano- scale precipitates and dislocations result in strong phonon scattering and thus ultralow lattice thermal conductivity. Consequently, we achieve an exceptional ZT of \(\sim 1.9\) at 873 K in p- type PbSe. This work demonstrates that a combination of band manipulation and microstructure engineering can be realized by tuning the composition, which is expected to be a general strategy for improving the thermoelectric performance in bulk materials.  
+
+<|ref|>text<|/ref|><|det|>[[148, 714, 850, 805]]<|/det|>
+Comment 2: The temperature- dependent of band- gap may be another evidence for the band convergence. The increase tendency of band gap become unobvious above \(\sim 500\mathrm{K}\) , which indicate that the heavy valence bands may dominates above this temperature. This phenomenon is also consistent with the temperature- dependent Hall measurement that \(\mathrm{R_H}\) peak appears around 500K (Figure S5).  
+
+<|ref|>text<|/ref|><|det|>[[147, 825, 829, 842]]<|/det|>
+Response: It is a good point. A related discussion has been added in our manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[148, 862, 850, 898]]<|/det|>
+Revision: The unobvious increase tendency of bandgap above \(\sim 500\mathrm{K}\) may be attributed to the band convergence, where the heavy valence bands dominate and the
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[148, 85, 712, 102]]<|/det|>
+position of heavy valence bands are almost temperature independent<sup>1</sup>.  
+
+<|ref|>text<|/ref|><|det|>[[148, 121, 848, 158]]<|/det|>
+1. Pei, Y. et al. Convergence of electronic bands for high performance bulk thermoelectrics. Nature 473, 66-69 (2011).  
+
+<|ref|>text<|/ref|><|det|>[[148, 177, 850, 232]]<|/det|>
+Comment 3: Mechanical property is important for the transport performance of materials. What is the effect on the mechanical property of PbSe with the introduction of \(\mathrm{AgInSe}_2\) ?  
+
+<|ref|>text<|/ref|><|det|>[[148, 251, 850, 306]]<|/det|>
+Response: Thanks for your comments. We calculated the bulk modulus \((K)\) as shown in Table S1. There is no significant effect on the mechanical property of PbSe with introducing \(\mathrm{AgInSe}_2\) because the bulk modulus has no obvious change.  
+
+<|ref|>text<|/ref|><|det|>[[148, 325, 850, 362]]<|/det|>
+Revision: The deduced Grüneisen parameters \((\gamma)\) and bulk modulus \((K)\) of LISS have no obvious change (Table S1).  
+
+<|ref|>text<|/ref|><|det|>[[147, 380, 850, 542]]<|/det|>
+Supplementary Table 1. Various parameters (longitudinal sound velocity \((v_{1})\) , transverse sound velocity \((v_{t})\) , average sound velocity \((v_{\mathrm{avg}})\) , Poisson ration \((v_{\mathrm{p}})\) , Grüneisen parameter \((\gamma)\) , and bulk modulus \((K)\) ) of \(\mathrm{Pb_{0.98}Na_{0.02}Se - x\% AgInSe_2}\) . The Poisson ration \((v_{\mathrm{p}})\) is calculated by \(v_{\mathrm{p}} = \frac{1 - 2(v_{t} / v_{t})^{2}}{2 - 2(v_{t} / v_{t})^{2}}\) , the Grüneisen parameter \((\gamma)\) is obtained using \(\gamma = \frac{3}{2}\left(\frac{1 + v_{p}}{2 - 3v_{p}}\right)\) and the bulk modulus \((K)\) is given by \(K = \rho \left(v_{t}^{2} - \frac{4}{3} v_{t}^{2}\right)\) \((\rho\) is the density of sample).  
+
+<|ref|>table<|/ref|><|det|>[[137, 564, 857, 737]]<|/det|>
+
+<table><tr><td>Sample</td><td>v1 (m/s)</td><td>v1 (m/s)</td><td>vavg (m/s)</td><td>vp</td><td>γ</td><td>K (GPa)</td></tr><tr><td>x=0</td><td>3165.6</td><td>1708.6</td><td>1907.1</td><td>0.294</td><td>1.74</td><td>48.9</td></tr><tr><td>x=0.5</td><td>3192.9</td><td>1726.9</td><td>1927.3</td><td>0.293</td><td>1.73</td><td>50.2</td></tr><tr><td>x=1</td><td>3214.7</td><td>1726.1</td><td>1927.4</td><td>0.297</td><td>1.75</td><td>51.2</td></tr><tr><td>x=1.5</td><td>3151.7</td><td>1715.4</td><td>1913.5</td><td>0.289</td><td>1.71</td><td>48.0</td></tr><tr><td>x=2</td><td>3217.4</td><td>1720.9</td><td>1922.1</td><td>0.299</td><td>1.77</td><td>50.9</td></tr><tr><td>x=2.05</td><td>3148.7</td><td>1718.3</td><td>1916.4</td><td>0.288</td><td>1.70</td><td>47.6</td></tr><tr><td>x=2.1</td><td>3179.8</td><td>1720.3</td><td>1919.9</td><td>0.293</td><td>1.73</td><td>48.3</td></tr><tr><td>x=2.15</td><td>3149.7</td><td>1718.8</td><td>1916.9</td><td>0.288</td><td>1.70</td><td>47.4</td></tr></table>  
+
+<|ref|>text<|/ref|><|det|>[[148, 754, 850, 790]]<|/det|>
+Comment 4: The Lorenz number was calculated assuming acoustic- phonon scattering dominates. How to prove this?  
+
+<|ref|>text<|/ref|><|det|>[[148, 809, 850, 900]]<|/det|>
+Response: Thanks for your comments. The carrier scattering mechanism can be revealed in the log \((\mu_{\mathrm{H}})\) - log (T) relation. A log \((\mu_{\mathrm{H}})\) - log (T) plot displays \(\mathrm{T}^{- 3 / 2}\) behavior for \(x = 2.05\) sample when \(\mathrm{T} < 600 \mathrm{K}\) (see below Figure), demonstrating that acoustic- phonon scattering dominates. The deviation from \(\mathrm{T}^{- 3 / 2}\) relation at high temperature is due to the increase of effective mass.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[148, 103, 850, 157]]<|/det|>
+Revision: The \(\kappa_{\mathrm{e}}\) was calculated by the Wiedemann- Franz relation, \(\kappa_{\mathrm{e}} = L\sigma T\) , where \(L\) (Figure S7a) is estimated by SPB model assuming acoustic phonon scattering dominates (Figure S6c).  
+
+<|ref|>image<|/ref|><|det|>[[150, 160, 847, 303]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[147, 308, 850, 417]]<|/det|>
+<center>Supplementary Figure 6. (a) Temperature dependence of Hall coefficient \((R_{\mathrm{H}})\) of \(\mathrm{Pb_{0.98}Na_{0.02}Se - 2.05\% AgInSe_2}\) . (b) Effective mass as a function of temperature for \(\mathrm{Pb_{0.98}Na_{0.02}Se - 2.05\% AgInSe_2}\) and \(\mathrm{Pb_{1 - x}Na_xSe}\) . (c) Temperature-dependent Hall mobility of \(\mathrm{Pb_{0.98}Na_{0.02}Se - 2.05\% AgInSe_2}\) , which displays a \(\mathrm{T}^{-3 / 2}\) behavior when \(\mathrm{T}< 600 \mathrm{K}\) , demonstrating that acoustic-phonon scattering dominates. The deviation from \(\mathrm{T}^{-3 / 2}\) relation at high temperature is due to the increase of effective mass. </center>  
+
+<|ref|>text<|/ref|><|det|>[[148, 436, 844, 454]]<|/det|>
+Comment 5: What is the uncertainty for the thermoelectric properties measurements?  
+
+<|ref|>text<|/ref|><|det|>[[147, 473, 851, 620]]<|/det|>
+Response: Thanks for your comments. The standard samples were measured and these measurement data are compared with the reference value. The uncertainties of thermoelectric parameters are given below. In Figure 1 and 2, the measurements of thermal diffusivity \((D)\) , electrical resistivity \((\rho)\) and Seebeck coefficient \((S)\) are well consistent with their reference values. The error of thermal diffusivity is less than \(2\%\) (Figure 1). The uncertainties of electrical resistivity \((\rho)\) and Seebeck coefficient \((S)\) are less than \(2\%\) and \(5\%\) , respectively (Figure 2). The combined uncertainty of all measurements for determining the \(ZT\) is less than \(20\%\) .  
+
+<|ref|>image<|/ref|><|det|>[[228, 623, 763, 785]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[147, 789, 849, 825]]<|/det|>
+<center>Figure 1. (a) Comparison of thermal diffusivity \((D)\) between measurement and reference for standard sample (Inconel). (b) The uncertainty of \(D\) . </center>
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[227, 85, 768, 413]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[147, 418, 850, 474]]<|/det|>
+<center>Figure 2. Comparison of (a) electrical resistivity \((\rho)\) and (c) Seebeck coefficient \((S)\) between measurement and standard sample (Constantan), respectively. The corresponding uncertainties are shown in (b) and (d). </center>
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[149, 85, 462, 101]]<|/det|>
+## Reviewer #2 (Remarks to the Author):  
+
+<|ref|>sub_title<|/ref|><|det|>[[149, 105, 310, 120]]<|/det|>
+## General comment:  
+
+<|ref|>text<|/ref|><|det|>[[148, 123, 851, 418]]<|/det|>
+In this manuscript, the authors investigated the thermoelectric properties of \(\mathrm{Pb_{0.98}Na_{0.02}Se - x\%AgInSe_2}\) (LISS). An exceptional figure- of- merit ZT of \(\sim 2.1\) at \(873\mathrm{K}\) was achieved. They performed a systematic study on the electrical/thermal transport properties and microstructures for this system. The introduction of \(\mathrm{AgInSe_2}\) enlarges the band gap, suppressing the bipolar effect. It is very important to achieve high thermoelectric performance at high temperature regime, but has been challenging to obtain. Interestingly, the incorporation of \(\mathrm{AgInSe_2}\) facilitates the convergence of multiple valence bands, resulting in high weighted mobility and large power factor. The presence of nano- scale \(\mathrm{AgInSe_2}\) precipitates and dislocations results in strong phonon scattering. As a result, a combination of band convergence and strong phonon scattering gives record- high thermoelectric performance in \(\mathrm{PbSe}\) . The local structure analysis by XAFS is very interesting, providing a microscopic perspective to understand the role of doped elements. This is a very solid work and it is suitable for the scope of Nature Communications. This work shows that \(\mathrm{PbSe}\) thermoelectrics can compete with the much expensive \(\mathrm{PbTe}\) . It is highly recommended to be published in Nature Communications after addressing minor points as given below:  
+
+<|ref|>text<|/ref|><|det|>[[149, 437, 848, 472]]<|/det|>
+Response: We thank the reviewer 2 for his/her positive comments and valuable suggestions, which is a publishable justification for our submission.  
+
+<|ref|>text<|/ref|><|det|>[[149, 493, 850, 547]]<|/det|>
+Comment 1: In the "Introduction" section, please cite related works when you mentioned the quality factor B (The lattice thermal conductivity is another important parameter for the thermoelectric performance indicated by the quality factor B).  
+
+<|ref|>text<|/ref|><|det|>[[149, 567, 848, 602]]<|/det|>
+Response: Thanks for your comments. References about quality factor B are cited in the sentence.  
+
+<|ref|>text<|/ref|><|det|>[[149, 622, 850, 658]]<|/det|>
+Revision: The lattice thermal conductivity is another important parameter for the thermoelectric performance indicated by the quality factor \(B\) ( \(B \propto \mu \nu / \kappa_{\mathrm{L}}\) ) \(^{30,31}\) .  
+
+<|ref|>text<|/ref|><|det|>[[149, 678, 850, 732]]<|/det|>
+30. Kang, S. D., Snyder G. J. Transport property analysis method for thermoelectric materials material: quality factor and the effective mass model. arXiv:1710.06896 [cond-mat.mtrl-sci] (2017).  
+
+<|ref|>text<|/ref|><|det|>[[149, 734, 848, 769]]<|/det|>
+31. Tan, G., Zhao, L.-D. & Kanatzidis, M. G. Rationally Designing High-Performance Bulk Thermoelectric Materials. Chem. Rev. 116, 12123-12149 (2016).  
+
+<|ref|>text<|/ref|><|det|>[[149, 789, 850, 861]]<|/det|>
+Comment 2: It is mentioned that "the tetragonal \(\mathrm{AgInSe_2}\) is perfectly inserted to the \(\mathrm{PbSe}\) matrix as nano- scale precipitates revealed by the transmission electron microscopy". This sentence can make readers confused because a part of Ag and In atoms occupies Pb sites.  
+
+<|ref|>text<|/ref|><|det|>[[149, 882, 761, 898]]<|/det|>
+Response: We appreciate your comments. This sentence has been modified.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[149, 103, 849, 140]]<|/det|>
+Revision: The sentence mentioned above is changed to "Nano- scale AgInSe₂ precipitates are revealed by the transmission electron microscopy (TEM)."  
+
+<|ref|>text<|/ref|><|det|>[[149, 158, 850, 213]]<|/det|>
+Comment 3: The effective masses increase with the introduction of AgInSe₂ as indicated by the Pisarenko plot. Please show the effective masses for each sample, which is more straightforward for the readers.  
+
+<|ref|>text<|/ref|><|det|>[[149, 232, 850, 269]]<|/det|>
+Response: Thanks for your good suggestions. The effective masses as a function of AgInSe₂ content are given in our manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[148, 287, 848, 325]]<|/det|>
+Revision: the effective mass \((m^{*})\) of LISS is largely increased from \(0.44 m_{e}\) to 0.81 \(m_{e}\) with the introduction of AgInSe₂ (Figure 4a, Figure S1b).  
+
+<|ref|>image<|/ref|><|det|>[[149, 330, 855, 539]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[148, 548, 850, 602]]<|/det|>
+Supplementary Figure 1. (a) Hall carrier concentrations and (b) density- of- states effective mass of \(\mathrm{Pb_{0.98}Na_{0.02}Se}\) - \(x\%\) AgInSe₂ (LISS) with increasing AgInSe₂ content at 303K.  
+
+<|ref|>text<|/ref|><|det|>[[148, 620, 850, 657]]<|/det|>
+Comment 4: The measurement of sound velocities is not mentioned in the experimental section.  
+
+<|ref|>text<|/ref|><|det|>[[148, 676, 850, 712]]<|/det|>
+Response: Thanks for your comments. The measurements of sound velocity have added in the experimental section.  
+
+<|ref|>text<|/ref|><|det|>[[148, 731, 850, 768]]<|/det|>
+Revision: Pulse- echo method was used to measure the speed of sound and the waveforms were recorded using a Tektronix TBS 1102 oscilloscope.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[148, 84, 850, 158]]<|/det|>
+Comment 5: The heat capacity \(\mathrm{(C_p)}\) calculated by the Dulong- Petit law will underestimate the thermal conductivity at high temperature. Estimating the \(\mathrm{C_p}\) by the empirical equation is more accurate. It is not necessary to use the \(\mathrm{C_p}\) value estimated by the Dulong- Petit law.  
+
+<|ref|>text<|/ref|><|det|>[[148, 177, 849, 231]]<|/det|>
+Response: We appreciate your good suggestions. We estimated the heat capacity using the empirical equation for all samples. The total thermal conductivity \((\kappa_{\mathrm{tot}})\) , lattice thermal conductivity \((\kappa_{\mathrm{L}})\) and the figure- of- merit \(ZT\) have been recalculated.  
+
+<|ref|>text<|/ref|><|det|>[[148, 250, 850, 305]]<|/det|>
+Revision: we recalculated the total thermal conductivity \((\kappa_{\mathrm{tot}})\) , lattice thermal conductivity \((\kappa_{\mathrm{L}})\) and the dimensionless figure- of- merit \(ZT\) . Accordingly, Figure 1, Figure 6 and Figure S8 have been revised.  
+
+<|ref|>image<|/ref|><|det|>[[175, 325, 822, 560]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[147, 567, 850, 657]]<|/det|>
+<center>Fig. 1 Multiple valence bands enable high \(ZT\) values in p-type PbSe. a Schmatic diagram of multi-bands \((\mathrm{L}, \Sigma , \Lambda)\) involvement in transport. The Brillouin zone shows that the degeneracies at the \(\mathrm{L}, \Sigma\) , and \(\Lambda\) points are 4, 12, and 8, respectively. b The activated third band \(\Lambda\) enables higher \(ZT\) values compared with the single-band and two-band PbSe-based materials. </center>
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[175, 92, 816, 476]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[147, 491, 850, 621]]<|/det|>
+<center>Fig. 6 Thermal transport properties and dimensionless figure-of-merit \(ZT\) as a function of temperature for \(\mathrm{Pb_{0.98}Na_{0.02}Se - x\% AgInSe_2}\) (LISS) compounds. a Total thermal conductivity. b Lattice thermal conductivity. Inset shows the room-temperature lattice thermal conductivities departure from the theoretical line calculated by the Callaway model. c The average sound velocity \((v_{\mathrm{avg}})\) versus lattice thermal conductivity \((\kappa_{\mathrm{L}})\) for LISS compounds at room temperature. d Temperature-dependent \(ZT\) for LISS samples. </center>
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[150, 88, 838, 488]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[147, 511, 850, 566]]<|/det|>
+<center>Supplementary Figure 8. Temperature-dependent (a) electrical conductivity, (b) Seebeck coefficient, (c) total thermal conductivity, and (d) dimensionless figure-of-merit \(ZT\) for several \(x = 2.05\) and \(x = 2.1\) samples, respectively. </center>
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[149, 85, 463, 101]]<|/det|>
+## Reviewer #3 (Remarks to the Author):  
+
+<|ref|>sub_title<|/ref|><|det|>[[149, 105, 310, 120]]<|/det|>
+## General comment:  
+
+<|ref|>text<|/ref|><|det|>[[148, 123, 850, 288]]<|/det|>
+The present manuscript provides interesting data describing the thermoelectric properties of PbSe doped with AgInSe2. This doping / alloying leads to a reduction in the thermal conductivity and an improvement of band convergence enabling a zT value slightly above 2. Thermoelectric materials are considered a viable option to improve energy conversion since they convert waste heat into electrical energy or enable efficient cooling. Hence identifying promising thermoelectric materials is a timely topic, which could be suitable for Nature Communications. Nevertheless, there are several reasons why the present manuscript does not meet my expectations for a manuscript to be published in Nature Communications.  
+
+<|ref|>text<|/ref|><|det|>[[149, 308, 848, 343]]<|/det|>
+Response: We appreciate your valuable comments and suggestions, which will strengthen our work. Hopefully, our revised manuscript could meet your expectations.  
+
+<|ref|>text<|/ref|><|det|>[[148, 363, 850, 583]]<|/det|>
+Comment 1: One of the main claims is that there is multi- band convergence. Yet, the nature of these band and how they could possibly be described and explained is missing. One of the main claims of the present manuscript is the idea that more than 3 bands can contribute, yet the proof for this claim and the explanation of the nature of these bands appears rather incomplete. I would like to see very strong and convincing evidence that indeed more than two bands contribute and what their nature is. To mention one option to provide such evidence: several groups have recently employed tight- binding methods to explain the band structure of related chalcogenides [1,2]. Such calculations could be performed to explore the potential nature and more importantly origin of the bands involved. I am not aware of any study that has claimed and proven so far that three bands can provide a contribution to the thermoelectric performance of PbSe.  
+
+<|ref|>text<|/ref|><|det|>[[148, 586, 588, 621]]<|/det|>
+[1] Chemistry of Materials 32 (22), 9771- 9779 (2020)  [2] Advanced Materials 30, 1801787 (2018)  
+
+<|ref|>text<|/ref|><|det|>[[148, 641, 850, 731]]<|/det|>
+Response: Thanks for your insightful comments. Our DFT calculations reveal that a third valence band \(\Lambda\) along \(\Gamma\) - L is activated. The large weighted mobility and effective mass also reflect the multi- band convergence indirectly. A comparison of the density- of- states effective mass for various p- type PbSe- based materials are shown in the Table 1 below.  
+
+<|ref|>text<|/ref|><|det|>[[148, 752, 850, 899]]<|/det|>
+Indeed, the tight- binding methods are powerful tool to understand the nature of electronic band structure. However, it is hard to employ tight- binding calculations for Ag- In co- doped PbSe since the supercells contain too many atoms. Instead, we calculated the atomic orbital projected band structure by DFT to understand their nature. The conduction and valence bands of PbSe system are dominated by the Pb- p and Se- p states, respectively, which is in line with the tight- binding calculations for PbTe [Brod, M. K., et al. Chem. Mater. 32, 9771- 9779 (2020)]. Owing to the rock- salt structure of PbSe, these p- bands are half- filled forming a \(\sigma\) - bond, which is
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[148, 85, 850, 177]]<|/det|>
+characteristic of metavalent bonding [Wuttig, M., et al. Adv. Mater. 30, e1803777 (2018)]. Similar to PbTe, the valence band maximum (L band) of PbSe is contributed by the p- states. The projected electronic band structure also implies that the third valence band \(\Lambda\) show a large contribution by the Ag 4d state and Se 4p state. This may explain the promoted band convergence in PbSe by alloying with AgInSe₂.  
+
+<|ref|>text<|/ref|><|det|>[[148, 196, 850, 306]]<|/det|>
+A similar electronic band structure can also be found in Ag- Sr co- doped PbSe system [Luo, Z. Z. et al. Angew. Chem. 2021, 133, 272 - 277]. As shown in Figure 2 below, the valence band 2 (Σ) and valence band 3 (Λ) are almost at the same energy level. The energy offset between valence band 1 (L) and the other two valence bands (Σ and Λ) is \(\sim 0.17 \mathrm{eV}\) . They found a strong band convergence behavior in this system. However, they didn't mention the underlying multi- band convergence behavior.  
+
+<|ref|>table<|/ref|><|det|>[[139, 377, 840, 586]]<|/det|>
+<|ref|>table_caption<|/ref|><|det|>[[147, 326, 848, 362]]<|/det|>
+Table 1. Density-of-states effective masses \((m^{*})\) for various p-type PbSe-based materials.   
+
+<table><tr><td>sample</td><td>m*(me) at 300K</td><td>m*(me) at 773K</td><td>reference</td></tr><tr><td>PbSe-Na-Ag-In</td><td>0.81</td><td>2.16</td><td>This work</td></tr><tr><td>PbSe-Na</td><td>0.28</td><td>0.7</td><td>1</td></tr><tr><td>PbSe-Ag-Sr</td><td>0.4</td><td>1.1</td><td>2</td></tr><tr><td>PbSe-Ag-Ba</td><td>0.4</td><td>1.0</td><td>2</td></tr><tr><td>PbSe-Na-Hg</td><td>0.45</td><td>1.3</td><td>3</td></tr><tr><td>PbSe-Cd-Na-Te</td><td>0.57</td><td></td><td>4</td></tr><tr><td>PbSe-Ag</td><td>0.35</td><td></td><td>5</td></tr><tr><td>PbSe-Na-Ca</td><td>0.56</td><td></td><td>6</td></tr><tr><td>PbSe-Na-Ba</td><td>0.56</td><td></td><td>6</td></tr><tr><td>PbSe-Na-Sr</td><td>0.48</td><td></td><td>6</td></tr></table>  
+
+<|ref|>text<|/ref|><|det|>[[147, 604, 850, 901]]<|/det|>
+1 Wang, H., Pei, Y., LaLonde, A. D. & Snyder, G. J. Heavily doped p- type PbSe with high thermoelectric performance: an alternative for PbTe. Adv. Mater. 23, 1366- 1370 (2011). 2 Luo, Z. Z. et al. Strong Valence Band Convergence to Enhance Thermoelectric Performance in PbSe with Two Chemically Independent Controls. Angew. Chem. Int. Ed. 60, 268- 273 (2021). 3 Hodges, J. M. et al. Chemical Insights into PbSe- x%HgSe: High Power Factor and Improved Thermoelectric Performance by Alloying with Discordant Atoms. J. Am. Chem. Soc. 140, 18115- 18123 (2018). 4 Tan, G., Zhao, L.- D. & Kanatzidis, M. G. Rationally Designing High- Performance Bulk Thermoelectric Materials. Chem. Rev. 116, 12123- 12149 (2016). 5 Wang, S. et al. Exploring the doping effects of Ag in p- type PbSe compounds with enhanced thermoelectric performance. J. Phys. D: Appl. Phys. 44, 475304 (2011). 6 Lee, Y. et al. High- performance tellurium- free thermoelectrics: all- scale
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[147, 85, 848, 121]]<|/det|>
+hierarchical structuring of p-type PbSe- MSe systems (M = Ca, Sr, Ba). J. Am. Chem. Soc. 135, 5152- 5160 (2013).  
+
+<|ref|>image<|/ref|><|det|>[[290, 150, 707, 465]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[147, 475, 849, 530]]<|/det|>
+<center>Figure 2 in reference [Luo, Z. Z. et al. Angew. Chem. 2021, 133, 272 - 277]. Electronic band structures and density-of-states (DOS) for Ag-doped PbSe (a, b), Ag-doped and SrSe-alloyed PbSe (c, d), and Ag-doped and BaSe-alloyed PbSe (e, f). </center>  
+
+<|ref|>text<|/ref|><|det|>[[147, 548, 851, 900]]<|/det|>
+Revision: The electronic band structures of Ag and In doped PbSe were calculated (Figure S2a, S2b) to understand their role in band manipulation. The Ag- doping and In- doping reflect p- type and n- type doping effect, respectively, which are consistent with previous experimental results<sup>42,43</sup>. Additionally, In- doping has a more important effect on decreasing energy offset ( \(\Delta E_{1 - 2}\) ) compared with the Ag- doping (Figure S2c), while Ag- doping plays a major role in enlarging the bandgap (Figure S2d). The orbital projected band structures reveal that the interaction between Pb- p and Se- p orbitals dominate the band structure (Figure S3a, S3b), which is consistent with previous study<sup>44</sup>. This is a typical feature of the metal- valent bonding system<sup>13,45,46</sup>. The tight binding calculations reveal that the cation states have important effect on the shape of valence band although their orbital projections are not obvious<sup>44</sup>. Indeed, the Ag- d orbitals play an important role in modulating the third valence band \(\Lambda\) along \(\Gamma\) - L (Figure S3c). A similar phenomenon was also observed in Ag- Sr co- dope PbSe system<sup>47</sup>. In addition, the cation- site doping can also contribute to the conduction band (Figure S3d) depending on the nature of cation states. Our results indicate that Ag- In co- doping enable multiple valence band convergence, verifying that the cation- site doping is an effective way to modulate the valence band in PbSe. Similar effects can be expected in other materials, such as PbTe and GeTe, by employing the same chemical bonding mechanism as PbSe.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[145, 102, 850, 156]]<|/det|>
+13. Wuttig, M., Deringer, V. L., Gonze, X., Bichara, C. & Raty, J. Y. Incipient metals: functional materials with a unique bonding mechanism. Adv. Mater. 30, e1803777 (2018).  
+
+<|ref|>text<|/ref|><|det|>[[147, 158, 850, 212]]<|/det|>
+42. Wang, S. et al. Exploring the doping effects of Ag in p-type PbSe compounds with enhanced thermoelectric performance. J. Phys. D: Appl. Phys. 44, 475304 (2011).  
+
+<|ref|>text<|/ref|><|det|>[[147, 214, 849, 268]]<|/det|>
+43. Androulakis, J., Lee, Y., Todorov, I., Chung, D.-Y. & Kanatzidis, M. High-temperature thermoelectric properties of n-type PbSe doped with Ga, In, and Pb. Phys. Rev. B 83 (2011).  
+
+<|ref|>text<|/ref|><|det|>[[147, 270, 849, 306]]<|/det|>
+44. Brod, M. K., Toriyama, M. Y. & Snyder, G. J. Orbital chemistry that leads to high valley degeneracy in PbTe. Chem. Mater. 32, 9771-9779 (2020).  
+
+<|ref|>text<|/ref|><|det|>[[147, 307, 850, 361]]<|/det|>
+45. Maier, S. et al. Discovering electron-transfer-driven changes in chemical bonding in lead chalcogenides (PbX, where X = Te, Se, S, O). Adv. Mater. 32, e2005533 (2020).  
+
+<|ref|>text<|/ref|><|det|>[[147, 363, 849, 399]]<|/det|>
+46. Raty, J. Y. et al. A quantum-mechanical map for bonding and properties in solids. Adv. Mater. 31, e1806280 (2019).  
+
+<|ref|>text<|/ref|><|det|>[[147, 400, 850, 454]]<|/det|>
+47. Luo, Z. Z. et al. Strong valence band convergence to enhance thermoelectric performance in PbSe with two chemically independent controls. Angew. Chem. Int. Ed. 60, 268-273 (2021).  
+
+<|ref|>image<|/ref|><|det|>[[185, 458, 808, 800]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[147, 808, 850, 863]]<|/det|>
+<center>Supplementary Figure 2. Electronic band structures of (a) \(\mathrm{Pb_{26}AgSe_{27}}\) and (b) \(\mathrm{Pb_{26}InSe_{27}}\) . (c) The energy offset \((\Delta E_{1:2})\) between L and \(\Sigma\) valence band. (d) Theoretical bandgaps \((E_{\mathrm{g}})\) for pristine, Ag-doped, In-doped and Ag-In co-doped PbSe. </center>
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[174, 88, 820, 450]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[147, 455, 851, 567]]<|/det|>
+<center>Supplementary Figure 3. The atomic orbital projected band structure of \(\mathrm{Pb_{25}AgInSe_{27}}\) . (a) The conduction band is dominated by Pb-p orbitals, while the valence band contain considerable Pb-s character. (b) The Se-p orbital primarily contributes to the valence band. (c) The Ag-d orbitals have a considerable contribution to the valence band. (d) There is distinct In-s character at the conduction band. </center>
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[148, 85, 850, 158]]<|/det|>
+Comment 2: Then, I am also concerned about the apparent disagreement between theory and experiment. The authors claim that their experiment shows an increase of band gap upon alloying with AgInSe₂. Yet, the DFT calculations presented in fig. 5b do not seem to support this conclusion.  
+
+<|ref|>text<|/ref|><|det|>[[148, 177, 850, 288]]<|/det|>
+Response: We are sorry for this confusion. It is not obvious to distinguish the bandgap from the electronic density- of- states (Figure 5b). Actually, our calculation is consistent with the experimental result that the bandgap increases with introducing AgInSe₂ in PbSe matrix (Figure 5a). The theoretical bandgap for pure PbSe, Ag- doped PbSe, In- doped PbSe and Ag- In co- doped PbSe are compared in Figure S2d shown below. A possible reason for this phenomenon is given in our manuscript.  
+
+<|ref|>image<|/ref|><|det|>[[150, 309, 843, 523]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[147, 530, 850, 604]]<|/det|>
+<center>Fig. 5 Electronic band structure. a Electronic band structure of Pb27Se27 (black) and Pb25AgInSe27 (red). b Electronic density of states (DOS) near the Fermi level for Pb27Se27 (black), Pb26AgSe27 (green), Pb26InSe27 (blue) and Pb25AgInSe27 (red), respectively. </center>
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[200, 85, 792, 411]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[147, 418, 850, 475]]<|/det|>
+<center>Supplementary Figure 2. Electronic band structures of (a) \(\mathrm{Pb_{26}AgSe_{27}}\) and (b) \(\mathrm{Pb_{26}InSe_{27}}\) . (c) The energy offset \((\Delta E_{1:2})\) between L and \(\Sigma\) valence band. (d) Theoretical bandgaps \((E_{\mathrm{g}})\) for pristine, Ag-doped, In-doped and Ag-In co-doped PbSe. </center>  
+
+<|ref|>text<|/ref|><|det|>[[147, 492, 851, 715]]<|/det|>
+Revision: The experimental bandgap is \(\sim 0.24 \mathrm{eV}\) for the pristine PbSe, while the bandgap increases obviously with the incorporation of \(\mathrm{AgInSe_2}\) and a large bandgap \(\sim 0.33 \mathrm{eV}\) is achieved for the PbSe - \(2\%\) \(\mathrm{AgInSe_2}\) sample (Figure 2d). The small bandgap of PbSe results from its unconventional chemical bonding mechanism (metavalent bonding). For a perfect half- filled p- band, the energy band structures resemble a metallic system. Yet, the bandgap opens due to a small Peierls distortion or charge transfer<sup>41</sup>. It is the charge transfer between Pb and Se that opens a small bandgap in PbSe given its perfect octahedral arrangements. DFT results show that the enlarged bandgap is mainly attributed to the incorporation of Ag. The eletronegativity difference between Ag and Te \((\sim 0.62)\) is larger than that between Pb and Te \((\sim 0.22)\) . Therefore, the substitution of Ag at Pb sites will strengthen the charge transfer between cation and anion, leading to an enlarged bandgap.  
+
+<|ref|>text<|/ref|><|det|>[[148, 732, 849, 788]]<|/det|>
+41. Yu, Y., Cagnoni, M., Cojocaru- Miré din, O. & Wuttig, M. Chalcogenide Thermoelectrics Empowered by an Unconventional Bonding Mechanism. Adv. Funct. Mater. 30, 1904862 (2019).
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[148, 85, 850, 196]]<|/det|>
+Comment 3: Finally, in recent years the thermoelectric properties in lead chalcogenides have been discussed in terms of the underlying bonding mechanism, which must be related to the corresponding band structure [1,3]. A discussion of the fundamental bonding mechanism relevant here is missing. Such a discussion is important since it can help to predict and explain which materials and changes of bonding can improve the performance of a given thermoelectric material.  
+
+<|ref|>text<|/ref|><|det|>[[148, 197, 588, 233]]<|/det|>
+[1] Chemistry of Materials 32 (22), 9771- 9779 (2020)  [3] Advanced Materials 32, 202005533 (2020)  
+
+<|ref|>text<|/ref|><|det|>[[149, 252, 850, 306]]<|/det|>
+Response: Thanks for your valuable suggestions. We have made discussions to understand the nature of electronic band structure and the bandgap behavior in chemical bonding perspective.  
+
+<|ref|>sub_title<|/ref|><|det|>[[149, 327, 230, 342]]<|/det|>
+## Revision:  
+
+<|ref|>text<|/ref|><|det|>[[149, 344, 849, 417]]<|/det|>
+Introduction section: Materials with disordered or complex crystal structure \(^{9,10}\) , giant anharmonicity \(^{11,12}\) , metavalent bonding \(^{13}\) , and lone pair electrons \(^{14}\) often exhibit intrinsic low lattice thermal conductivity, which are promising candidates for thermoelectric applications.  
+
+<|ref|>text<|/ref|><|det|>[[148, 436, 850, 603]]<|/det|>
+Bandgap: The small bandgap of PbSe results from its unconventional chemical bonding mechanism (metavalent bonding). For a perfect half- filled p- band, the energy band structures resemble a metallic system. Yet, the bandgap opens due to a small Peierls distortion or charge transfer \(^{41}\) . It is the charge transfer between Pb and Se that opens a small bandgap in PbSe given its perfect octahedral arrangements. DFT results show that the enlarged bandgap is mainly attributed to the incorporation of Ag. The eletronegativity difference between Ag and Te ( \(\sim 0.62\) ) is larger than that between Pb and Te ( \(\sim 0.22\) ). Therefore, the substitution of Ag at Pb sites will strengthen the charge transfer between cation and anion, leading to an enlarged bandgap.  
+
+<|ref|>text<|/ref|><|det|>[[148, 622, 850, 825]]<|/det|>
+Electronic band structure: We calculated the orbital projected band structures to understand the nature of electronic band structure in chemical bonding perspective. The orbital projected band structures reveal that the interaction between Pb- p and Te- p orbitals dominate the band structure (Figure S3a, S3b), which is consistent with previous study \(^{46}\) . This is a typical feature of the metavalent bonding system \(^{13,45,46}\) . The Ag- d orbitals play an important role in modulating the third valence band \(\Lambda\) along \(\Gamma\) - L (Figure S3c). Our results indicate that Ag- In co- doping enable multiple valence band convergence, verifying that the cation- site doping is an effective way to modulate the valence band in PbSe. Similar effects can be expected in other materials, such as PbTe and GeTe, by employing the same chemical bonding mechanism as PbSe. A more detailed discussion is shown above when we answering the comment 1.  
+
+<|ref|>text<|/ref|><|det|>[[149, 845, 849, 899]]<|/det|>
+Temperature- dependent bandgap: Clearly, the bandgap increases with rising temperature, which is also verified experimentally (Figure 5d). As revealed by Brod et al. \(^{44}\) , there is sufficient interaction between Pb- p and Te- p (Se- p in our case) to
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[148, 84, 850, 178]]<|/det|>
+provide the molecular orbitals with the proper s- type symmetry to place the VBM at L point. The weak s- p hybridization is a small addition to this effect. The thermal expansion will lead to a reduction of orbital overlap between p- orbitals \(^{41}\) as well as a weakened s- p hybridization \(^{48}\) . As a result, the energy of VBM (L point) in the electronic band structure decreases \(^{49}\) , resulting in an enlarged bandgap \((E_{\mathrm{g}})\) .  
+
+<|ref|>text<|/ref|><|det|>[[147, 195, 850, 530]]<|/det|>
+13. Wuttig, M., Deringer, V. L., Gonze, X., Bichara, C. & Raty, J. Y. Incipient metals: functional materials with a unique bonding mechanism. Adv. Mater. 30, e1803777 (2018).  
+41. Yu, Y., Cagnoni, M., Cojocaru-Miré din, O. & Wuttig, M. Chalcogenide Thermoelectrics Empowered by an Unconventional Bonding Mechanism. Adv. Funct. Mater. 30, 1904862 (2019).  
+44. Brod, M. K., Toriyama, M. Y. & Snyder, G. J. Orbital chemistry that leads to high valley degeneracy in PbTe. Chem. Mater. 32, 9771-9779 (2020).  
+45. Maier, S. et al. Discovering electron-transfer-driven changes in chemical bonding in lead chalcogenides (PbX, where X = Te, Se, S, O). Adv. Mater. 32, e2005533 (2020).  
+46. Raty, J. Y. et al. A quantum-mechanical map for bonding and properties in solids. Adv. Mater. 31, e1806280 (2019).  
+48. Zeier, W. G. et al. Thinking like a chemist: intuition in thermoelectric materials. Angew. Chem. Int. Ed. 55, 6826-6841 (2016).  
+49. Cagnoni, M., Fuhren, D. & Wuttig, M. Thermoelectric performance of IV-VI compounds with octahedral-like coordination: a chemical-bonding perspective. Adv. Mater., e1801787 (2018).
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[119, 83, 328, 98]]<|/det|>
+## REVIEWERS' COMMENTS  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 112, 420, 128]]<|/det|>
+## Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 142, 853, 173]]<|/det|>
+The authors have answered all the raised questions from these three reviewers. I am satisfied with the response. So, I suggest to accept this paper to publish in Nature Communications.  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 201, 420, 216]]<|/det|>
+## Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[118, 231, 864, 290]]<|/det|>
+The manuscript has been properly revised to address all the comments by the reviewers. Now it can be published as it is. This work is a milestone for PbSe thermoelectrics, which possibly can outperform PbTe thermoelectrics in the near future.  
+
+<|ref|>sub_title<|/ref|><|det|>[[119, 318, 420, 333]]<|/det|>
+## Reviewer #3 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[119, 348, 820, 393]]<|/det|>
+In the response to the questions and comments from the different reviewers, the authors have addressed all questions and concerns adequately. Hence, the manuscript is acceptable in its present form.
+
+<--- Page Split --->

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+[
+  {
+    "type": "image",
+    "img_path": "images/Supplementary_Figure_4.jpg",
+    "caption": "Supplementary Fig. 4 The relationship between length and resistance of PAni fibres with increasing lengths from 1 cm to 10 cm.",
+    "footnote": [],
+    "bbox": [],
+    "page_idx": 0
+  }
+]

peer_reviews/supplementary_0_Peer Review File__01cfd239b63d0b5b3bd8fba2f46c4523116e4ea7471e771fa2f189c14b26428e/supplementary_0_Peer Review File__01cfd239b63d0b5b3bd8fba2f46c4523116e4ea7471e771fa2f189c14b26428e.mmd

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+
+# nature portfolio  
+
+Peer Review File  
+
+Scalable production of ultrafine polyaniline fibres for tactile organic electrochemical transistors  
+
+![](images/Supplementary_Figure_4.jpg)
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+REVIEWER COMMENTS</B>  
+
+Reviewer #1 (Remarks to the Author):  
+
+The research work reported in the paper represents a significant breakthrough in flexible, polymer- based electronic applications, particularly, (1) improved electrical performance, (2) a pathway for high- volume production, and (3) high mechanical performance. The fibers developed could be used for many other applications for flexible, wearable electronics beyond the examples highlighted in the paper. The production approach shows the promise for future commercial applications. The mechanical properties (strength, modulus) of the fibers are in the very high range, demonstrating the potential for many demanding applications. The paper is very well written, with a clear presentation of the state- of- the- art status, motivations, approached, and mechanisms. The conclusions are reasonable and supported by data presented.  
+
+Comment regarding equation for D_AB^O: What is its source? From literature or by data fitting? Variables in equation are defined but units should also be given.  
+
+Minor edits: less one => less than one; larger than of conventional => larger than that of conventional  
+
+Reviewer #2 (Remarks to the Author):  
+
+## Summary:  
+
+The authors report a method to develop PANI fibers using a wet spinning approach with small diameters \(< 5 \text{um}\) , good mechanical strength, and electrical properties amenable for use in e- textile applications. The reported method leveraged predoping with camphor sulfonic acid, and careful solvent choice of the coagulation bath. The authors demonstrate that choice of solvent dramatically impacts the surface morphology of the fibers, with NMP and DMF providing smooth and thin fibers with stability in air for four weeks. The authors then speculate that the use of a "good" solvent prevented quick solidification, and increased interfacial pressure during solvent exchange, and therefore allowed stretching of the fiber to reduce diameter and enhance morphology. The fibers showed good mechanical performance compared to other non- blended conjugated polymer- based fibers, which was attributed to the decrease in diameter and structural defects.  
+
+The fibers were then used to create a microcapacitor, which was characterized by CV and GCD. The capacitance, power density, energy density, and stability, demonstrated reliable electrochemical performance. The charge storage capacity was determined and rivals that of other similar platforms. As
+
+<--- Page Split --->
+
+such the fiber was used to make a solid- state OECT with a silver wire as the gate electrode, the fiber as the channel, and a PVA- H3PO4 gel, all wrapped in a polyurethane protective coating. The OECT was characterized and demonstrated low power consumption. The OECT was then demonstrated to respond to mechanical deformation through the pressure induced change in ion penetration. The favorable mechanical and electronic properties, as well as the scalability of these fibers will be of interest to the scientific community and warrants publication in Nature Communications. Please respond to the following:  
+
+1) What exactly do the authors mean by "good" or "bad" solvent? Solution viscosity? The speed at which the PANI solidifies/coagulates, preventing slenderization? PANI solubility? 2nd virial coefficient (A2)? Diffusivity?  
+
+2) Why do the differences in morphology when using a "good" or "bad" solvent lead to differences in stability?  
+
+3) How reproducible and uniform are the properties of these fibers?  
+
+4) For the fiber based OECT, an insulating PU was used to encapsulate the Ag wire, how does the capacitance of the PU compare with the double layer capacitance near the fiber/solid-state electrolyte interface? How is the gate voltage distributing from the gate to the channel?  
+
+5) Can the authors make a table to compare the basic electrical properties of their OECT with previously reported fiber based OECTs  
+
+## Figures  
+
+- When referring to the 5.4 km of UFPF, the text says Fig. 1e but should refer to Fig. 1g.  
+
+- Clearly indicate that the panel next to Fig. 3e is a zoom in.  
+
+- Add a legend to Fig. 2e to indicate the meaning of color  
+
+- For Fig. 5e, considering the relative position between the Ag wire and PU layer, why will a change in friction alter the position of Ag inside the PU?  
+
+## Methods  
+
+- What molecular weight of PANI powder was used?  
+
+- Was there a particular choice behind using camphor sulfonic acid? Others have used AMPSA, sulfuric acid, HClO4, and NMP.  
+
+- When referring to Raman "de-doping" peaks, explain what bonds you are looking at and give a citation.  
+
+- How did you perform your Rheology?
+
+<--- Page Split --->
+
+## Point by point responses to the reviewer comments:  
+
+## Reviewer 1:  
+
+1. Reviewer's Comment: The research work reported in the paper represents a significant breakthrough in flexible, polymer-based electronic applications, particularly, (1) improved electrical performance, (2) a pathway for high-volume production, and (3) high mechanical performance. The fibers developed could be used for many other applications for flexible, wearable electronics beyond the examples highlighted in the paper. The production approach shows the promise for future commercial applications. The mechanical properties (strength, modulus) of the fibers are in the very high range, demonstrating the potential for many demanding applications. The paper is very well written, with a clear presentation of the state-of-the-art status, motivations, approached, and mechanisms. The conclusions are reasonable and supported by data presented.  
+
+Our response: We thank the reviewer for the positive comments.  
+
+2. Reviewer's Comment regarding equation for \(D_{AB}^0\) : What is its source? From literature or by data fitting? Variables in equation are defined but units should also be given. Our response: We thank the reviewer for kind concern. The equation for \(D_{AB}^0\) is derived from Ref. 25 (Smart Structures and Materials 2001: Electroactive Polymer Actuators and Devices. 4329, 59-71, 2001). We have also added the units of every variable (highlighted in Line 174-177).  
+
+3. Reviewer's Comment: Minor edits: less one => less than one; larger than of conventional => larger than that of conventional.  
+
+Our response: We thank the reviewer for critically reading our manuscript and pointing out our mistakes. We have corrected all the edits (highlighted in Line 34, 81 and 282).  
+
+## Reviewer 2:  
+
+1. Reviewer's Comment: The authors report a method to develop PANI fibers using a wet spinning approach with small diameters (< 5 um), good mechanical strength, and electrical
+
+<--- Page Split --->
+
+properties amenable for use in e- textile applications. The reported method leveraged pre- doping with camphor sulfonic acid, and careful solvent choice of the coagulation bath. The authors demonstrate that choice of solvent dramatically impacts the surface morphology of the fibers, with NMP and DMF providing smooth and thin fibers with stability in air for four weeks. The authors then speculate that the use of a "good" solvent prevented quick solidification, and increased interfacial pressure during solvent exchange, and therefore allowed stretching of the fiber to reduce diameter and enhance morphology. The fibers showed good mechanical performance compared to other non- blended conjugated polymer- based fibers, which was attributed to the decrease in diameter and structural defects.  
+
+The fibers were then used to create a micro capacitor, which was characterized by CV and GCD. The capacitance, power density, energy density, and stability, demonstrated reliable electrochemical performance. The charge storage capacity was determined and rivals that of other similar platforms. As such the fiber was used to make a solid- state OECT with a silver wire as the gate electrode, the fiber as the channel, and a \(\mathrm{PVA - H_3PO_4}\) gel, all wrapped in a polyurethane protective coating. The OECT was characterized and demonstrated low power consumption. The OECT was then demonstrated to respond to mechanical deformation through the pressure induced change in ion penetration. The favorable mechanical and electronic properties, as well as the scalability of these fibers will be of interest to the scientific community and warrants publication in Nature Communications.  
+
+Our response: We thank the reviewer for the positive comments.  
+
+2. Reviewer's Comment: What exactly do the authors mean by "good" or "bad" solvent? Solution viscosity? The speed at which the PANI solidifies/coagulates, preventing slenderization? PANI solubility? 2nd virial coefficient (A2)? Diffusivity? Our response: We thank the reviewer for the important comment. From the perspective of physical interpretation, "good" and "poor" solvents of PANI are determined by the intermolecular interactions between PANi chains and solvent molecules. In a good solvent, interactions between PANi chains solvent molecules are energetically favorable, and will
+
+<--- Page Split --->
+
+cause PAni chains to expand and disperse well. In a poor solvent, PAni- PAni interactions are preferred and cause the PAni chains to disperse poor. Consequently, PAni molecules disperse good in good solvents, and disperse poor in poor solvents. We have added the discussions in the revised manuscript (highlighted in Line 137- 140).  
+
+3. Reviewer's Comment: Why do the differences in morphology when using a "good" or "bad" solvent lead to differences in stability?  
+
+Our response: We thank the reviewer for the crucial questions. The morphology does not directly determine the differences in stability. According to our SEM observations and X- ray diffraction analysis, the fibres produced in good solvents behaved higher degree of orientation and crystallization. The higher degree of crystallization protects the doping bonding in PAni chains from the attack of ambientes at the molecular level. As a result, the fibres produced in good solvents could show better stability in air. To avoid the misleading, we have rearranged the related discussions in the revised manuscript (highlighted in Line 145- 150).  
+
+4. Reviewer's Comment: How reproducible and uniform are the properties of these fibers? Our response: We thank the reviewer for the kind comment. We discuss the uniformity of PAni fibres from the perspective of electrical and mechanical properties. In the case of electrical properties, because we used pre-doped PAni solutions as the raw dopes, the PAni fibres show uniform charge distribution throughout the fibre length. To confirm this point, we added a measurement to record the relationship between length and resistance. As shown in Supplementary Fig. 4, the value of resistance increases linearly with increasing length from 1 cm to 10 cm, demonstrating the favorable uniformity of electrical properties. In the case of mechanical properties, we provided the mechanical tensile tests of 5 fibres from 5 different batches, the results are shown in Response Tab. 1 for the information. The tensile strength and strain are generally stable, demonstrating the reliable uniformity of mechanical properties. We have added the discussions in the revised manuscript (highlighted in Line 111).
+
+<--- Page Split --->
+![PLACEHOLDER_6_0]
+
+<center>Supplementary Fig. 4 The relationship between length and resistance of PAni fibres with increasing lengths from 1 cm to 10 cm. </center>  
+
+<table><tr><td colspan="3">Response Tab. 1: The mechanical tests of PAni fibre from 5 batches</td></tr><tr><td>Batches</td><td>Tensile strength (MPa)</td><td>Strain (%)</td></tr><tr><td>Batch 1</td><td>1117.8</td><td>4.23</td></tr><tr><td>Batch 2</td><td>1073.2</td><td>3.03</td></tr><tr><td>Batch 3</td><td>1076.1</td><td>3.83</td></tr><tr><td>Batch 4</td><td>1127.8</td><td>4.13</td></tr><tr><td>Batch 5</td><td>1009.1</td><td>3.13</td></tr></table>  
+
+5. Reviewer's Comment: For the fiber based OECT, an insulating PU was used to encapsulate the Ag wire, how does the capacitance of the PU compare with the double layer capacitance near the fiber/solid-state electrolyte interface? How is the gate voltage distributing from the gate to the channel?  
+
+Our response: We thank the reviewer for the kind comments. We do not have the suitable devices at the micrometer scale to measure those parameters. However, PU is a dielectric layer which are difficult to be doped, so the capacitance of PU layer should be much smaller than that of fibre/electrolyte interface. From the gate to the channel, there are mainly three parts: the gate/electrolyte interface, gel electrolyte, and electrolyte/fibre interface. Among of them, the used gel electrolyte, PVA- \(\mathsf{H}_3\mathsf{PO}_4\) gel, is almost insulating according to our
+
+<--- Page Split --->
+
+electrical test (with a resistance beyond 10 MΩ along with the gate bias direction). Thus, the gate bias is mainly divided into gate/electrolyte voltage and electrolyte/channel voltage. Kindly for your information, please refer to Ref. 54 (IEEE Electron Device Lett. 42, 46- 49, 2020). We have added the discussions in the revised manuscript (highlighted in Line 275- 277).  
+
+6. Reviewer's Comment: Can the authors make a table to compare the basic electrical properties of their OECT with previously reported fiber based OECTs?  
+
+Our response: We thank the reviewer for the kind suggestions. We have added the table to compare the electrical properties of our device to other fibre-based OECTs in the revised manuscript (highlighted in Line 287- 288).  
+
+<table><tr><td colspan="5">Supplementary Tab. 1: The electrical properties in fibre-based OECTs</td></tr><tr><td>Ref No.</td><td>Channel</td><td>On/Off ratio</td><td>Drive (V)</td><td>gm (μS)</td></tr><tr><td>This work</td><td>PAni</td><td>103</td><td>0.6</td><td>60</td></tr><tr><td>Ref. S1</td><td>PPy/PVA/PE</td><td>2.6x102</td><td>3</td><td>/</td></tr><tr><td>Ref. S2</td><td>PPy</td><td>104</td><td>2</td><td>/</td></tr><tr><td>Ref. S3</td><td>CNT</td><td>102</td><td>1</td><td>1350</td></tr><tr><td>Ref. S4</td><td>PPy/Graphene</td><td>102</td><td>2</td><td>/</td></tr><tr><td>Ref. S5</td><td>PEDOT/PSS</td><td>103</td><td>1</td><td>1000</td></tr></table>  
+
+7. Reviewer's Comment: When referring to the 5.4 km of UFPF, the text says Fig. 1e but should refer to Fig. 1g.  
+
+Our response: We have corrected it in the revised manuscript (highlighted in Line 114).  
+
+8. Reviewer's Comment: Clearly indicate that the panel next to Fig. 3e is a zoom in. Our response: We thank the kind suggestions of the reviewer. We have added the label in Fig. 3e in the revised manuscript.  
+
+9. Reviewer's Comment: Add a legend to Fig. 2e to indicate the meaning of color. Our response: We thank the reviewer for the kind suggestion. We have added the legend
+
+<--- Page Split --->
+
+in the revised manuscript (highlighted in Line 130- 132).  
+
+10. Reviewer's Comment: For Fig. 5e, considering the relative position between the Ag wire and PU layer, why will a change in friction alter the position of Ag inside the PU? Our response: We thank the reviewer for the kind comment. Our OECT is a polymer-based soft device. The Ag gate is located in the upper PU layer, close to the friction action interface. It feels like we rub our skins using fingers. The action of friction will incur repeated horizonal movement of PU layer, which drives the movement of Ag gate, as illustrated in Fig. 5e. We have added the discussions in the revised manuscript (highlighted in Line 314- 318).  
+
+11. Reviewer's Comment: What molecular weight of PANI powder was used? Our response: The molecular weight of PANI used is 91.1106. We are not sure the value of average chain length. It was purchased from Aladdin, and the CAS number is 25233-30-1. We have added the information of chemicals in the revised manuscript (highlighted in Line 354).  
+
+12. Reviewer's Comment: Was there a particular choice behind using camphor sulfonic acid? Others have used AMPSA, sulfuric acid, HClO4, and NMP.  
+
+Our response: We thank the reviewer for the kind comment. The use of camphor sulfonic acid plays two roles: sulfonic groups enhance the dispersibility of PANI molecules and the proton improves the transport performance along PANI chains. Although we do not try other dopants due to the lack of storage in our lab, other dopants with above features could also work in principle.  
+
+13. Reviewer's Comment: When referring to Raman "de-doping" peaks, explain what bonds you are looking at and give a citation.  
+
+Our response: We thank the reviewer for the kind suggestion. We have added the explanations and some citations in the revised manuscript (highlighted in Line 154- 155).  
+
+14. Reviewer's Comment: How did you perform your Rheology?
+
+<--- Page Split --->
+
+Our response: We thank the reviewer for the kind comment. The test of viscosity was conducted by the viscometer (NDJ- 5S/9S/8S). We prepared a series of PANi gels by extruding PANi solutions in different solvents. Then, the probe of viscometer inserted into PANi gels after soaking in solvents, and the viscosity was recorded. To monitor the real state of gels as far as possible, we controlled the shear speed of probes at a very low value from 10 to 60 Rev. We have summarized and added the explanations in the revised manuscript (highlighted in Line 351- 353).
+
+<--- Page Split --->
+
+REVIEWERS' COMMENTS  
+
+Reviewer #1 (Remarks to the Author):  
+
+The revised manuscript has addressed my concerns in my earlier review. I have no further comment.
+
+<--- Page Split --->
+
+# Point by point responses to the reviewer comments:  
+
+# Reviewer 1:  
+
+1. Reviewer's Comment: The revised manuscript has addressed my concerns in my earlier review. I have no further comment.  
+
+Our response: We thank the reviewer for the positive comments.
+
+<--- Page Split --->

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@@ -0,0 +1,239 @@
+<|ref|>title<|/ref|><|det|>[[100, 40, 506, 90]]<|/det|>
+# nature portfolio  
+
+<|ref|>text<|/ref|><|det|>[[106, 110, 373, 139]]<|/det|>
+Peer Review File  
+
+<|ref|>text<|/ref|><|det|>[[108, 161, 855, 219]]<|/det|>
+Scalable production of ultrafine polyaniline fibres for tactile organic electrochemical transistors  
+
+<|ref|>image<|/ref|><|det|>[[93, 732, 262, 780]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[271, 732, 880, 784]]<|/det|>
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 91, 321, 106]]<|/det|>
+REVIEWER COMMENTS</B>  
+
+<|ref|>text<|/ref|><|det|>[[115, 147, 393, 163]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[114, 203, 877, 367]]<|/det|>
+The research work reported in the paper represents a significant breakthrough in flexible, polymer- based electronic applications, particularly, (1) improved electrical performance, (2) a pathway for high- volume production, and (3) high mechanical performance. The fibers developed could be used for many other applications for flexible, wearable electronics beyond the examples highlighted in the paper. The production approach shows the promise for future commercial applications. The mechanical properties (strength, modulus) of the fibers are in the very high range, demonstrating the potential for many demanding applications. The paper is very well written, with a clear presentation of the state- of- the- art status, motivations, approached, and mechanisms. The conclusions are reasonable and supported by data presented.  
+
+<|ref|>text<|/ref|><|det|>[[115, 406, 820, 441]]<|/det|>
+Comment regarding equation for D_AB^O: What is its source? From literature or by data fitting? Variables in equation are defined but units should also be given.  
+
+<|ref|>text<|/ref|><|det|>[[115, 480, 860, 497]]<|/det|>
+Minor edits: less one => less than one; larger than of conventional => larger than that of conventional  
+
+<|ref|>text<|/ref|><|det|>[[115, 567, 393, 583]]<|/det|>
+Reviewer #2 (Remarks to the Author):  
+
+<|ref|>sub_title<|/ref|><|det|>[[115, 624, 191, 639]]<|/det|>
+## Summary:  
+
+<|ref|>text<|/ref|><|det|>[[114, 652, 882, 833]]<|/det|>
+The authors report a method to develop PANI fibers using a wet spinning approach with small diameters \(< 5 \text{um}\) , good mechanical strength, and electrical properties amenable for use in e- textile applications. The reported method leveraged predoping with camphor sulfonic acid, and careful solvent choice of the coagulation bath. The authors demonstrate that choice of solvent dramatically impacts the surface morphology of the fibers, with NMP and DMF providing smooth and thin fibers with stability in air for four weeks. The authors then speculate that the use of a "good" solvent prevented quick solidification, and increased interfacial pressure during solvent exchange, and therefore allowed stretching of the fiber to reduce diameter and enhance morphology. The fibers showed good mechanical performance compared to other non- blended conjugated polymer- based fibers, which was attributed to the decrease in diameter and structural defects.  
+
+<|ref|>text<|/ref|><|det|>[[115, 844, 872, 899]]<|/det|>
+The fibers were then used to create a microcapacitor, which was characterized by CV and GCD. The capacitance, power density, energy density, and stability, demonstrated reliable electrochemical performance. The charge storage capacity was determined and rivals that of other similar platforms. As
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[114, 89, 880, 218]]<|/det|>
+such the fiber was used to make a solid- state OECT with a silver wire as the gate electrode, the fiber as the channel, and a PVA- H3PO4 gel, all wrapped in a polyurethane protective coating. The OECT was characterized and demonstrated low power consumption. The OECT was then demonstrated to respond to mechanical deformation through the pressure induced change in ion penetration. The favorable mechanical and electronic properties, as well as the scalability of these fibers will be of interest to the scientific community and warrants publication in Nature Communications. Please respond to the following:  
+
+<|ref|>text<|/ref|><|det|>[[114, 255, 883, 309]]<|/det|>
+1) What exactly do the authors mean by "good" or "bad" solvent? Solution viscosity? The speed at which the PANI solidifies/coagulates, preventing slenderization? PANI solubility? 2nd virial coefficient (A2)? Diffusivity?  
+
+<|ref|>text<|/ref|><|det|>[[114, 321, 860, 356]]<|/det|>
+2) Why do the differences in morphology when using a "good" or "bad" solvent lead to differences in stability?  
+
+<|ref|>text<|/ref|><|det|>[[114, 367, 616, 384]]<|/det|>
+3) How reproducible and uniform are the properties of these fibers?  
+
+<|ref|>text<|/ref|><|det|>[[114, 396, 870, 450]]<|/det|>
+4) For the fiber based OECT, an insulating PU was used to encapsulate the Ag wire, how does the capacitance of the PU compare with the double layer capacitance near the fiber/solid-state electrolyte interface? How is the gate voltage distributing from the gate to the channel?  
+
+<|ref|>text<|/ref|><|det|>[[114, 461, 875, 496]]<|/det|>
+5) Can the authors make a table to compare the basic electrical properties of their OECT with previously reported fiber based OECTs  
+
+<|ref|>sub_title<|/ref|><|det|>[[115, 537, 170, 552]]<|/det|>
+## Figures  
+
+<|ref|>text<|/ref|><|det|>[[115, 564, 745, 581]]<|/det|>
+- When referring to the 5.4 km of UFPF, the text says Fig. 1e but should refer to Fig. 1g.  
+
+<|ref|>text<|/ref|><|det|>[[115, 593, 544, 609]]<|/det|>
+- Clearly indicate that the panel next to Fig. 3e is a zoom in.  
+
+<|ref|>text<|/ref|><|det|>[[115, 621, 528, 637]]<|/det|>
+- Add a legend to Fig. 2e to indicate the meaning of color  
+
+<|ref|>text<|/ref|><|det|>[[115, 649, 854, 684]]<|/det|>
+- For Fig. 5e, considering the relative position between the Ag wire and PU layer, why will a change in friction alter the position of Ag inside the PU?  
+
+<|ref|>sub_title<|/ref|><|det|>[[115, 725, 183, 740]]<|/det|>
+## Methods  
+
+<|ref|>text<|/ref|><|det|>[[115, 753, 492, 770]]<|/det|>
+- What molecular weight of PANI powder was used?  
+
+<|ref|>text<|/ref|><|det|>[[115, 781, 861, 816]]<|/det|>
+- Was there a particular choice behind using camphor sulfonic acid? Others have used AMPSA, sulfuric acid, HClO4, and NMP.  
+
+<|ref|>text<|/ref|><|det|>[[115, 827, 875, 844]]<|/det|>
+- When referring to Raman "de-doping" peaks, explain what bonds you are looking at and give a citation.  
+
+<|ref|>text<|/ref|><|det|>[[115, 856, 397, 872]]<|/det|>
+- How did you perform your Rheology?
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[148, 86, 590, 103]]<|/det|>
+## Point by point responses to the reviewer comments:  
+
+<|ref|>sub_title<|/ref|><|det|>[[149, 128, 249, 143]]<|/det|>
+## Reviewer 1:  
+
+<|ref|>text<|/ref|><|det|>[[147, 154, 853, 425]]<|/det|>
+1. Reviewer's Comment: The research work reported in the paper represents a significant breakthrough in flexible, polymer-based electronic applications, particularly, (1) improved electrical performance, (2) a pathway for high-volume production, and (3) high mechanical performance. The fibers developed could be used for many other applications for flexible, wearable electronics beyond the examples highlighted in the paper. The production approach shows the promise for future commercial applications. The mechanical properties (strength, modulus) of the fibers are in the very high range, demonstrating the potential for many demanding applications. The paper is very well written, with a clear presentation of the state-of-the-art status, motivations, approached, and mechanisms. The conclusions are reasonable and supported by data presented.  
+
+<|ref|>text<|/ref|><|det|>[[149, 432, 653, 450]]<|/det|>
+Our response: We thank the reviewer for the positive comments.  
+
+<|ref|>text<|/ref|><|det|>[[147, 487, 853, 644]]<|/det|>
+2. Reviewer's Comment regarding equation for \(D_{AB}^0\) : What is its source? From literature or by data fitting? Variables in equation are defined but units should also be given. Our response: We thank the reviewer for kind concern. The equation for \(D_{AB}^0\) is derived from Ref. 25 (Smart Structures and Materials 2001: Electroactive Polymer Actuators and Devices. 4329, 59-71, 2001). We have also added the units of every variable (highlighted in Line 174-177).  
+
+<|ref|>text<|/ref|><|det|>[[148, 682, 852, 728]]<|/det|>
+3. Reviewer's Comment: Minor edits: less one => less than one; larger than of conventional => larger than that of conventional.  
+
+<|ref|>text<|/ref|><|det|>[[148, 737, 852, 783]]<|/det|>
+Our response: We thank the reviewer for critically reading our manuscript and pointing out our mistakes. We have corrected all the edits (highlighted in Line 34, 81 and 282).  
+
+<|ref|>sub_title<|/ref|><|det|>[[149, 822, 249, 837]]<|/det|>
+## Reviewer 2:  
+
+<|ref|>text<|/ref|><|det|>[[149, 849, 852, 894]]<|/det|>
+1. Reviewer's Comment: The authors report a method to develop PANI fibers using a wet spinning approach with small diameters (< 5 um), good mechanical strength, and electrical
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[147, 88, 853, 357]]<|/det|>
+properties amenable for use in e- textile applications. The reported method leveraged pre- doping with camphor sulfonic acid, and careful solvent choice of the coagulation bath. The authors demonstrate that choice of solvent dramatically impacts the surface morphology of the fibers, with NMP and DMF providing smooth and thin fibers with stability in air for four weeks. The authors then speculate that the use of a "good" solvent prevented quick solidification, and increased interfacial pressure during solvent exchange, and therefore allowed stretching of the fiber to reduce diameter and enhance morphology. The fibers showed good mechanical performance compared to other non- blended conjugated polymer- based fibers, which was attributed to the decrease in diameter and structural defects.  
+
+<|ref|>text<|/ref|><|det|>[[147, 366, 853, 662]]<|/det|>
+The fibers were then used to create a micro capacitor, which was characterized by CV and GCD. The capacitance, power density, energy density, and stability, demonstrated reliable electrochemical performance. The charge storage capacity was determined and rivals that of other similar platforms. As such the fiber was used to make a solid- state OECT with a silver wire as the gate electrode, the fiber as the channel, and a \(\mathrm{PVA - H_3PO_4}\) gel, all wrapped in a polyurethane protective coating. The OECT was characterized and demonstrated low power consumption. The OECT was then demonstrated to respond to mechanical deformation through the pressure induced change in ion penetration. The favorable mechanical and electronic properties, as well as the scalability of these fibers will be of interest to the scientific community and warrants publication in Nature Communications.  
+
+<|ref|>text<|/ref|><|det|>[[149, 673, 653, 690]]<|/det|>
+Our response: We thank the reviewer for the positive comments.  
+
+<|ref|>text<|/ref|><|det|>[[147, 718, 852, 891]]<|/det|>
+2. Reviewer's Comment: What exactly do the authors mean by "good" or "bad" solvent? Solution viscosity? The speed at which the PANI solidifies/coagulates, preventing slenderization? PANI solubility? 2nd virial coefficient (A2)? Diffusivity? Our response: We thank the reviewer for the important comment. From the perspective of physical interpretation, "good" and "poor" solvents of PANI are determined by the intermolecular interactions between PANi chains and solvent molecules. In a good solvent, interactions between PANi chains solvent molecules are energetically favorable, and will
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[147, 88, 852, 191]]<|/det|>
+cause PAni chains to expand and disperse well. In a poor solvent, PAni- PAni interactions are preferred and cause the PAni chains to disperse poor. Consequently, PAni molecules disperse good in good solvents, and disperse poor in poor solvents. We have added the discussions in the revised manuscript (highlighted in Line 137- 140).  
+
+<|ref|>text<|/ref|><|det|>[[147, 218, 852, 264]]<|/det|>
+3. Reviewer's Comment: Why do the differences in morphology when using a "good" or "bad" solvent lead to differences in stability?  
+
+<|ref|>text<|/ref|><|det|>[[147, 274, 853, 488]]<|/det|>
+Our response: We thank the reviewer for the crucial questions. The morphology does not directly determine the differences in stability. According to our SEM observations and X- ray diffraction analysis, the fibres produced in good solvents behaved higher degree of orientation and crystallization. The higher degree of crystallization protects the doping bonding in PAni chains from the attack of ambientes at the molecular level. As a result, the fibres produced in good solvents could show better stability in air. To avoid the misleading, we have rearranged the related discussions in the revised manuscript (highlighted in Line 145- 150).  
+
+<|ref|>text<|/ref|><|det|>[[147, 515, 853, 867]]<|/det|>
+4. Reviewer's Comment: How reproducible and uniform are the properties of these fibers? Our response: We thank the reviewer for the kind comment. We discuss the uniformity of PAni fibres from the perspective of electrical and mechanical properties. In the case of electrical properties, because we used pre-doped PAni solutions as the raw dopes, the PAni fibres show uniform charge distribution throughout the fibre length. To confirm this point, we added a measurement to record the relationship between length and resistance. As shown in Supplementary Fig. 4, the value of resistance increases linearly with increasing length from 1 cm to 10 cm, demonstrating the favorable uniformity of electrical properties. In the case of mechanical properties, we provided the mechanical tensile tests of 5 fibres from 5 different batches, the results are shown in Response Tab. 1 for the information. The tensile strength and strain are generally stable, demonstrating the reliable uniformity of mechanical properties. We have added the discussions in the revised manuscript (highlighted in Line 111).
+
+<--- Page Split --->
+<|ref|>image<|/ref|><|det|>[[286, 85, 710, 341]]<|/det|>
+<|ref|>image_caption<|/ref|><|det|>[[147, 350, 850, 395]]<|/det|>
+<center>Supplementary Fig. 4 The relationship between length and resistance of PAni fibres with increasing lengths from 1 cm to 10 cm. </center>  
+
+<|ref|>table<|/ref|><|det|>[[148, 420, 848, 622]]<|/det|>
+
+<table><tr><td colspan="3">Response Tab. 1: The mechanical tests of PAni fibre from 5 batches</td></tr><tr><td>Batches</td><td>Tensile strength (MPa)</td><td>Strain (%)</td></tr><tr><td>Batch 1</td><td>1117.8</td><td>4.23</td></tr><tr><td>Batch 2</td><td>1073.2</td><td>3.03</td></tr><tr><td>Batch 3</td><td>1076.1</td><td>3.83</td></tr><tr><td>Batch 4</td><td>1127.8</td><td>4.13</td></tr><tr><td>Batch 5</td><td>1009.1</td><td>3.13</td></tr></table>  
+
+<|ref|>text<|/ref|><|det|>[[147, 644, 852, 747]]<|/det|>
+5. Reviewer's Comment: For the fiber based OECT, an insulating PU was used to encapsulate the Ag wire, how does the capacitance of the PU compare with the double layer capacitance near the fiber/solid-state electrolyte interface? How is the gate voltage distributing from the gate to the channel?  
+
+<|ref|>text<|/ref|><|det|>[[147, 756, 852, 912]]<|/det|>
+Our response: We thank the reviewer for the kind comments. We do not have the suitable devices at the micrometer scale to measure those parameters. However, PU is a dielectric layer which are difficult to be doped, so the capacitance of PU layer should be much smaller than that of fibre/electrolyte interface. From the gate to the channel, there are mainly three parts: the gate/electrolyte interface, gel electrolyte, and electrolyte/fibre interface. Among of them, the used gel electrolyte, PVA- \(\mathsf{H}_3\mathsf{PO}_4\) gel, is almost insulating according to our
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[147, 89, 852, 219]]<|/det|>
+electrical test (with a resistance beyond 10 MΩ along with the gate bias direction). Thus, the gate bias is mainly divided into gate/electrolyte voltage and electrolyte/channel voltage. Kindly for your information, please refer to Ref. 54 (IEEE Electron Device Lett. 42, 46- 49, 2020). We have added the discussions in the revised manuscript (highlighted in Line 275- 277).  
+
+<|ref|>text<|/ref|><|det|>[[147, 247, 852, 293]]<|/det|>
+6. Reviewer's Comment: Can the authors make a table to compare the basic electrical properties of their OECT with previously reported fiber based OECTs?  
+
+<|ref|>text<|/ref|><|det|>[[147, 302, 852, 377]]<|/det|>
+Our response: We thank the reviewer for the kind suggestions. We have added the table to compare the electrical properties of our device to other fibre-based OECTs in the revised manuscript (highlighted in Line 287- 288).  
+
+<|ref|>table<|/ref|><|det|>[[147, 380, 849, 610]]<|/det|>
+
+<table><tr><td colspan="5">Supplementary Tab. 1: The electrical properties in fibre-based OECTs</td></tr><tr><td>Ref No.</td><td>Channel</td><td>On/Off ratio</td><td>Drive (V)</td><td>gm (μS)</td></tr><tr><td>This work</td><td>PAni</td><td>103</td><td>0.6</td><td>60</td></tr><tr><td>Ref. S1</td><td>PPy/PVA/PE</td><td>2.6x102</td><td>3</td><td>/</td></tr><tr><td>Ref. S2</td><td>PPy</td><td>104</td><td>2</td><td>/</td></tr><tr><td>Ref. S3</td><td>CNT</td><td>102</td><td>1</td><td>1350</td></tr><tr><td>Ref. S4</td><td>PPy/Graphene</td><td>102</td><td>2</td><td>/</td></tr><tr><td>Ref. S5</td><td>PEDOT/PSS</td><td>103</td><td>1</td><td>1000</td></tr></table>  
+
+<|ref|>text<|/ref|><|det|>[[147, 658, 852, 705]]<|/det|>
+7. Reviewer's Comment: When referring to the 5.4 km of UFPF, the text says Fig. 1e but should refer to Fig. 1g.  
+
+<|ref|>text<|/ref|><|det|>[[147, 715, 830, 734]]<|/det|>
+Our response: We have corrected it in the revised manuscript (highlighted in Line 114).  
+
+<|ref|>text<|/ref|><|det|>[[147, 761, 852, 835]]<|/det|>
+8. Reviewer's Comment: Clearly indicate that the panel next to Fig. 3e is a zoom in. Our response: We thank the kind suggestions of the reviewer. We have added the label in Fig. 3e in the revised manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[147, 863, 852, 909]]<|/det|>
+9. Reviewer's Comment: Add a legend to Fig. 2e to indicate the meaning of color. Our response: We thank the reviewer for the kind suggestion. We have added the legend
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[147, 90, 583, 107]]<|/det|>
+in the revised manuscript (highlighted in Line 130- 132).  
+
+<|ref|>text<|/ref|><|det|>[[147, 135, 853, 348]]<|/det|>
+10. Reviewer's Comment: For Fig. 5e, considering the relative position between the Ag wire and PU layer, why will a change in friction alter the position of Ag inside the PU? Our response: We thank the reviewer for the kind comment. Our OECT is a polymer-based soft device. The Ag gate is located in the upper PU layer, close to the friction action interface. It feels like we rub our skins using fingers. The action of friction will incur repeated horizonal movement of PU layer, which drives the movement of Ag gate, as illustrated in Fig. 5e. We have added the discussions in the revised manuscript (highlighted in Line 314- 318).  
+
+<|ref|>text<|/ref|><|det|>[[148, 376, 853, 505]]<|/det|>
+11. Reviewer's Comment: What molecular weight of PANI powder was used? Our response: The molecular weight of PANI used is 91.1106. We are not sure the value of average chain length. It was purchased from Aladdin, and the CAS number is 25233-30-1. We have added the information of chemicals in the revised manuscript (highlighted in Line 354).  
+
+<|ref|>text<|/ref|><|det|>[[148, 533, 852, 580]]<|/det|>
+12. Reviewer's Comment: Was there a particular choice behind using camphor sulfonic acid? Others have used AMPSA, sulfuric acid, HClO4, and NMP.  
+
+<|ref|>text<|/ref|><|det|>[[147, 588, 853, 719]]<|/det|>
+Our response: We thank the reviewer for the kind comment. The use of camphor sulfonic acid plays two roles: sulfonic groups enhance the dispersibility of PANI molecules and the proton improves the transport performance along PANI chains. Although we do not try other dopants due to the lack of storage in our lab, other dopants with above features could also work in principle.  
+
+<|ref|>text<|/ref|><|det|>[[148, 746, 852, 792]]<|/det|>
+13. Reviewer's Comment: When referring to Raman "de-doping" peaks, explain what bonds you are looking at and give a citation.  
+
+<|ref|>text<|/ref|><|det|>[[148, 802, 852, 848]]<|/det|>
+Our response: We thank the reviewer for the kind suggestion. We have added the explanations and some citations in the revised manuscript (highlighted in Line 154- 155).  
+
+<|ref|>text<|/ref|><|det|>[[148, 886, 660, 903]]<|/det|>
+14. Reviewer's Comment: How did you perform your Rheology?
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[147, 88, 853, 275]]<|/det|>
+Our response: We thank the reviewer for the kind comment. The test of viscosity was conducted by the viscometer (NDJ- 5S/9S/8S). We prepared a series of PANi gels by extruding PANi solutions in different solvents. Then, the probe of viscometer inserted into PANi gels after soaking in solvents, and the viscosity was recorded. To monitor the real state of gels as far as possible, we controlled the shear speed of probes at a very low value from 10 to 60 Rev. We have summarized and added the explanations in the revised manuscript (highlighted in Line 351- 353).
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 300, 106]]<|/det|>
+REVIEWERS' COMMENTS  
+
+<|ref|>text<|/ref|><|det|>[[115, 146, 394, 163]]<|/det|>
+Reviewer #1 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[112, 203, 850, 220]]<|/det|>
+The revised manuscript has addressed my concerns in my earlier review. I have no further comment.
+
+<--- Page Split --->
+<|ref|>title<|/ref|><|det|>[[148, 86, 590, 103]]<|/det|>
+# Point by point responses to the reviewer comments:  
+
+<|ref|>title<|/ref|><|det|>[[148, 128, 250, 143]]<|/det|>
+# Reviewer 1:  
+
+<|ref|>text<|/ref|><|det|>[[148, 155, 852, 199]]<|/det|>
+1. Reviewer's Comment: The revised manuscript has addressed my concerns in my earlier review. I have no further comment.  
+
+<|ref|>text<|/ref|><|det|>[[148, 211, 653, 227]]<|/det|>
+Our response: We thank the reviewer for the positive comments.
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__022065845b0149dd1bde836e14da3448f70c4603767a53533c6448baf134c8c3/images_list.json

@@ -0,0 +1,10 @@
+[
+  {
+    "type": "image",
+    "img_path": "images/Figure_1.jpg",
+    "caption": "Figure 1: RMSE calculated from Figure 4A of Solomon et al., 2004",
+    "footnote": [],
+    "bbox": [],
+    "page_idx": 0
+  }
+]

peer_reviews/supplementary_0_Peer Review File__022065845b0149dd1bde836e14da3448f70c4603767a53533c6448baf134c8c3/supplementary_0_Peer Review File__022065845b0149dd1bde836e14da3448f70c4603767a53533c6448baf134c8c3.mmd

@@ -0,0 +1,499 @@
+
+# nature portfolio  
+
+Peer Review File  
+
+Crowding results from optimal integration of visual targets with contextual information  
+
+![](images/Figure_1.jpg)
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+## REVIEWER COMMENTS  
+
+Reviewer #1 (Remarks to the Author):  
+
+This is an outstanding manuscript. The authors propose a novel and fascinating connection between crowding and serial dependence, two extensively studied areas of perception and cognition. They thoroughly test their idea psychophysically and with modeling. The results support the hypothesis and this will stimulate a lot of future research. I know this hypothesis will be provocative in the field, and not everyone will agree (it's a fairly contentious field), but this is a strength; the manuscript is exceptionally well balanced and approaches the issues in a most constructive way. The crowding field has been somewhat stagnant for years, and the authors' novel connection is much needed inspiration for researchers to pursue new directions. I expect this paper will motivate a great flurry of new experiments. I have a few minor suggestions below, but these are just requested clarifications, nothing major. Given the broad connections this manuscript makes across fields and the novelty of the idea and results, the manuscript certainly merits publication in Nature Comms.  
+
+Minor points:  
+
+Flanker Similarity and the (unmentioned) Diagnostic criteria for crowding. There's a nod to these diagnostic criteria (eg, Whitney & Levi, 2011) but not a direct statement. The critical spacing one is key of course (p. 6)—and the model does a great job predicting that—but it's not the only one. Another key characteristic directly addressed in the MS is similarity. This is relevant here bc the prior literature had little explanation for why "similarity" matters in the way that it does (eg similarity modulates crowding and dissimilarity releases crowding). The authors' idea of a connection between serial dependence and crowding, and their model, is very powerful and important in part bc it provides that "why". In future work it will be interesting to test if other diagnostic criteria like inner- outer flanker asymmetry, upper lower visual field diff, etc also hold. This isn't necessary here but readers may wonder and the authors could prompt that question and help motivate the important follow up research.  
+
+P2, "tasks like or face recognition" delete "or" and perhaps add a reference here. Maybe Farzin et al 2009 (for faces) or the cited reviews (if this is a generic statement about objects).  
+
+P3, "qualitatively and qualitatively". Perhaps one of these was intended to be "quantitatively"?  
+
+Fig 1b. Using red outline and blue outline around the respective panels (or at least red and blue color somehow in those two panels) would help readers follow the correspondence between all the Figs; red always indicates high reliability target. Might as well start using that rule in fig 1b.  
+
+P.4. "...Formally the modeling section" should be "formally in the..."  
+
+P5. "...With difference between..." should have "the" or an "s" after "difference"  
+
+Fig 2, abscissa. Add clarification that this axis is "difference" in orientation. It's not absolute orientation, right?  
+
+Fig 2. Where would isolated (single) targets be on this graph?  
+
+P.11. Is the "signature" of the "signature function" the derivative-of-Gaussian shape in the SD literature? If so, perhaps mention that or explain what is meant by "signature"  
+
+P 12. The first sentence of the "causal inference model" section. That first sentence is too difficult to parse or understand. Not just because the word "form" probably isn't intended. Rephrasing could help a lot.
+
+<--- Page Split --->
+
+P12. "...the weight assigned of is the..."Rephrase, please.  
+
+Aside from these very minor points, this is an excellent manuscript.  
+
+Reviewer #2 (Remarks to the Author):  
+
+This is a fascinating manuscript, with novel findings that present a new perspective on a widely studied phenomenon. The authors examine visual crowding, the disruptive effect of clutter on object recognition. A large body of research has depicted this effect as the 'fundamental bottleneck on object recognition' in peripheral vision especially. As a result, we know a great deal about the way this process affects object recognition and the potential mechanisms. What is much less clear is why crowding occurs in the first place. This manuscript presents an interesting answer to this question by considering its usefulness.  
+
+The broad approach here is to compare crowding with properties of 'serial dependence', the effect whereby judgements of a stimulus are influenced by the presentation of other stimuli in prior trials. With this comparison, the authors ask whether crowding can be considered to be an efficient/optimal process, rather than reflecting a disruptive bottleneck. Several predictions are made in this case, all of which are ultimately argued to be supported by the data. The authors ask observers to judge the orientation of shapes made from an outline of dots. They first observe greater biases and higher response scatter with "low reliability" near- circular target stimuli that are more difficult to judge, compared with "high reliability" elliptical targets. Second, they note that response scatter is greatest when the orientation of the flankers is close to the target, with a decrease as dissimilarity decreases (that is, performance improves as crowding increases, showing its efficiency). Finally, the pattern of biases follows the mean orientation of flankers rather than an independent combination, which is used to justify a higher- level model. The manuscript is well written and engaging, and presents a provocative view of a widely studied process. If the findings here are true then this presents an important aspect of our understanding. I do have a number of issues with the manuscript as it stands, however.  
+
+## 1. The pattern of response scatter  
+
+The main issue concerns the second finding - that response scatter is greatest (i.e. performance is worst) when the orientation of the flankers is most similar to the target. This finding is a key aspect of the proposal that crowding is efficient/optimal, since errors decrease as the strength of crowding increases. If true however, this finding is inconsistent with a large literature on the effect of target- flanker similarity in crowding. More typically, crowding is greatest when target and flanker elements are most similar to one another, decreasing as their dissimilarity increases (the opposite of the current observation). This has been found for a range of stimulus properties including contrast polarity, color, spatial frequency, and direction (Kooi, Toet, Tripathy, & Levi, 1994; Chung, Levi, & Legge, 2001; Gheri, Morgan, & Solomon, 2007), and in particular for orientation judgements (Andriessen & Bouma, 1976; Wilkinson, Wilson, & Ellemberg, 1997), similar to those used in the present study. In those latter studies, flankers that share similar orientations to the target induce the most crowding, with less crowding as the orientation of the flankers rotates away. Given that a major premise of the current study rests on the opposite finding, this discrepancy needs explanation and/or further exploration. The authors do in fact cite some of these studies to begin the manuscript, describing the patterns above, but the discrepancy with the current results is subsequently ignored. How can this discrepancy be explained, and how does this fit with the central arguments regarding the efficiency/optimality of crowding?  
+
+There seem to me at least two possibilities to explain the discrepancy. One is that the authors have not fully measured the range of possible target- flanker differences in orientation. Targets are
+
+<--- Page Split --->
+
+presented at either 35 or 55 degrees rotation, with flankers that differ from these values by up to \(\pm 45\) degrees. Response scatter peaks at the highest values measured \((\pm 45\) degrees). It is however possible then that these values may drop again as the differences further increase, up to their maximum of 90 degrees from the target orientation. It is typically these 90 degree values that are compared in order to show target- flanker similarity effects (Andriessen & Bouma, 1976; Wilkinson, Wilson, & Ellemberg, 1997), and I suspect that if the measurements continued here that performance would drop again. Indeed – patterns of this nature have been reported in a prior study (Solomon, Felisberti, & Morgan, 2004). There, orientation sensitivity is high when flankers are similarly oriented to the target, drops for orientations up to \(\pm 45\) degrees, and then increases again as the rotation continues to 90 degrees. The same may be true of the present stimuli if a larger range of orientations were tested. Would that not alter the interpretations regarding the efficiency of this process?  
+
+A second possibility is the eccentricity – the authors present their stimuli 26 degrees from fixation. Prior observations of target- flanker similarity have tended to use lower eccentricities. Given that some properties of crowding change with eccentricity, e.g. the response biases (Mareschal, Morgan, & Solomon, 2010) and of course the well- known effects of spatial extent (Bouma, 1970), it could be that the present results are something that only arises in the far periphery. Were this the case, however, the question remains – why is efficiency evident in the present results and not these other studies?  
+
+To summarize, the results regarding response scatter appear to follow the opposite pattern to a range of well- established and replicated findings in the literature. The premise of the paper rests heavily on this observation. The authors need to demonstrate that this pattern is reliable by extending the range of their measurements in some way and/or by addressing this discrepancy with prior results. If the current results show efficiency, what does that say about all of these other results? If crowding is only efficient in these limited circumstances, is it really efficient?  
+
+## 2. The lack of an unflanked baseline  
+
+Part of the issue of interpretation with the above response scatter data also relates to the lack of an unflanked baseline. Typically, performance with flankers is used to measure crowding, with an unflanked baseline (with an isolated element) used to measure uncrowded performance. The authors here take their baseline using flanked performance, using flankers with orientations at the extremes of their range (p14). Given the odd pattern of response scatter (as above), this assumption is problematic. If unflanked performance is more like the values with a flanker difference of 0 degrees, then performance would go from largely unaffected with the 0 degree flankers to impaired with the \(\pm 45\) degree flankers, rather than from improved with the 0 degree flankers to unaffected with the \(\pm 45\) degree flankers, as the authors argue. Does that not change the interpretation of the results and their efficiency/optimality substantially?  
+
+## 3. The distinction with 'low-level' pooling models of crowding  
+
+The authors contrast their findings with 'low level pooling models', which do not seem to me to be at odds with the present results. This distinction begins in the abstract (e.g. on line 2), where low level models are contrasted with their 'alternative hypothesis', and continues throughout, e.g. on p15 of the discussion, where it is argued that pooling models cannot explaining the effects of flanker orientation. Later on however, the authors describe their model as a pooling process (p17). The mechanism proposed for their model, with large receptive fields in areas like V4 (p16), also sounds very similar to ideas raised in various pooling models. For instance, processes of 'population pooling' have attributed the crowding of orientation signals to pooling within receptive fields in area V2 or V4 (van den Berg, Roerdink, & Cornelissen, 2010; Harrison & Bex, 2015). Similar arguments are also made by 'high dimensional' pooling models (Rosenholtz, Yu, & Keshvari, 2019). In fact, patterns of bias that are very similar to those in Figure 2A of this manuscript have been reported previously and accounted for via pooling processes (Greenwood & Parsons, 2020). In this latter case, the model accounts for target- flanker similarity effects via variations in the weights applied to the flankers. I don't see why this is
+
+<--- Page Split --->
+
+inconsistent with the variations shown in the current study.  
+
+The authors distinguish between two possible models, both of which seem like pooling models to me. One is a 'low level' version in which the interactions happen independently between each flanker and the target, linked with a feedforward local process. The other involves a broader integration in which the flankers have a combined influence on the target, linked with recurrent feedback interactions. The latter does not seem wholly distinct from the operation of the most recent population pooling models (Harrison & Bex, 2015; Greenwood & Parsons, 2020) described above however. In those cases, the flankers affect the target through their combination within a single population response. My feeling is that the results of Experiment 2 in the present study would be entirely consistent with these models - when flanker orientations vary independently, their combined population response would have a shifted mean that would tend to alter the subsequent judgements related to the target. If so, then I do not think these results are inconsistent with pooling, nor do they provide clear evidence for feedback. This is not to take away from the novelty of these findings, however - I agree that the results provide clear evidence that the flankers do not interact independently with the target. The distinction between the two models presented here is certainly interesting, but their physiological basis is clearly overstated.  
+
+## 4. Effects of target reliability  
+
+The first result reported in the manuscript is that biases are greater and response scatter higher with "low reliability" near- circular target stimuli that are more difficult to judge, compared with "high reliability" elliptical targets. This effect is attributed to reliability, and explained via a Bayesian framework. Its relation to similar results with alternative explanations is unexplored, however. Most notably, crowding is strongest with flankers of high luminance contrast (Chung, Levi, & Legge, 2001; Pelli, Palomares, & Majaj, 2004). Lowering the target contrast can also increase crowding (Felisberti, Solomon, & Morgan, 2005). Assimilative biases related to orientation judgements are also increased when noise is added to stimuli (Mareschal, Morgan, & Solomon, 2010). Can all of these effects be understood via reliability? It seems to me there is an alternative explanation that crowding is determined by the strength of the target signal, relative to the strength of the flanker signal(s). Could these effects, including those of the present study, be understood as signal strength rather than reliability per se?  
+
+## 5. The relation to serial dependence  
+
+Much is made of the similarities between serial dependence and crowding, which I agree is a fascinating link to make. The arguments for efficiency in this context also sound to me like arguments made more broadly in vision for the principles of redundancy reduction (Attneave, 1954), including for processes like adaptation (Clifford, 2002) and surround suppression (Rao & Ballard, 1999). Could the similarities here in fact indicate a broader link in the form of a "canonical computation" across all of visual perception? I wonder if the strong link to serial dependence is a little short- sighted in this sense.  
+
+## 5. The neural basis of crowding  
+
+The idea of crowding relating to higher cortical areas like V4 is attributed to Pelli & Tillman (p2), but this idea derives from earlier work (Motter & Simoni, 2007; Motter, 2009). Others have also linked crowding with receptive field sizes in areas like V2 (He, Wang, & Fang, 2019).  
+
+## 6. Stimulus details  
+
+Was the rotation of flankers taken from the target orientation on each trial, such that the \(\pm 45\) degree range differed in terms of absolute orientations for the 35 and 55 degree targets?
+
+<--- Page Split --->
+
+Additionally, can we be sure that the judgements made by observers concern the orientation of these stimuli, rather than another property? Given the dotted nature of the stimuli used in the present task, perhaps observers are not judging orientation, but rather another property like the position of the outermost dots in the elements. This could allow a kind of relative position or Vernier judgement. Prior studies have tended to use line elements or Gabors in this context – if true, could this explain the difference with the studies of target- flanker similarity described above?  
+
+## References  
+
+Andriessen, J. J., & Bouma, H. (1976). Eccentric vision: Adverse interactions between line segments. Vision Research, 16(1), 71- 78. Attneave, F. (1954). Some informational aspects of visual perception. Psychological Review, 61(3), 183- 193. Bouma, H. (1970). Interaction effects in parafoveal letter recognition. Nature, 226, 177- 178. Chung, S. T. L., Levi, D. M., & Legge, G. E. (2001). Spatial- frequency and contrast properties of crowding. Vision Research, 41, 1833- 1850. Clifford, C. W. G. (2002). Perceptual adaptation: Motion parallels orientation. Trends in Cognitive Sciences, 6(3), 136- 143. Felisberti, F. M., Solomon, J. A., & Morgan, M. J. (2005). The role of target salience in crowding. Perception, 34(7), 823- 833. Gheri, C., Morgan, M. J., & Solomon, J. A. (2007). The relationship between search efficiency and crowding. Perception, 36(12), 1779- 1787. Greenwood, J. A., & Parsons, M. J. (2020). Dissociable effects of visual crowding on the perception of color and motion. Proceedings of the National Academy of Sciences of the United States of America, 117(14), 8196- 8202. Harrison, W. J., & Bex, P. J. (2015). A Unifying Model of Orientation Crowding in Peripheral Vision. Current Biology, 25(24), 3213- 3219. He, D., Wang, Y., & Fang, F. (2019). The critical role of V2 population receptive fields in visual orientation crowding. Current Biology, 29(13), 2229- 2236. e2223. Kooi, F. L., Toet, A., Tripathy, S. P., & Levi, D. M. (1994). The effect of similarity and duration on spatial interaction in peripheral vision. Spatial Vision, 8(2), 255- 279. Mareschal, I., Morgan, M. J., & Solomon, J. A. (2010). Cortical distance determines whether flankers cause crowding or the tilt illusion. Journal of Vision, 10(8):13, 1- 14. Motter, B. C. (2009). Central V4 Receptive Fields Are Scaled by the V1 Cortical Magnification and Correspond to a Constant- Sized Sampling of the V1 Surface. Journal of Neuroscience, 29(18), 5749- 5757. Motter, B. C., & Simoni, D. A. (2007). The roles of cortical image separation and size in active visual search performance. Journal of Vision, 7(2(6)), 1- 15. Pelli, D. G., Palomares, M., & Majaj, N. J. (2004). Crowding is unlike ordinary masking: Distinguishing feature integration from detection. Journal of Vision, 4(12), 1136- 1169. Rao, R. P. N., & Ballard, D. H. (1999). Predictive coding in the visual cortex: A functional interpretation of some extra- classical receptive- field effects. Nature Neuroscience, 2(1), 79- 87. Rosenholtz, R., Yu, D., & Keshvari, S. (2019). Challenges to pooling models of crowding: Implications for visual mechanisms. Journal of Vision, 19(7), 1- 25. Solomon, J. A., Felisberti, F. M., & Morgan, M. J. (2004). Crowding and the tilt illusion: Toward a unified account. Journal of Vision, 4, 500- 508. van den Berg, R., Roerdink, J. B. T. M., & Cornelissen, F. W. (2010). A Neurophysiologically Plausible Population Code Model for Feature Integration Explains Visual Crowding. PLoS Computational Biology, 6(1), e1000646. Wilkinson, F., Wilson, H. R., & Ellemberg, D. (1997). Lateral interactions in peripherally viewed texture arrays. Journal of the Optical Society of America. A, Optics, Image Science, and Vision, 14(9), 2057- 2068.
+
+<--- Page Split --->
+
+Reviewer #3 (Remarks to the Author):  
+
+Cicchini and colleagues put forward the hypothesis that crowding is results from Bayes- optimal integration of visual targets with spatial context. The authors identify four features of their empirical data that are consistent with Bayes- optimal integration: (1) Crowding is strongest for reliable flankers and unreliable targets, (2) Crowding depends on flanker- target similarity (here orientation), (3) precision of orientation judgments increases with increasing flanker- target similarity, and (4) Crowding depends on similarity of targets to average flanker orientation, not individual flanker orientations. The authors present two ideal observer models (a Bayesian ideal observer, and a causal inference model), which can reproduce the above features of the empirical data.  
+
+While I find the hypothesis that crowding results from optimal integration intriguing, I am somewhat reserved when it comes to the evidence provided in the current study. I am also not convinced that the behavioral benefits described here can be ascribed to crowding rather than ensemble perception. Please find my detailed points below.  
+
+1. The ideal observer models only provide adequate fits when equipped with scaling parameters that account for "sub-optimal" behavior. The required scaling is not negligible, rescaling the optimal integration weights by \(\sim 40\) to \(50\%\) . Therefore, it is not clear whether the observers' behavior is at all optimal, beyond resembling some qualitative features of the data. The authors could make a much stronger case when quantitatively accounting for the sub-optimal behavior. For instance, how strong would regression to the mean of orientation judgments need to be (put forward by the authors as an explanation of sub-optimality) in order to match this scaling? Is this consistent with the empirical data?  
+
+2. Related to point 1, it is not clear in how far the empirical features are exclusively accounted for by Bayes-optimal integration versus other forms of (non-optimal) integration. As the authors note in their discussion feature 1 (orientation uncertainty) could be captured by obligatory integration models. Feature 2 (flanker-target similarity) could be explained by interference between similarly tuned, and therefore more strongly interconnected neural populations. For feature 4 (global vs local context), it seems that one could develop an alternative optimal observer that integrates local instead of global context. That is, I do not understand which optimality consideration would strictly dictate global versus local integration.  
+
+I believe most of these concerns of whether the behavioral features really arise from optimal integration could be mitigated by improving point 1 above, i.e. providing a more detailed quantitative explanation of behavior, rather than absorbing a considerable mismatch between predictions and data into one or two unexplained "sub-optimality" parameters.  
+
+3. It is not clear whether the behavioral benefits examined in this experiment are due to crowding or ensemble perception. While these appear to be at least partially distinct phenomena, they can co-occur ("Reexamining the possible benefits of visual crowding: dissociating crowding from ensemble percepts" Bulakowski et al., 2011). Cicchini et al. test the influence of target-distractor distance, and demonstrate that bias depends on distance, as expected for a crowding effect. However, I would contest that increasing flanker-target distance also alters the ensemble, and can therefore also impact ensemble perception. Perhaps one way to address this issue would be to test whether or not similar integration effects occur for more foveally presented stimuli, i.e. in the absence of crowding (albeit under matched conditions of visual uncertainty). If they do, the current observations would perhaps be better explained as resulting from ensemble perception, while crowding merely co-occurs in the current setup.  
+
+Minor comments:  
+
+Figure 5B. Minimum scatter appears to occur at 0 deg, while the ideal observer models predict the
+
+<--- Page Split --->
+
+minimum to occur at 15 degrees. I am curious whether the authors have any explanation/speculation of why the bias and variance data diverge in this aspect.  
+
+In their introduction the authors state "Crowding impacts on many important daily tasks, such as face recognition and reading [...]" I would be curious how the authors reconcile this view that crowding appears to negatively impact perception in real world scenarios ("daily tasks") with their optimal integration theory.
+
+<--- Page Split --->
+
+## GENERAL  
+
+We thank the editor and particularly the reviewers for their time and very helpful advice. We have taken all the suggestions on board, definitely resulting in an improved manuscript. We trust it is now acceptable for publication.  
+
+We have marked the changes in blue on the revised manuscript.  
+
+David Burr  
+
+For the authors  
+
+## REVIEWER COMMENTS  
+
+Reviewer #1 (Remarks to the Author):  
+
+This is an outstanding manuscript. The authors propose a novel and fascinating connection between crowding and serial dependence, two extensively studied areas of perception and cognition. They thoroughly test their idea psychophysically and with modeling. The results support the hypothesis and this will stimulate a lot of future research. I know this hypothesis will be provocative in the field, and not everyone will agree (it's a fairly contentious field), but this is a strength; the manuscript is exceptionally well balanced and approaches the issues in a most constructive way. The crowding field has been somewhat stagnant for years, and the authors' novel connection is much needed inspiration for researchers to pursue new directions. I expect this paper will motivate a great flurry of new experiments. I have a few minor suggestions below, but these are just requested clarifications, nothing major. Given the broad connections this manuscript makes across fields and the novelty of the idea and results, the manuscript certainly merits publication in Nature Comms.  
+
+We thank the reviewer for their kind words, and agree that the findings will be contentious, but hopefully stimulale useful research.  
+
+Minor points:  
+
+Flanker Similarity and the (unmentioned) Diagnostic criteria for crowding. There's a nod to these diagnostic criteria (eg, Whitney & Levi, 2011) but not a direct statement. The critical spacing one is key of course (p. 6)—and the model does a great job predicting that—but it's not the only one. Another key characteristic directly addressed in the MS is similarity. This is relevant here bc the prior literature had little explanation for why "similarity" matters in the way that it does (eg similarity modulates crowding and dissimilarity releases crowding). The authors' idea of a connection between serial dependence and crowding, and their model, is very powerful and important in part bc it provides that "why". In future work it will be interesting to test if other diagnostic criteria like inner- outer flanker asymmetry, upper lower visual field diff, etc also hold. This isn't necessary here but readers may wonder and the authors could prompt that question and help motivate the important follow up research.  
+
+Thanks for this important suggestion. We now mention the diagnosis criteria more clearly in introduction, and pick it up again in discussion.  
+
+P2, "tasks like or face recognition" delete "or" and perhaps add a reference here. Maybe Farzin et al 2009 (for faces) or the cited reviews (if this is a generic statement about objects).  
+
+P3, "qualitatively and qualitatively". Perhaps one of these was intended to be "quantitatively"?  
+
+Fig 1b. Using red outline and blue outline around the respective panels (or at least red and blue color somehow in those two panels) would help readers follow the correspondence between all the Figs; red always indicates high reliability target. Might as well start using that rule in fig 1b.  
+
+## Excellent idea  
+
+P.4. "...Formally the modeling section" should be "formally in the..."  
+
+P5. "...With difference between..." should have "the" or an "s" after "difference"  
+
+Fig 2, abscissa. Add clarification that this axis is "difference" in orientation. It's not absolute orientation, right?  
+
+Fig 2. Where would isolated (single) targets be on this graph?
+
+<--- Page Split --->
+
+Unfortunately we did not measure the effects without flankers.  
+
+P.11. Is the "signature" of the "signature function" the derivative- of- Gaussian shape in the SD literature? If so, perhaps mention that or explain what is meant by "signature"  
+
+P 12. The first sentence of the "causal inference model" section. That first sentence is too difficult to parse or understand. Not just because the word "form" probably isn't intended. Rephrasing could help a lot.  
+
+P12. "...the weight assigned of is the..."Rephrase, please.  
+
+Aside from these very minor points, this is an excellent manuscript.  
+
+Thank you very much, all the minor points have all been dealt with.  
+
+Reviewer #2 (Remarks to the Author):  
+
+This is a fascinating manuscript, with novel findings that present a new perspective on a widely studied phenomenon. The authors examine visual crowding, the disruptive effect of clutter on object recognition. A large body of research has depicted this effect as the 'fundamental bottleneck on object recognition' in peripheral vision especially. As a result, we know a great deal about the way this process affects object recognition and the potential mechanisms. What is much less clear is why crowding occurs in the first place. This manuscript presents an interesting answer to this question by considering its usefulness.  
+
+Thank you for the kind words. Thank you also for the very detailed help you have given, providing useful references and encouraging us to make clearer our ideas.  
+
+The broad approach here is to compare crowding with properties of 'serial dependence', the effect whereby judgements of a stimulus are influenced by the presentation of other stimuli in prior trials. With this comparison, the authors ask whether crowding can be considered to be an efficient/optimal process, rather than reflecting a disruptive bottleneck. Several predictions are made in this case, all of which are ultimately argued to be supported by the data. The authors ask observers to judge the orientation of shapes made from an outline of dots. They first observe greater biases and higher response scatter with "low reliability" near- circular target stimuli that are more difficult to judge, compared with "high reliability" elliptical targets. Second, they note that response scatter is greatest when the orientation of the flankers is close to the target, with a decrease as dissimilarity decreases (that is, performance improves as crowding increases, showing its efficiency). Finally, the pattern of biases follows the mean orientation of flankers rather than an independent combination, which is used to justify a higher- level model. The manuscript is well written and engaging, and presents a provocative view of a widely studied process. If the findings here are true then this presents an important aspect of our understanding. I do have a number of issues with the manuscript as it stands, however.  
+
+## 1. The pattern of response scatter  
+
+The main issue concerns the second finding - that response scatter is greatest (i.e. performance is worst) when the orientation of the flankers is most similar to the target. This finding is a key aspect of the proposal that crowding is efficient/optimal, since errors decrease as the strength of crowding increases. If true however, this finding is inconsistent with a large literature on the effect of target- flanker similarity in crowding. More typically, crowding is greatest when target and flanker elements are most similar to one another, decreasing as their dissimilarity increases (the opposite of the current observation). This has been found for a range of stimulus properties including contrast polarity, color, spatial frequency, and direction (Kooi, Toet, Tripathy, & Levi, 1994; Chung, Levi, & Legge, 2001; Gheri, Morgan, & Solomon, 2007), and in particular for orientation judgements (Andriessen & Bouma, 1976; Wilkinson, Wilson, & Eilenberg, 1997), similar to those used in the present study. In those latter studies, flankers that share similar orientations to the target induce the most crowding, with less crowding as the orientation of the flankers rotates away. Given that a major premise of the current study rests on the opposite finding, this discrepancy needs explanation and/or further exploration. The authors do in fact cite some of these studies to begin the manuscript, describing the patterns above, but the discrepancy with the current results is subsequently ignored. How can this discrepancy be explained, and how does this fit with the central arguments regarding the efficiency/optimality of crowding?  
+
+This important comment shows that we need to do a better job explaining our results and ideas. Firstly, we assume that the first sentence was a typo - response scatter is least (precision greatest) when orientations coincide (where crowding is greatest). That may seem counter- intuitive, but it depends on how you measure crowding. Typically it is percent correct, or perhaps contrast sensitivity. We are saying that RMS Errors (which comprise both accuracy and precision) are reduced, because although average accuracy decreases (strong bias), the increased precision more than offsets the bias, resulting in lower RMSE (radial distance in Fig 2C). However, other performance measures need not necessarily improve. For example, simple measures of accuracy (whether the reproduction was near veridical) would be low when the bias is high, as the orientation
+
+<--- Page Split --->
+
+judgement would seldom be "correct"; improved precision would not increase accuracy, and possibly decrease it, all responses become more tightly grouped around the incorrect bias. The only paper to measure separately bias and precision that we know of is Solomon, Felisberti and Morgan (JoV 2004: thanks for the pointer), and they report results very similar to ours (their Figure 4A). Also their data indicate a reduction in RMS Error (although they did not describe their results that way), shown in this figure below derived from their data. We have tried to make the explanations clearer in the results and discussion sections, and have added a brief paragraph discussing the apparent paradox.  
+
+![PLACEHOLDER_10_0]
+
+<center>Figure 1: RMSE calculated from Figure 4A of Solomon et al., 2004 </center>  
+
+There seem to me at least two possibilities to explain the discrepancy. One is that the authors have not fully measured the range of possible target- flanker differences in orientation. Targets are presented at either 35 or 55 degrees rotation, with flankers that differ from these values by up to \(\pm 45\) degrees. Response scatter peaks at the highest values measured ( \(\pm 45\) degrees). It is however possible then that these values may drop again as the differences further increase, up to their maximum of 90 degrees from the target orientation. It is typically these 90 degree values that are compared in order to show target- flanker similarity effects (Andriessen & Bouma, 1976; Wilkinson, Wilson, & Ellemberg, 1997), and I suspect that if the measurements continued here that performance would drop again. Indeed - patterns of this nature have been reported in a prior study (Solomon, Felisberti, & Morgan, 2004). There, orientation sensitivity is high when flankers are similarly oriented to the target, drops for orientations up to \(\pm 45\) degrees, and then increases again as the rotation continues to 90 degrees. The same may be true of the present stimuli if a larger range of orientations were tested. Would that not alter the interpretations regarding the efficiency of this process?  
+
+See above response for the explanation of apparent discrepancy. It is interesting that precision (but not bias) improved for \(90^{\circ}\) flankers in Solomon et al. Unfortunately we did not measure out that far. However, it would not change our story, as we are interested in the range where the flankers cause crowding by biasing results; in that range there is a clear trade- off between accuracy and precision for both our and their data. Our data show maximum bias at a lower orientation than theirs (about \(20^{\circ}\) compared with their \(45^{\circ}\) ), so it did not seem necessary to measure beyond \(45^{\circ}\) .  
+
+A second possibility is the eccentricity - the authors present their stimuli 26 degrees from fixation. Prior observations of target- flanker similarity have tended to use lower eccentricities. Given that some properties of crowding change with eccentricity, e.g. the response biases (Mareschal, Morgan, & Solomon, 2010) and of course the well- known effects of spatial extent (Bouma, 1970), it could be that the present results are something that only arises in the far periphery. Were this the case, however, the question remains - why is efficiency evident in the present results and not these other studies?  
+
+It is true that we used a large eccentricity, to maximize the effects, but our results agree with the only other study where efficiency can be calculated (Solomon et al, above figure), who worked at the much lower eccentricity of \(3.7^{\circ}\) (now mentioned).  
+
+To summarize, the results regarding response scatter appear to follow the opposite pattern to a range of well- established and replicated findings in the literature. The premise of the paper rests heavily on this observation. The authors need to demonstrate that this pattern is reliable by extending the range of their measurements in some way and/or by addressing this discrepancy with prior results. If the current results show efficiency, what does that say about all of these other results? If crowding is only efficient in these limited circumstances, is it really efficient?
+
+<--- Page Split --->
+
+We hope the above comments address this seeming paradox to the reviewer's satisfaction.  
+
+## 2. The lack of an unflanked baseline  
+
+Part of the issue of interpretation with the above response scatter data also relates to the lack of an unflanked baseline. Typically, performance with flankers is used to measure crowding, with an unflanked baseline (with an isolated element) used to measure uncrowded performance. The authors here take their baseline using flanked performance, using flankers with orientations at the extremes of their range (p14). Given the odd pattern of response scatter (as above), this assumption is problematic. If unflanked performance is more like the values with a flanker difference of 0 degrees, then performance would go from largely unaffected with the 0 degree flankers to impaired with the \(\pm 45\) degree flankers, rather than from improved with the 0 degree flankers to unaffected with the \(\pm 45\) degree flankers, as the authors argue. Does that not change the interpretation of the results and their efficiency/optimality substantially?  
+
+Unfortunately we did not measure a baseline. We did not think this is a major problem, but in retrospect it would have been useful to do so.  
+
+## 3. The distinction with 'low-level' pooling models of crowding  
+
+The authors contrast their findings with 'low level pooling models', which do not seem to me to be at odds with the present results. This distinction begins in the abstract (e.g. on line 2), where low level models are contrasted with their 'alternative hypothesis', and continues throughout, e.g. on p15 of the discussion, where it is argued that pooling models cannot explaining the effects of flanker orientation. Later on however, the authors describe their model as a pooling process (p17). The mechanism proposed for their model, with large receptive fields in areas like V4 (p16), also sounds very similar to ideas raised in various pooling models. For instance, processes of 'population pooling' have attributed the crowding of orientation signals to pooling within receptive fields in area V2 or V4 (van den Berg, Roerdink, & Cornelissen, 2010; Harrison & Bex, 2015). Similar arguments are also made by 'high dimensional' pooling models (Rosenholtz, Yu, & Keshavar, 2019). In fact, patterns of bias that are very similar to those in Figure 2A of this manuscript have been reported previously and accounted for via pooling processes (Greenwood & Parsons, 2020). In this latter case, the model accounts for target- flanker similarity effects via variations in the weights applied to the flankers. I don't see why this is inconsistent with the variations shown in the current study.  
+
+The authors distinguish between two possible models, both of which seem like pooling models to me. One is a 'low level' version in which the interactions happen independently between each flanker and the target, linked with a feedforward local process. The other involves a broader integration in which the flankers have a combined influence on the target, linked with recurrent feedback interactions. The latter does not seem wholly distinct from the operation of the most recent population pooling models (Harrison & Bex, 2015; Greenwood & Parsons, 2020) described above however. In those cases, the flankers affect the target through their combination within a single population response. My feeling is that the results of Experiment 2 in the present study would be entirely consistent with these models - when flanker orientations vary independently, their combined population response would have a shifted mean that would tend to alter the subsequent judgements related to the target. If so, then I do not think these results are inconsistent with pooling, nor do they provide clear evidence for feedback. This is not to take away from the novelty of these findings, however - I agree that the results provide clear evidence that the flankers do not interact independently with the target. The distinction between the two models presented here is certainly interesting, but their physiological basis is clearly overstated.  
+
+Indeed most models of crowding since Morgan's Nature paper involve some sort of obligatory pooling. The two novelty of what we propose are that the pooling is "intelligent", occurring when it leads to improved efficiency (measured by RMS Error); and that it is relatable to serial dependence, allowing cross- fertilization of the two fields. No previous model that we are aware of predicts this. Certainly there are elements in common, especially with our preferred model involving compulsory (unintelligent) pooling of signals; but the key difference is that this integrated information is not to final output but is combined intelligently with the more local signal. We have tried to make that clearer in the text.  
+
+## 4. Effects of target reliability  
+
+The first result reported in the manuscript is that biases are greater and response scatter higher with "low reliability" near- circular target stimuli that are more difficult to judge, compared with "high reliability" elliptical targets. This effect is attributed to reliability, and explained via a Bayesian framework. Its relation to similar results with alternative explanations is unexplored, however. Most notably, crowding is strongest with flankers of high luminance contrast (Chung, Levi, & Legge, 2001; Pelli, Palomares, & Majaj, 2004). Lowering the target contrast can also increase crowding (Felisberti, Solomon, & Morgan, 2005). Assimilative biases related to orientation judgements are also increased when noise is added to stimuli (Mareschal, Morgan, & Solomon, 2010). Can all of these effects be understood via reliability? It seems to me there is an alternative explanation that crowding is determined by the strength of the target signal, relative to the strength of the
+
+<--- Page Split --->
+
+flanker signal(s). Could these effects, including those of the present study, be understood as signal strength rather than reliability per se?  
+
+This is a good point, and we have now added those references. Technically, reliability is the inverse of the variance of the underlying noise distribution. Variance will certainly be affected by contrast and by noise in the way suggested (these have been the standard techniques of manipulating reliability in the multi- sensory literature), so yes, signal strength will increase reliability. However, given that our modelling is quantitatively based on reliability of flanker and target, we prefer to remain within that framework.  
+
+It is also interesting to note that the stimuli we employ (ellipses defined by dots), on the other hand allow manipulating reliability while keeping more basic parameters (contrast, visibility etc) as matched as possible. This suggests that the framework we chose could be a more general one and could encompass those special cases reported in the literature  
+
+## 5. The relation to serial dependence  
+
+Much is made of the similarities between serial dependence and crowding, which I agree is a fascinating link to make. The arguments for efficiency in this context also sound to me like arguments made more broadly in vision for the principles of redundancy reduction (Attneave, 1954), including for processes like adaptation (Clifford, 2002) and surround suppression (Rao & Ballard, 1999). Could the similarities here in fact indicate a broader link in the form of a "canonical computation" across all of visual perception? I wonder if the strong link to serial dependence is a little short- sighted in this sense.  
+
+This is a great idea, but we feel it goes beyond this paper. We would prefer to link crowding to a specific and well studied phenomenon, like serial dependence, rather than trying to push our claims too far at this stage. But we expect that the idea of canonical calculation will prove to be correct.  
+
+## 5. The neural basis of crowding  
+
+The idea of crowding relating to higher cortical areas like V4 is attributed to Pelli & Tillman (p2), but this idea derives from earlier work (Motter & Simoni, 2007; Motter, 2009). Others have also linked crowding with receptive field sizes in areas like V2 (He, Wang, & Fang, 2019).  
+
+Thanks for this feedback, we now have acknowledged these earlier scholars.  
+
+## 6. Stimulus details  
+
+Was the rotation of flankers taken from the target orientation on each trial, such that the \(\pm 45\) degree range differed in terms of absolute orientations for the 35 and 55 degree targets?  
+
+Yes it was.  
+
+Additionally, can we be sure that the judgements made by observers concern the orientation of these stimuli, rather than another property? Given the dotted nature of the stimuli used in the present task, perhaps observers are not judging orientation, but rather another property like the position of the outermost dots in the elements. This could allow a kind of relative position or Vernier judgement. Prior studies have tended to use line elements or Gabors in this context - if true, could this explain the difference with the studies of target- flanker similarity described above?  
+
+If we understand correctly the reviewer is asking us to consider the possibility that observers are not judging orientation of the main axis of the ellipse but rather the relative position of the outermost dot of the target object. This quantity could provide a rough proxy for orientation in that when the target exceeds (i.e. it is more to the right) the flankers likely the target is more horizontal (and vice versa if more inmost). If this was the case however in the "rounded target - slim flankers" the target would not exceed the flankers in this metric and reports should lean towards vertical. Conversely in the "slim target - rounded flanker", condition. These two predictions however are not met as there are no substantial biases.  
+
+Another possibility is that observers were judging the relative orientation (or position) of the two outmost dots of each stimulus. This would enable for instance judging the absolute orientation of the object (whereas previous hypothesis only would inform on relative orientation respect to the flanker). However, this mechanism cannot account for the clear difference of precision between the two types of stimuli.
+
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+
+## References  
+
+Andriessen, J. J., & Bouma, H. (1976). Eccentric vision: Adverse interactions between line segments. Vision Research, 16(1), 71- 78. Attneave, F. (1954). Some informational aspects of visual perception. Psychological Review, 61(3), 183- 193. Bouma, H. (1970). Interaction effects in parafoveal letter recognition. Nature, 226, 177- 178. Chung, S. T. L., Levi, D. M., & Legge, G. E. (2001). Spatial- frequency and contrast properties of crowding. Vision Research, 41, 1833- 1850. Clifford, C. W. G. (2002). Perceptual adaptation: Motion parallels orientation. Trends in Cognitive Sciences, 6(3), 136- 143. Felisberti, F. M., Solomon, J. A., & Morgan, M. J. (2005). The role of target salience in crowding. Perception, 34(7), 823- 833. Gheri, C., Morgan, M. J., & Solomon, J. A. (2007). The relationship between search efficiency and crowding. Perception, 36(12), 1779- 1787. Greenwood, J. A., & Parsons, M. J. (2020). Dissociable effects of visual crowding on the perception of color and motion. Proceedings of the National Academy of Sciences of the United States of America, 117(14), 8196- 8202. Harrison, W. J., & Bex, P. J. (2015). A Unifying Model of Orientation Crowding in Peripheral Vision. Current Biology, 25(24), 3213- 3219. He, D., Wang, Y., & Fang, F. (2019). The critical role of V2 population receptive fields in visual orientation crowding. Current Biology, 29(13), 2229- 2236. e2223. Kooi, F. L., Toet, A., Tripathy, S. P., & Levi, D. M. (1994). The effect of similarity and duration on spatial interaction in peripheral vision. Spatial Vision, 8(2), 255- 279. Mareschal, I., Morgan, M. J., & Solomon, J. A. (2010). Cortical distance determines whether flankers cause crowding or the tilt illusion. Journal of Vision, 10(8):13, 1- 14. Motter, B. C. (2009). Central V4 Receptive Fields Are Scaled by the V1 Cortical Magnification and Correspond to a Constant- Sized Sampling of the V1 Surface. Journal of Neuroscience, 29(18), 5749- 5757. Motter, B. C., & Simoni, D. A. (2007). The roles of cortical image separation and size in active visual search performance. Journal of Vision, 7(2(6)), 1- 15. Pelli, D. G., Palomares, M., & Majaj, N. J. (2004). Crowding is unlike ordinary masking: Distinguishing feature integration from detection. Journal of Vision, 4(12), 1136- 1169. Rao, R. P. N., & Ballard, D. H. (1999). Predictive coding in the visual cortex: A functional interpretation of some extra- classical receptive- field effects. Nature Neuroscience, 2(1), 79- 87. Rosenholtz, R., Yu, D., & Keshvari, S. (2019). Challenges to pooling models of crowding: Implications for visual mechanisms. Journal of Vision, 19(7), 1- 25. Solomon, J. A., Felisberti, F. M., & Morgan, M. J. (2004). Crowding and the tilt illusion: Toward a unified account. Journal of Vision, 4, 500- 508. van den Berg, R., Roerdink, J. B. T. M., & Cornelissen, F. W. (2010). A Neurophysiologically Plausible Population Code Model for Feature Integration Explains Visual Crowding. PLoS Computational Biology, 6(1), e1000646. Wilkinson, F., Wilson, H. R., & Ellemberg, D. (1997). Lateral interactions in peripherally viewed texture arrays. Journal of the Optical Society of America. A, Optics, Image Science, and Vision, 14(9), 2057- 2068.  
+
+## Reviewer #3 (Remarks to the Author):  
+
+Cicchini and colleagues put forward the hypothesis that crowding is results from Bayes- optimal integration of visual targets with spatial context. The authors identify four features of their empirical data that are consistent with Bayes- optimal integration: (1) Crowding is strongest for reliable flankers and unreliable targets, (2) Crowding depends on flanker- target similarity (here orientation), (3) precision of orientation judgments increases with increasing flanker- target similarity, and (4) Crowding depends on similarity of targets to average flanker orientation, not individual flanker orientations. The authors present two ideal observer models (a Bayesian ideal observer, and a causal inference model), which can reproduce the above features of the empirical data.  
+
+While I find the hypothesis that crowding results from optimal integration intriguing, I am somewhat reserved when it comes to the evidence provided in the current study. I am also not convinced that the behavioral benefits described here can be ascribed to crowding rather than ensemble perception. Please find my detailed points below.  
+
+1. The ideal observer models only provide adequate fits when equipped with scaling parameters that account for "sub-optimal" behavior. The required scaling is not negligible, rescaling the optimal integration weights by \(\sim 40\) to \(50\%\) . Therefore, it is not clear whether the observers' behavior is at all optimal, beyond resembling some qualitative features of the data. The authors could make a much stronger case when quantitatively accounting for the sub-optimal behavior. For instance, how strong would regression to the mean of orientation judgments need to be (put forward by the authors as an explanation of sub-optimality) in order to match this scaling? Is this consistent with the empirical data?
+
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+
+Thanks for this important suggestion. We were trying to keep our models as simple as possible to be more accessible to the reader, but the calculation proposed is quite simple. Regression to the mean compresses the output by about \(30\%\) , which accounts for much of the underestimation. We now need a scaling factor of only 0.7 to fit the data. The main features of the models is that the same models that fit well serial dependence capture the DoG pattern of results (to a scaling factor), but we agree it is more impressive when the scaling factor is close to unity.  
+
+2. Related to point 1, it is not clear in how far the empirical features are exclusively accounted for by Bayes-optimal integration versus other forms of (non-optimal) integration. As the authors note in their discussion feature 1 (orientation uncertainty) could be captured by obligatory integration models. Feature 2 (flanker-target similarity) could be explained by interference between similarly tuned, and therefore more strongly interconnected neural populations. For feature 4 (global vs local context), it seems that one could develop an alternative optimal observer that integrates local instead of global context. That is, I do not understand which optimality consideration would strictly dictate global versus local integration.  
+
+I believe most of these concerns of whether the behavioral features really arise from optimal integration could be mitigated by improving point 1 above, i.e. providing a more detailed quantitative explanation of behavior, rather than absorbing a considerable mismatch between predictions and data into one or two unexplained "suboptimality" parameters.  
+
+We trust that the improved absolute fit goes some way towards addressing the referee's concerns. We do of course accept that mechanisms of the type suggested could be involved, such as interconnections between similarly tuned neural populations (which we now mention, pointing out they are not consistent with the second experiment). However, we believe that our models put considerable constraints on how these mechanisms act. We also believe that it is constructive to relate crowding to serial dependence, which is currently being studied intensely, prompting cross- fertilization of ideas and discussions between the two important fields of research.  
+
+3. It is not clear whether the behavioral benefits examined in this experiment are due to crowding or ensemble perception. While these appear to be at least partially distinct phenomena, they can co-occur ("Reexamining the possible benefits of visual crowding: dissociating crowding from ensemble percepts" Bulakowski et al., 2011). Cicchini et al. test the influence of target-distractor distance, and demonstrate that bias depends on distance, as expected for a crowding effect. However, I would contest that increasing flanker-target distance also alters the ensemble, and can therefore also impact ensemble perception. Perhaps one way to address this issue would be to test whether or not similar integration effects occur for more favorably presented stimuli, i.e. in the absence of crowding (albeit under matched conditions of visual uncertainty). If they do, the current observations would perhaps be better explained as resulting from ensemble perception, while crowding merely co-occurs in the current setup.  
+
+Thanks for pointing us to this interesting paper, of which we were unaware. It is interesting that ensemble perception and crowding follow partially different rules, opening the way for interesting experiments. In our experiment we only measured crowding (judging orientation of a single target, rather than the average). It would certainly be interesting to do the converse (ensemble judgements) to see if the two phenomena followed similar rules. If they do not, it would be further proof that the two are at least partially different phenomena. This is of course a large new study (which the second author may pursue for his PhD), but we do now mention ensemble perception, and cite the important study mentioned. Thank you  
+
+## Minor comments:  
+
+Figure 5B. Minimum scatter appears to occur at 0 deg, while the ideal observer models predict the minimum to occur at 15 degrees. I am curious whether the authors have any explanation/speculation of why the bias and variance data diverge in this aspect.  
+
+Indeed, the prediction was off. However, we had previous calculated variance of the aggregate participant together, so different individual biases added artificially to the variance. We now remove the individual biases from the calculation of scatter (essentially calculating variance separately for each participant and averaging), and the result is far closer to the predicted \(15^{\circ}\) (see new figure 5B). We explain this procedure in the methods.  
+
+In their introduction the authors state "Crowding impacts on many important daily tasks, such as face recognition and reading [...]" I would be curious how the authors reconcile this view that crowding appears to negatively impact perception in real world scenarios ("daily tasks") with their optimal integration theory.  
+
+This is a very good point, thank you. Perhaps it goes a bit beyond the scope of this study, but we now add a paragraph discussing how optimizing for one aspect (minimal RMS Errors) may lead to sub- optimal behaviour of others (such as face recognition), as occurs in other forms of Bayesian optimization.
+
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+
+## REVIEWER COMMENTS  
+
+Reviewer #1 (Remarks to the Author):  
+
+The authors have thoroughly addressed all of the reviewer concerns, and the manuscript is acceptable for publication.  
+
+Reviewer #2 (Remarks to the Author):  
+
+As in the first submission, this is a fascinating manuscript, with novel findings that present a new perspective on the widely studied phenomenon of crowding. The revisions have done much to clarify the contributions of this work and the nature of the associated analyses. My major issue was previously with the findings regarding response scatter and their relation to prior work, which is now addressed to some extent in the revised manuscript. Many of the other issues have also been resolved. However, there remains one major issue in particular that has been completely ignored in the revisions, along with some minor issues. It is clear to me that this paper presents a novel and useful viewpoint to the literature, but it is especially important that these claims be supported by evidence. At the moment it is still not clear to me that this is the case.  
+
+## 1. The lack of an unflanked baseline  
+
+The major shortcoming with the revised manuscript is the lack of an unflanked performance baseline - the authors seek to measure crowding (recognition of a target when surrounded by flankers) but never measure performance in the absence of crowding (a target without flankers). This issue was raised in the first submission, to which the authors responded that it "would have been useful", but this shortcoming was neither measured nor is its absence acknowledged in the revised manuscript. This absence leads to problematic assumptions about the data, a potentially problematic implementation within the models, and problematic statements about the nature of crowding.  
+
+The authors assert at many points that "crowding improves overall performance" (p6), and that they observe "improved perceptual performance" (p16), "a reduction in response scatter [and] total RMS error" (p16), "to improve performance" (p17) and "improved performance" (p20). However, without the measurement of an unflanked baseline where crowding is absent (i.e. measurement of orientation perception for an isolated target without flankers), it is not clear what this improvement is relative to.  
+
+In the absence of this measurement, the authors have assumed that the most extreme values of target- flanker difference that were tested correspond to the performance baseline. As noted in my first review, this is not at all clear. Prior studies have shown that the reduction in crowding with increased dissimilarity between target and flankers does not often return performance to the unflanked baseline. This can be seen for instance in the cited studies on orientation judgements (Wilkinson, Wilson, & Ellemberg, 1997; Solomon, Felisberti, & Morgan, 2004) - a target surrounded by orthogonal flankers is not recognized as well as a target presented on its own. In the data of Wilkinson et al, for instance, the threshold elevations (from unflanked) can remain as high as 2.5 times the unflanked baseline, even with dissimilar flanker elements. It is not possible to assume that crowding is absent so long as the flankers remain present, in other words.  
+
+If the authors were able to demonstrate that the response scatter with similar flankers (near the middle of Figure 2b) is indeed better than unflanked performance, this would be a convincing demonstration that the presence of crowding improves performance relative to its absence (with a target in isolation). Otherwise, what we are looking at is an improvement in performance when crowding is strong vs. when it is reduced. A far more problematic outcome would be that that the unflanked baseline may in fact yield very low values of response scatter, which would correspond more closely to the values with similar flankers (near the middle). In this case, all of the performance
+
+<--- Page Split --->
+
+with flankers would in fact be a decrement/impairment, rather than an improvement. In other words, the apparent benefits of crowding that are described here would simply be a case of the authors arbitrarily relabelling "up" as "down". Without these measurements, we can never know. The question of optimality is never truly addressed until we do.  
+
+This is not simply a quibble about stimulus details - it it is a key aspect of the measurements being performed here. Without these measurements, the way in which performance with these circumstances is unclear, as reflected in the lack of a meaningful performance comparison in the statements quoted above (i.e. is it that crowding improves performance relative to uncrowded performance, or simply that strong crowding is better than reduced crowding?).  
+
+These assumptions regarding performance also carry through to the modelling. On p15 the authors use the extreme points of the dataset as a baseline to estimate optimal sensory reliability. This in turn carries through to key operations of the model (equation 12). If the unflanked reliability is in fact lower than this point, can crowding truly be said to be optimal? Precision (when calculated as in this study) is certainly better when crowded biases are strongest (with small target- flanker differences) compared to when they are reduced, but the authors cannot say whether this is optimal compared to the absence of crowding altogether.  
+
+## 2. Assumptions in the model and optimality  
+
+Much has been improved in the discussion of how these findings relate to prior reports of impaired performance in tasks like letter and face recognition (p19). It seems to me however that a key aspect of this discrepancy relates to the assumptions of the model. The text on p11 states that "ideal responses...can be expressed as a linear weighted combination of [the] target and flankers...". But this is surely suboptimal when the task is to judge the orientation of the target and to ignore the flankers (as in prior studies on letter/face recognition etc). The updated description of the causal inference model makes this assumption more explicit where it is stated (p13) that "...an optimal blend [assumes] that the two curves originate from the same cause" (with a similar comment on p14). With this assumption in place, I could see how these interactions are optimal, and indeed given the structural regularities of the visual scene (that similar orientations, colors, etc are likely to be found together) this is perhaps a sensible assumption for the most part, as exploited by texture/statistical models of peripheral vision and crowding (Freeman & Simoncelli, 2011; Rosenholtz, Yu, & Keshvari, 2019). But it is problematic in tasks where the observer must recognize the target, ignoring the flankers. Although performance may then be optimal given this assumption, it is clearly not optimal given the task being required of observers in (most) crowding paradigms. In these cases, where the task is clearly very different (identify the target face amongst flankers, for instance), is there not therefore an over- application of this principle and thus a suboptimality? I suspect that this again relates back to the lack of an unflanked baseline in the authors' measurements, and the missing perspective that arises from this.  
+
+## 3. The findings regarding response scatter  
+
+The pattern of results obtained with response scatter are much more clear now. However, the authors now describe the discrepancy between the current and previous studies as being due to their separate measurement of bias and precision (p18). The work of Solomon, Felisberti, and Morgan (2004) is cited in this context as 'the only study to our knowledge' to have similarly measured bias and precision. A range of other studies have used this approach however, some of which report patterns that do not quite fit with that observed here, to my eye. For instance, Glen and Dakin (2013) report sensitivity and biases for orientation crowding, while Greenwood and Parsons (2020) measured crowded biases and precision/thresholds for color and motion. The patterns there do not quite match those of the current study, with the least precision arising when flankers are most similar to the target, though it could indeed be the case that on the whole the combination of bias and precision leads to a pattern of response scatter similar to that of the current work.
+
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+
+## 4. Relation to pooling models  
+
+4. Relation to pooling modelsThe discussion of the relationship to pooling models is much improved in the introduction and modelling sections. But there is still a statement in the discussion (p16) that the findings are 'difficult to reconcile' with pooling models. On the contrary, effects of target-flanker similarity are simulated by pooling models both in Greenwood and Parsons (2020) and in the context of texture pooling models (Rosenholtz, Yu, & Keshvari, 2019). Again, these approaches do not seem so dissimilar to that employed in the current study.  
+
+## 5. Missing references  
+
+5. Missing referencesAt points the authors make reference to prior literature without citing the studies being referred to, particularly in the new additions to the manuscript. This occurs on p2 ("Crowding is stronger in the upper than the lower visual field, and for radial than for tangential flankers") and p16 ("the myriad of experiments showing that similarities in shape...cause maximum crowding"). It is particularly unclear to me what studies the latter refers to.  
+
+## 6. Biases  
+
+6. BiasesIt would help the clarity of the manuscript to have the direction of biases explained somewhere, e.g. on p6 to explain that the errors in Figure 2 follow the orientation of the flankers, and that they presumably go in the opposite direction with some separations in Figure 3.  
+
+## References  
+
+Freeman, J., & Simoncelli, E. P. (2011). Metamers of the ventral stream. Nature Neuroscience, 14, 1195- 1201. Glen, J. C., & Dakin, S. C. (2013). Orientation- crowding within contours. Journal of Vision, 13, 1- 11. Greenwood, J. A., & Parsons, M. J. (2020). Dissociable effects of visual crowding on the perception of color and motion. Proceedings of the National Academy of Sciences of the United States of America, 117, 8196- 8202. Rosenholtz, R., Yu, D., & Keshvari, S. (2019). Challenges to pooling models of crowding: Implications for visual mechanisms. Journal of Vision, 19, 1- 25. Solomon, J. A., Felisberti, F. M., & Morgan, M. J. (2004). Crowding and the tilt illusion: Toward a unified account. Journal of Vision, 4, 500- 508. Wilkinson, F., Wilson, H. R., & Ellemberg, D. (1997). Lateral interactions in peripherally viewed texture arrays. Journal of the Optical Society of America. A, Optics, Image Science, and Vision, 14, 2057- 2068.  
+
+Reviewer #3 (Remarks to the Author):  
+
+I am still not entirely sure whether Nature Communications is the best outlet, as the study puts forward an intriguing idea, but leaves open many questions that would require more data. However, I agree with the authors and fellow reviewers that this is an interesting research direction.  
+
+I appreciate the authors' attempt to minimize the scaling factor, by quantitatively accounting for known suboptimalities due to the oblique bias. I am still not entirely convinced that we are dealing with optimal integration, or whether part of the required scaling is due to suboptimal integration, which would limit the authors' claim of crowding resulting from optimal integration. If not done in the current manuscript, this will be an important point for future studies.  
+
+I realize that I have been somewhat unclear about my previous point about crowding vs ensemble
+
+<--- Page Split --->
+
+perception. My main question was whether the spatial integration observed in the current experiments could perhaps be more general than just occurring for peripheral targets surrounded by flankers (i.e., the conditions under which crowding occurs). For instance, would a similar bias occur for very noisy \*foveal\* target stimuli surrounded by flankers, in the absence of crowding. That is, would observers generally rely on a weighted average of an ensemble, when visual information about the target is very poor. In the current experiments, crowding might increase the uncertainty/reliability of the target, which might prompt observers to integrate information of the spatial context. In this case, optimal integration would be the \*consequence\* of crowding, not the \*cause\* of crowding. If I understand the authors correctly, they claim that optimal integration is the cause of the crowding phenomenon. I would appreciate if the authors could clarify this point.  
+
+I also concur Reviewer #2 that an unflanked baseline (or orthogonal flankers) would help to clarify whether maximally similar flankers indeed enhance performance (better than baseline), or whether they are least detrimental (equal or worse performance than baseline).
+
+<--- Page Split --->
+
+Please find enclosed the response to the reviewers and the novel submission with all changes flagged in blue.  
+
+Reviewer #1 (Remarks to the Author):  
+
+The authors have thoroughly addressed all of the reviewer concerns, and the manuscript is acceptable for publication.  
+
+Reviewer #2 (Remarks to the Author):  
+
+As in the first submission, this is a fascinating manuscript, with novel findings that present a new perspective on the widely studied phenomenon of crowding. The revisions have done much to clarify the contributions of this work and the nature of the associated analyses. My major issue was previously with the findings regarding response scatter and their relation to prior work, which is now addressed to some extent in the revised manuscript. Many of the other issues have also been resolved. However, there remains one major issue in particular that has been completely ignored in the revisions, along with some minor issues. It is clear to me that this paper presents a novel and useful viewpoint to the literature, but it is especially important that these claims be supported by evidence. At the moment it is still not clear to me that this is the case.  
+
+## 1. The lack of an unflanked baseline  
+
+The major shortcoming with the revised manuscript is the lack of an unflanked performance baseline - the authors seek to measure crowding (recognition of a target when surrounded by flankers) but never measure performance in the absence of crowding (a target without flankers). This issue was raised in the first submission, to which the authors responded that it "would have been useful", but this shortcoming was neither measured nor is its absence acknowledged in the revised manuscript. This absence leads to problematic assumptions about the data, a potentially problematic implementation within the models, and problematic statements about the nature of crowding.  
+
+We have now measured baselines for the rounded targets condition (the main one, that leads to the strongest effects). We also measure with orthogonal flankers. The results are shown in Figure 2 as hollow squares/diamonds. The two thresholds are similar to each other, and worse than all the other flanker conditions. We hope this is sufficient for publication now.  
+
+The authors assert at many points that "crowding improves overall performance" (p6), and that they observe "improved perceptual performance" (p16), "a reduction in response scatter [and] total RMS error" (p16), "to improve performance" (p17) and "improved performance" (p20). However, without the measurement of an unflanked baseline where crowding is absent (i.e. measurement of
+
+<--- Page Split --->
+
+orientation perception for an isolated target without flankers), it is not clear what this improvement is relative to.  
+
+In the absence of this measurement, the authors have assumed that the most extreme values of target- flanker difference that were tested correspond to the performance baseline. As noted in my first review, this is not at all clear. Prior studies have shown that the reduction in crowding with increased dissimilarity between target and flankers does not often return performance to the unflanked baseline. This can be seen for instance in the cited studies on orientation judgements (Wilkinson, Wilson, & Ellemberg, 1997; Solomon, Felisberti, & Morgan, 2004) – a target surrounded by orthogonal flankers is not recognized as well as a target presented on its own. In the data of Wilkinson et al, for instance, the threshold elevations (from unflanked) can remain as high as 2.5 times the unflanked baseline, even with dissimilar flanker elements. It is not possible to assume that crowding is absent so long as the flankers remain present, in other words.  
+
+If the authors were able to demonstrate that the response scatter with similar flankers (near the middle of Figure 2b) is indeed better than unflanked performance, this would be a convincing demonstration that the presence of crowding improves performance relative to its absence (with a target in isolation). Otherwise, what we are looking at is an improvement in performance when crowding is strong vs. when it is reduced. A far more problematic outcome would be that that the unflanked baseline may in fact yield very low values of response scatter, which would correspond more closely to the values with similar flankers (near the middle). In this case, all of the performance with flankers would in fact be a decrement/impairment, rather than an improvement. In other words, the apparent benefits of crowding that are described here would simply be a case of the authors arbitrarily relabelling "up" as "down". Without these measurements, we can never know. The question of optimality is never truly addressed until we do.  
+
+This is not simply a quibble about stimulus details – it it is a key aspect of the measurements being performed here. Without these measurements, the way in which performance with these circumstances is unclear, as reflected in the lack of a meaningful performance comparison in the statements quoted above (i.e. is it that crowding improves performance relative to uncrowded performance, or simply that strong crowding is better than reduced crowding?).  
+
+These assumptions regarding performance also carry through to the modelling. On p15 the authors use the extreme points of the dataset as a baseline to estimate optimal sensory reliability. This in turn carries through to key operations of the model (equation 12). If the unflanked reliability is in fact lower than this point, can crowding truly be said to be optimal? Precision (when calculated as in this study) is certainly better when crowded biases are strongest (with small target- flanker differences) compared to when they are reduced, but the authors cannot say whether this is optimal compared to the absence of crowding altogether.  
+
+We accept your arguments, thank you, and have added the baseline to the main condition. We agree that this strengthens the manuscript.
+
+<--- Page Split --->
+
+## 2. Assumptions in the model and optimality  
+
+Much has been improved in the discussion of how these findings relate to prior reports of impaired performance in tasks like letter and face recognition (p19). It seems to me however that a key aspect of this discrepancy relates to the assumptions of the model. The text on p11 states that "ideal responses...can be expressed as a linear weighted combination of [the] target and flankers...". But this is surely suboptimal when the task is to judge the orientation of the target and to ignore the flankers (as in prior studies on letter/face recognition etc). The updated description of the causal inference model makes this assumption more explicit where it is stated (p13) that "...an optimal blend [assumes] that the two curves originate from the same cause" (with a similar comment on p14). With this assumption in place, I could see how these interactions are optimal, and indeed given the structural regularities of the visual scene (that similar orientations, colors, etc are likely to be found together) this is perhaps a sensible assumption for the most part, as exploited by texture/statistical models of peripheral vision and crowding (Freeman & Simoncelli, 2011; Rosenholtz, Yu, & Keshvari, 2019). But it is problematic in tasks where the observer must recognize the target, ignoring the flankers. Although performance may then be optimal given this assumption, it is clearly not optimal given the task being required of observers in (most) crowding paradigms. In these cases, where the task is clearly very different (identify the target face amongst flankers, for instance), is there not therefore an over- application of this principle and thus a suboptimality? I suspect that this again relates back to the lack of an unflanked baseline in the authors' measurements, and the missing perspective that arises from this.  
+
+We now stress that optimization of basic features while optimal strictly speaking, may still impact negatively in higher recognition processes. Indeed it is not uncommon that optimal processes lead to illusions such as the ventriloquist effect and the hollow face illusion  
+
+## 3. The findings regarding response scatter  
+
+The pattern of results obtained with response scatter are much more clear now. However, the authors now describe the discrepancy between the current and previous studies as being due to their separate measurement of bias and precision (p18). The work of Solomon, Felisberti, and Morgan (2004) is cited in this context as 'the only study to our knowledge' to have similarly measured bias and precision. A range of other studies have used this approach however, some of which report patterns that do not quite fit with that observed here, to my eye. For instance, Glen and Dakin (2013) report sensitivity and biases for orientation crowding, while Greenwood and Parsons (2020) measured crowded biases and precision/thresholds for color and motion. The patterns there do not quite match those of the current study, with the least precision arising when flankers are most similar to the target, though it could indeed be the case that on the whole the combination of bias and precision leads to a pattern of response scatter similar to that of the current work.  
+
+Thank you, we were not aware of these studies. We now reference them and point out the differences in results (possibly due to differences in the experimental techniques). But we also take the opportunity to add a penultimate paragraph mentioning that our results may not generalize
+
+<--- Page Split --->
+
+beyond orientation, encouraging experiments along these lines for other features such as motion and color. Thank you for prompting this caveat.  
+
+## 4. Relation to pooling models  
+
+The discussion of the relationship to pooling models is much improved in the introduction and modelling sections. But there is still a statement in the discussion (p16) that the findings are 'difficult to reconcile' with pooling models. On the contrary, effects of target- flanker similarity are simulated by pooling models both in Greenwood and Parsons (2020) and in the context of texture pooling models (Rosenholtz, Yu, & Keshvari, 2019). Again, these approaches do not seem so dissimilar to that employed in the current study.  
+
+Thank you. We have toned down our claims of novelty, but do still want to stress the major difference, of flexible pooling.  
+
+## 5. Missing references  
+
+At points the authors make reference to prior literature without citing the studies being referred to, particularly in the new additions to the manuscript. This occurs on p2 ("Crowding is stronger in the upper than the lower visual field, and for radial than for tangential flankers") and p16 ("the myriad of experiments showing that similarities in shape...cause maximum crowding"). It is particularly unclear to me what studies the latter refers to.  
+
+Thank you we now added relevant references  
+
+## 6. Biases  
+
+It would help the clarity of the manuscript to have the direction of biases explained somewhere, e.g. on p6 to explain that the errors in Figure 2 follow the orientation of the flankers, and that they presumably go in the opposite direction with some separations in Figure 3.  
+
+Thank you we now do.  
+
+## References  
+
+Freeman, J., & Simoncelli, E. P. (2011). Metamers of the ventral stream. Nature Neuroscience, 14, 1195- 1201.  
+
+Glen, J. C., & Dakin, S. C. (2013). Orientation- crowding within contours. Journal of Vision, 13, 1- 11.
+
+<--- Page Split --->
+
+Greenwood, J. A., & Parsons, M. J. (2020). Dissociable effects of visual crowding on the perception of color and motion. Proceedings of the National Academy of Sciences of the United States of America, 117, 8196- 8202.  
+
+Rosenholtz, R., Yu, D., & Keshvari, S. (2019). Challenges to pooling models of crowding: Implications for visual mechanisms. Journal of Vision, 19, 1- 25.  
+
+Solomon, J. A., Felisberti, F. M., & Morgan, M. J. (2004). Crowding and the tilt illusion: Toward a unified account. Journal of Vision, 4, 500- 508.  
+
+Wilkinson, F., Wilson, H. R., & Ellemberg, D. (1997). Lateral interactions in peripherally viewed texture arrays. Journal of the Optical Society of America. A, Optics, Image Science, and Vision, 14, 2057- 2068.  
+
+Reviewer #3 (Remarks to the Author):  
+
+I am still not entirely sure whether Nature Communications is the best outlet, as the study puts forward an intriguing idea, but leaves open many questions that would require more data. However, I agree with the authors and fellow reviewers that this is an interesting research direction.  
+
+I appreciate the authors' attempt to minimize the scaling factor, by quantitatively accounting for known suboptimalities due to the oblique bias. I am still not entirely convinced that we are dealing with optimal integration, or whether part of the required scaling is due to suboptimal integration, which would limit the authors' claim of crowding resulting from optimal integration. If not done in the current manuscript, this will be an important point for future studies.  
+
+I realize that I have been somewhat unclear about my previous point about crowding vs ensemble perception. My main question was whether the spatial integration observed in the current experiments could perhaps be more general than just occurring for peripheral targets surrounded by flankers (i.e., the conditions under which crowding occurs). For instance, would a similar bias occur for very noisy foveal target stimuli surrounded by flankers, in the absence of crowding. That is, would observers generally rely on a weighted average of an ensemble, when visual information about the target is very poor. In the current experiments, crowding might increase the uncertainty/reliability of the target, which might prompt observers to integrate information of the spatial context. In this case, optimal integration would be the consequence of crowding, not the cause of crowding. If I understand the authors correctly, they claim that optimal integration is the cause of the crowding phenomenon. I would appreciate if the authors could clarify this point.  
+
+We thank the referee for making their point more clear. It is certainly a good point, which we are not able to address at this stage (other than showing in Figure 3 that the assimilative effects disappear at large separations). We do now mention that this is an open question meriting further research (in the penultimate paragraph).
+
+<--- Page Split --->
+
+I also concur Reviewer #2 that an unflanked baseline (or orthogonal flankers) would help to clarify whether maximally similar flankers indeed enhance performance (better than baseline), or whether they are least detrimental (equal or worse performance than baseline).  
+
+We too were convinced, and did the extra measurements (open symbols in Figure 2).
+
+<--- Page Split --->
+
+## REVIEWERS' COMMENTS  
+
+Reviewer #2 (Remarks to the Author):  
+
+The authors have responded comprehensively to issues raised in the previous rounds of submission, and my major issues with the manuscript are resolved. The new baseline measurements (with an additional condition including orthogonally- oriented flankers) are a convincing addition, providing a clear reference for the arguments that crowding can improve the precision of the shape judgements measured by the authors.  
+
+As I have said in earlier rounds, this is a fascinating manuscript whose findings present a new perspective on a widely studied phenomenon. I do not agree with everything that is said, but the authors have sufficiently qualified their statements to the extent that the ideas are fully available for the reader to decide. I am sure this will inspire a great deal of future research.  
+
+Reviewer #3 (Remarks to the Author):  
+
+The authors have addressed the remaining issues. I have no further comments.
+
+<--- Page Split --->

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+
+# nature portfolio  
+
+Peer Review File  
+
+Changes in limiting factors for forager population dynamics in Europe across the Last Glacial- Interglacial Transition  
+
+![PLACEHOLDER_0_0]
+  
+
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+
+Reviewers' Comments:  
+
+Reviewer #1:  
+
+Remarks to the Author:  
+
+This study aims to identify the limiting climate factors of hunter- gatherer population density in Europe from the LGM to early Holocene, which is a tempting research question that, if answered convincingly, could provide clues in resilience and adaptation strategies of ancient human societies. The authors built statistical models (quantile Generalised Additive Models) of population density versus each of the 18 climate variables based on contemporary hunter- gatherer dataset, and hindcasted population density using paleoclimate outputs from a climate model. This workflow and statistical techniques are not new (e.g. Tallavaara et al. 2015 pnas), but selecting the factor that predicts the lowest population density across space and time, based on the concept of limiting factor, provides a fresh angle of view. However, I have some major concerns about the robustness of the analysis and significance of current results, as outlined below. Based on these, I cannot recommend its publication, at least not in its current form.  
+
+Robustness of results:  
+
+Mean temperature of the Warmest Month (MwM) is identified a major limiting factor during all critical periods (Fig. 4), but MwM is the variable that has the most severe non- analogy problem comparing present- day climate space and past climates (Fig. 2 and Supplementary material S2). Although mentioned at Lines 126- 128, considering the strong relevance to the main findings, the risk of an unreliable extrapolation out of the range of data used to fit the statistical model is higher than acknowledged here.  
+
+In addition, it is not clear to me why the authors chose 90th percentile of population density to do the hindcast. First, the results, in principle, would not be comparable to previous estimates in literature. Second, does the resulted limiting factors change dependent on the choice of the percentile? Although the general shape of the population density versus climate relationship looks similar across different percentiles for each individual climate factor (Lines 311- 312), it is the relative magnitudes between all predicted densities by these factors that ultimately selects the limiting factor. Thus, it is not straightforward whether your results are sensitive to the choice of percentiles.  
+
+Regarding the comparison between hindcasted population density and the archeological population proxy (Fig. 3), I would not say they are "in line with" each other (Lines 139- 140). The black curve in Fig. 3 starts to increase already since 18 ka, which is relatively flat in the red curve; the red curve increases significantly during GI1 and GS1, whereas it is stale in the black curve.  
+
+By the way, at Lines 101- 103, why do you separate higher and lower predictive accuracy by a threshold of "explained deviances \(< 0.79\) " According to Table 1, these predictors are all so close.  
+
+About mechanism insights:  
+
+From the results it is hard to infer mechanisms regarding how the identified limiting factor has constrained population density. This is limited by the fact that only temperature and precipitation and their variants were used as predictors, without direct information about productivity; whereas climate impacts population density via indirect effects on ecosystem attributes like NPP (e.g. Freeman et al. 2020, doi:10.1016/j.jas.2020.105168). Throughout the text the authors have tried to relate some of the factors to environmental productivity, but it was highly speculative. Let me take Lines 185- 192 as an example. During 14.7ka to 11.7ka, the importance of ET decreases while importance of MwM and temperature seasonality increases. But all three variables are linked to NPP (and possibly other aspects of the ecosystem). From these changes one still cannot judge what process is taking effect in the end.  
+
+Given this, why not use NPP as a predictor in the first place? Data availability for the hindcast should not be a problem since simulated NPP for the past 21,000 years are publicly accessible from some climate models already.  
+
+Significance of the results in archaeological perspective: I commend the authors' effort to put the (more of ecology- oriented) results into archaeological
+
+<--- Page Split --->
+
+context, but currently it is still limited in qualitative descriptions scattered in the text. If the authors could achieve a more systematic compilation of archaeological records regarding how these societies have tackled with the limiting climate factors and find a consistency with your hindcasted results in space and time, it would add much merit to this study, with broader significance and impacts.  
+
+Aside from the above concerns, the organization of Results and discussion needs to be improved. Adding sub- headings would help. Besides, descriptions of the results in the text should be more careful. Currently they are sometimes inconsistent with the table or figures. For example, at Lines 104- 110, it says seasonal temperature variables are among the lowest explained deviance, which is not the case as listed in Table 1.  
+
+A minor point is that the uncertainties/biases in the paleoclimate outputs of the CCSM3 climate model should be discussed.  
+
+Code availability: though not mandatory, it is strongly encouraged to make the code readily available so as to enable reproduction of the results.  
+
+Table 1: A conceptual confusion: MCM is not "extreme events". Same for MWM, PDM, and PWM. Extreme events are events that occur with low frequency, not the regular seasonal maxima or minima.  
+
+There are a few careless errors in the manuscript, for example:  
+
+Line 266: "16 climatic predictors": there are 18 climate variables in Table 1.  
+
+Line 289: it says "We use a subsample of 159 hunter- gatherers populations..." in the Reporting summary, while here it says "127 populations".  
+
+Table 1: Acronym of "Precipitation of the Wettest Month" should be PWM, not PDM.  
+
+Figure caption of Fig. 3: "Minimum temperature of the Coldest Month" should be "Mean...", and "Maximum temperature of the Warmest Month" should be "Mean..."  
+
+Figure caption of Supplementary material S1: it is not "the six most important environmental factors". Please check carefully throughout the paper.  
+
+Reviewer #2:  
+
+Remarks to the Author:  
+
+This is truly thought- provoking, highly interesting and novel contribution to the hunter- gatherer ecology. I very much like the approach of applying the analysis of ecological limiting factors, for the first time, to prehistoric hunter- gatherers. Below I have highlighted few issues that you could consider to revise to further improve the paper.  
+
+Best regards, Miikka Tallavaara  
+
+1.  
+
+Given that many of the climate predictors are highly correlated (as you also write in the manuscript), they will convey partly the same information. This can potentially make it difficult to differentiate the importance between different climatic variables as limiting factors. I would suggest that you add some more justification for using large number correlated variables in the analysis. Or, alternatively, consider reducing the dimensionality of the data.  
+
+2.  
+
+Partly related to the item 1, you could provide justification for using univariate instead of multivariate approach. The effect of a predictor variable can change (sometimes dramatically) when controlled for the effects of other variables by adding them to the model. Therefore, you should explain why you rely on univariate approach, or, alternatively, try to add the best predictor candidates in the same model
+
+<--- Page Split --->
+
+and see how the results would change.  
+
+3.  
+
+Binford's data is notorious for spatial auto- correlation, especially because in particular areas, he has basically split closely living ecologically, demographically and culturally similar groups into smaller units even though one might consider many of those belonging to the same ethnic group. This can lead to inflated performance metrics in traditional cross- validation schemes. The idea of cross- validation is to test the model with data that the model has not seen before, so in the presence of spatial auto- correlation, test data can be "too" similar to training data. Therefore, your performance metrics are quite likely "too good" and I suggest that you could use some kind of spatial block cross- validation scheme, such as h- block cross validation. See, e.g.  
+
+Salonen, J.S., et al, 2016. Calibrating aquatic microfossil proxies with regression- tree ensembles:  
+
+Cross- validation with modern chironomid and diatom data. The Holocene 26, 1040- 1048.  
+
+https://doi.org/10.1177/0959683616632881 https://quantpalaeo.wordpress.com/2013/12/15/h- block- cross- validation- of- transfer- functions/ https://cran.r- project.org/web/packages/blockCV/vignettes/BlockCV_for_SDM.html  
+
+4.  
+
+Related to the above issue, on page 3 you write that no single environmental variable explained more than \(81\%\) of the population density variation among ethnographic foraging societies. It might be because of my ignorance of quantile regression, but I'm not sure if you can really say that quantile regression model can explain some percentage of the variation in a response variable. So clarify this and explain what the explained deviance is measuring in your quantile regressions, is it the goodness of fit of the 90th quantile or what?  
+
+I'm also not sure if one can directly compare your performance metrics (explained deviance) to e.g. our metrics (R2) (Tallavaara et al. 2015). Besides, our R2 e.g. for multivariate GAM is clearly smaller (0.6), not marginally better, than any your values. After a lot of exploration with Binford's data, I also think that it is really difficult to push the R2 of (multivariate) population density models well above 0.7 unless you really overfit the model.  
+
+5.  
+
+On page 5, you provide the modelled population size estimates for Europe, which seems to be pretty high. The LGM estimate is twice as large as our previous estimate (which has been argued to be way too large by some) despite we having larger geographical area. However, am I right that your estimates are actually maximum estimates based on the modelled 90th quantile? Whatever the case, this needs to be stated clearly in the text and in the relevant figure captions.  
+
+6.  
+
+I might have missed it somehow, but which of the many univariate models you are using when estimating the population size or average density (including figures 3 and 4a- e)? Or is it ensemble of all models? This is nevertheless important information and if it is missing, you should clearly provide the information in the text and also to relevant figure captions.  
+
+7.  
+
+Mean temperature of the warmest month seems to be one of the most important limiting factors of hunter- gatherer density in Europe (Table 1, Figure 4). It is therefore interesting that its impact on maximum hunter- gatherer density is negative between 22kyBP and 8kyBP (figure 5B). The figure 1 shows that between 10 and 15 C the 90th quantile of MWM is decreasing, because of couple of outlier points. These kind of "edge effects" are a known problem in GAMs and therefore there are different kinds of constrained GAMs available:  
+
+https://www.researchgate.net/publication/271740857_Shape_constrained_additive_models https://arxiv.org/pdf/1812.07696. pdf
+
+<--- Page Split --->
+
+It is nevertheless quite unrealistic to assume that increase of MwM would have had negative impact on forager density from the LGM to Mid Holocene and I therefore suggest that you either try to use more conservative smoothing parameter value to get rid of wiggles or switch to constrained GAM, although I dont know if there are quantile versions available for such techniques. The negative (but unrealistic) effect of MwM is at least one of the reasons why MwM appears to increase its importance as a limiting factor over time in Europe.  
+
+8.  
+
+On page 5, you describe your results so that during the LGM the northern limit of human range would have been in central France and southern Germany. However, my reading of figure 4 is that the whole of France would have been within the human range. You use one individual/100km2 as threshold for human occupancy, which is pretty high given that lowest densities in ethnographic data are 0.2- 0.25 individuals/100km2. But even with your threshold, the occupied area seems to be clearly bigger than you describe in the text. Why this discrepancy? I would suggest that you bravely stand behind your results and describe them as they are :-)  
+
+Reviewer #3:  
+
+Remarks to the Author:  
+
+The manuscript focuses on the relation between the environmental factors explored here and population density. One central assumption that is adopted in this paper is that for foragers, demographic and environmental changes correlate strongly. And that there are causal relations between different environmental variables and human responses through time and Space. They then focus on limiting environmental factor which are defined as the variable predicting the lowest population density at a given place and time and assume that one of these limiting factors, or a combination of several, limited the scarcest recourse, and in turn regulate population sizes and densities. They then identify the dominant climatic constraints for hunter- gatherer population densities and then hindcast their changing dynamics in Europe for the period between 20kyBP to 8kyBP. They detect spaciotemporal variations in these factors in relation to the assessed demographic data for human groups which suggests that European Upper Palaeolithic hunter- gatherers at various regions and periods needed to overcome very different adaptive challenges.  
+
+The paper is overall well written and the introduction and Results and Discussion are detailed, and cite a lot of relevant and up to date sources. Moreover, the main caveats associated with their data sources and analyses are mentioned and discussed  
+
+I would like to raise three issues which I think can be handled in the revised manuscript.  
+
+One is that while I agree that environmental changes seem to have been the main driving force behind evident demographic patterns in the case of human populations and various other species, as the authors indicate, there are also adaptive capacities of humans to buffer and manage at least to some extent, environmental changes and corresponding resource fluctuations. The cited paper by Filho et al. 2021, documents how several African communities deferentially adapted to climate changes. If we assume that at least some of the human groups, during the Last Glacial Maximum and post- LGM period had similar adaptive capacities, it follows that their population sizes, densities an even settlement patterns, will not only reflect a 'passive' causal relationship with a specific climatic limiting factor, but also a unique human capacity to buffer and perhaps even overcome some limitation. Some examples include shelters, fire, and projectile technology. Moreover, one of the main mechanisms is mobility and mainly dispersals to refugia with better resources and climatic conditions.  
+
+A second issue is the reliance on ethnographic data. The authors cite the article by Bird a& Codding
+
+<--- Page Split --->
+
+2021, about the Promise and peril of ecological and evolutionary modelling using cross- cultural datasets. While the authors of this paper claim that the promise outweighs the peril. It will be useful for the authors to mention in more details, the potential caveats of drawing the analogy between present day and Upper Palaeolithic hunter- gatherers, since various papers argued that the former are not really a good proxy for the latter.  
+
+A third issue is that on page 6, Figure 3, they refer to the date of recolonization of Europe to be 17 kyBP. This is no longer regarded, on the basis of archaeological data, as being the date of onset of the process, as new results indicate that it started around 19 kyBP- see the paper by Maier et al. 2020: https://doi.org/10.1007/s41982- 019- 00045- 1  
+
+In sum, the paper is informative and balanced but the above- mentioned points are raised as the way some of the text is worded, it seems that the underlying approach is that human demography is not only affected by environmental shifts, and more specifically climatic changes, but is directly caused only by these. In which case, the assessment of which specific limiting factor exerted the most impact on a given human populations at a given location and time is indeed informative and interesting. But it should be made clearer that the paper does not test the specific role of human cultural capacities, to buffer and even overcome some limiting factors. Moreover, the spatiotemporal variations are expected to be a reflection of the fact that indeed limiting factors varied and that hunter- gatherers needed to overcome different adaptive challenges, but they cannot shed light on how they actually adapted, or alternatively failed to adapt, to these changes.  
+
+## Minor comments  
+
+Some of the figures need to be improved in terms of colors and legends.:  
+
+Figure 1. What are the abscissa? It is not clear from the figure legend.  
+
+Figure 3, Change color for Maximum temperature of the Warmest Month.  
+
+Figure 4, side panel legend, should be Population density and not population size It is also difficult to understand the panels, what is the difference between each side and the colors are difficult to detect at this scale.  
+
+Figure 5, is it assessing population size or population density?
+
+<--- Page Split --->
+
+## Final responses:  
+
+R1c1: Mean temperature of the Warmest Month (MWM) is identified a major limiting factor during all critical periods (Fig. 4), but MWM is the variable that has the most severe non- analogy problem comparing present- day climate space and past climates (Fig. 2 and Supplementary material S2). Although mentioned at Lines 126- 128, considering the strong relevance to the main findings, the risk of an unreliable extrapolation out of the range of data used to fit the statistical model is higher than acknowledged here.  
+
+To acknowledge the points made by the reviewer regarding the non- analogy problem with the variable, Mean temperature of the Warmest Month, we remove this variable from the pool of factors used to assess population densities and limiting factors. We explain this in the text in L137- 140 and L401- 403. We do not consider that removing this variable is a significant problem for our analyses. We reason that our objective is not to determine how a specific variable determines population densities but on the possible processes (as we specify in Table 1) by which climate can determine population densities.  
+
+R1c2: It is not clear why the authors chose 9oth percentile of population density to do the hindcast. First, the results, in principle, would not be comparable to previous estimates in literature. Second, does the resulted limiting factors change dependent on the choice of the percentile? Although the general shape of the population density versus climate relationship looks similar across different percentiles for each individual climate factor (Lines 311- 312), it is the relative magnitudes between all predicted densities by these factors that ultimately selects the limiting factor. Thus, it is not straightforward whether your results are sensitive to the choice of percentiles.  
+
+The reviewer's point regarding the need to justify why we chose the 9oth percentile in our analyses is welcomed. We have done all the analyses using the 10th, 50th, and 9oth percentile in this revision. As we now clarify in the text (L62- 63; L365- 359; L407- 412), our goal is not to quantify the population size on each evaluated grid but to indicate what are the potential climatic limiting factors and which could be the expected values (maxi- mum/average/minimum) given this climatic limit. Furthermore, our results and discussion focus on how observed deviations from these estimates can be used to generate hypotheses to how different societies have (or not) tackled these climatic limits, allowing them to have larger population sizes.  
+
+R1c3: Regarding the comparison between hindcasted population density and the archaeological population proxy (Fig. 3), I would not say they are "in line with" each other (Lines 139- 140). The black curve in Fig. 3 starts to increase already since 18 ka, which is relatively flat in the red curve; the red curve increases significantly during GI1 and GS1, whereas it is stale in the black curve.  
+
+While we now acknowledge the nuanced description of the trends by the reviewer in the text (L172- 176), we consider that a perfect match on the timing of events cannot be expected as these are variables representing trends at two different resolutions. Having
+
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+said that, the archaeological population proxy and our population density estimates show a strong correlation (rho = - 0.7) when aggregated at the same temporal resolution as the archaeological population proxy. We now make this point explicit in our text (L172- 176).  
+
+R1c4: By the way, at Lines 101- 103, why do you separate higher and lower predictive accuracy by a threshold of "explained deviances < 0.79"? According to Table 1, these predictors are all so close.  
+
+We do make this distinction anymore. Now we acknowledge that there are differences in the predictive accuracy between variables, but that accuracy amongst predictors is somewhat similar (L122- 124).  
+
+R1c5: From the results it is hard to infer mechanisms regarding how the identified limiting factor has constrained population density. This is limited by the fact that only temperature and precipitation and their variants were used as predictors, without direct information about productivity; whereas climate impacts population density via indirect effects on ecosystem attributes like NPP (e.g. Freeman et al. 2020, doi:10.1016/j.jas.2020.105168). Throughout the text the authors have tried to relate some of the factors to environmental productivity, but it was highly speculative. Let me take Lines 185- 192 as an example. During 14.7ka to 11.7ka, the importance of ET decreases while importance of MWM and temperature seasonality increases. But all three variables are linked to NPP (and possibly other aspects of the ecosystem). From these changes one still cannot judge what process is taking effect in the end. Given this, why not use NPP as a predictor in the first place? Data availability for the hindcast should not be a problem since simulated NPP for the past 21,000 years are publicly accessible from some climate models already.  
+
+To address the comment, we have done two things:  
+
+First, we no use Net Primary Productivity (NPP) in our work as a predictor. Using the Miami model, we calculate this variable (Lieth, 1972, as described in Table 1). We use this modelling approach instead of other possible NPP products as we want to reduce the potential biases that could come from using environmental datasets from alternative sources. As we do this, NPP as a predictor shows that it is not a significant factor. Second, we now refer to Effective Temperature and Potential Evapotranspiration as factors determining energy availability in a broad context (Table 1). NPP relates only to a variable indicating the energy available to hunter- gatherers from primary producers.  
+
+R1c6: I commend the authors' effort to put the (more of ecology- oriented) results into archaeological context, but currently it is still limited in qualitative descriptions scattered in the text. If the authors could achieve a more systematic compilation of archaeological records regarding how these societies have tackled with the limiting climate factors and find a consistency with your hindcasted results in space and time, it would add much merit to this study, with broader significance and impacts.
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+Thank you for this comment – naturally, we love to expand on this particular issue. We now provide an extended discussion of how the archaeological record explicitly links to the identified limiting factors and how different forager groups overcame these. We also provide additional references relating to pyrotechnology, shelter, energy capture, etc. (e.g. L223- 226 and L262- 271).  
+
+R1c7: Aside from the above concerns, the organization of Results and discussion needs to be improved. Adding sub- headings would help. Besides, descriptions of the results in the text should be more careful. Currently they are sometimes inconsistent with the table or figures. For example, at Lines 104- 110, it says seasonal temperature variables are among the lowest explained deviance, which is not the case as listed in Table 1.  
+
+As suggested, we have added subheadings to the Results and discussion section to provide a clear outline of our study results and their implication and relevance. We have now addressed all inconsistencies between the tables, figures and text.  
+
+R1c8: A minor point is that the uncertainties/biases in the paleoclimate outputs of the CCSM3 climate model should be discussed.  
+
+We would like to evaluate and discuss how CCSM3 SynTrace paleoclimate simulations uncertainties propagate to our population density models and definition of limiting factors. However, the used downscaled and debiased paleoclimatic simulations do not contain uncertainty estimates, and this is a point we acknowledge in our manuscript methods (L394- 397).  
+
+R1c9: Code availability: though not mandatory, it is strongly encouraged to make the code readily available so as to enable reproduction of the results.  
+
+We have now made the code and data used in this study available through a project GitHub site: https://github.com/AlejoOrdonez/PaleoPopDen. This is now part of the data availability statement.  
+
+R1c10: Table 1: A conceptual confusion: MCM is not "extreme events". Same for MWM, PDM, and PWM. Extreme events are events that occur with low frequency, not the regular seasonal maxima or minima.  
+
+We have now renamed these as annual limits.  
+
+R1c11: Line 266: "16 climatic predictors": there are 18 climate variables in Table 1. We have now change these to the current number of predictors.  
+
+R1c12: Line 289: it says "We use a subsample of 159 hunter- gatherers populations..." in the Reporting summary, while here it says "127 populations".  
+
+We have corrected this to there is consistency with the Reporting summary.  
+
+R1c13: Table 1: Acronym of "Precipitation of the Wettest Month" should be PWM, not PDM.  
+
+We have corrected this as suggested.
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+R1c14: Figure caption of Fig. 3: "Minimum temperature of the Coldest Month" should be "Mean...", and "Maximum temperature of the Warmest Month" should be "Mean..." We have changed the figure layout to include all used variables and ensure the titles and legend match Table 1  
+
+R1c15: Figure caption of Supplementary material S1: it is not "the six most important environmental factors".  
+
+We have removed this figure as all regressions are now shown in the main text.  
+
+R2c1. Given that many of the climate predictors are highly correlated (as you also write in the manuscript), they will convey partly the same information. This can potentially make it difficult to differentiate the importance between different climatic variables as limiting factors. I would suggest that you add some more justification for using large number correlated variables in the analysis. Or, alternatively, consider reducing the dimensionality of the data.  
+
+While we acknowledge in our study that the level of correlation between predictors is high, this level of relationship amongst predictors allows us to "[justify] our grouping of individual variables within groups of possible explanatory mechanisms (as listed in Table 1)" (L69- 74; L124- 129; and L326- 327). We also provide further justifications for this in our response to the reviewer's flowing point.  
+
+R2c2. Partly related to the item 1, you could provide justification for using univariate instead of multivariate approach. The effect of a predictor variable can change (sometimes dramatically) when controlled for the effects of other variables by adding them to the model. Therefore, you should explain why you rely on univariate approach, or, alternatively, try to add the best predictor candidates in the same model and see how the results would change.  
+
+As we now explicitly state in our text, "we are not aiming at determining the best combination of variables to predict population density, but rather at determining the limiting effect of a given environmental driver" (L69- 74). This perspective aligns with the core idea of limiting factors behind the current study.  
+
+R2c3. Binford's data is notorious for spatial auto- correlation, especially because in particular areas, he has basically split closely living ecologically, demographically and culturally similar groups into smaller units even though one might consider many of those belonging to the same ethnic group. This can lead to inflated performance metrics in traditional cross- validation schemes. The idea of cross- validation is to test the model with data that the model has not seen before, so in the presence of spatial auto- correlation, test data can be "too" similar to training data. Therefore, your performance metrics are quite likely "too good" and I suggest that you could use some kind of spatial block cross- validation scheme, such as h- block cross validation.
+
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+As suggested, we have used an h- block cross- validation approach (L68; L377- 383; L398- 401) to determine, for each qGAM model, its' performance and use these multiple models to control for model specification variability in our estimates of Population Density.  
+
+R2c4. Related to the above issue, on page 3 you write that no single environmental variable explained more than \(81\%\) of the population density variation among ethnographic foraging societies. It might be because of my ignorance of quantile regression, but I'm not sure if you can really say that quantile regression model can explain some percentage of the variation in a response variable. So clarify this and explain what the explained deviance is measuring in your quantile regressions, is it the goodness of fit of the 90th quantile or what?  
+
+You are right in your assessment that quantile GAMs cannot provide an estimate of the "percentage of the variation in a response variable" (i.e., R2). Our values here refer to the 50th percentile qGAM (or a traditional GAM), a point that was not clear in the original submission. For these, it is possible to determine an R2 value. This revision ensures that the point is explicitly made in the text (L374- 377). In both the main text and the method section (L110- 120; Table 1), we now also describe the model deviance.  
+
+R2c5. I'm also not sure if one can directly compare your performance metrics (explained deviance) to e.g. our metrics (R2) (Tallavaara et al. 2015). Besides, our R2 e.g. for multivariate GAM is clearly smaller (0.6), not marginally better, than any your values. After a lot of exploration with Binford's data, I also think that it is really difficult to push the R2 of (multivariate) population density models well above 0.7 unless you really overfit the model.  
+
+We agree that a proper comparison between the performance of our models and those in other publications is not so straightforward. Therefore decided to omit this statement in the revised text.  
+
+R2c6. On page 5, you provide the modelled population size estimates for Europe, which seems to be pretty high. The LGM estimate is twice as large as our previous estimate (which has been argued to be way too large by some) despite we having larger geographical area. However, am I right that your estimates are actually maximum estimates based on the modelled 90th quantile? Whatever the case, this needs to be stated clearly in the text and in the relevant figure captions.  
+
+We are aware of this, and it is a result of us using the 90th percentile model when describing these trends - as accurately pointed out in the comments. This revision states that "Taken at face value, these figures are gross overestimations of actual sustained and demographically viable human land- use across this timeframe" (L169- 170). Furthermore, we state in the text that our goal is NOT to predict population density but rather to show the limiting effects of climate on this important variable (L74- 76). Therefore, it makes sense to consider maximum (90th- percentile), average (50th- percentile), and minimum (10th- percentile) values as descriptors of these possible
+
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+limits. These are clarifications we also make when describing our population size/density estimates (L154- 169; L177- 180).  
+
+R2c7. I might have missed it somehow, but which of the many univariate models you are using when estimating the population size or average density (including figures 3 and 4a- e)? Or is it ensemble of all models? This is nevertheless important information and if it is missing, you should clearly provide the information in the text and also to relevant figure captions.  
+
+This information was only in the methods in the original submission (L386- 397), and now it is part of the main text (L84- 89) and the relevant legends.  
+
+R2c8. Mean temperature of the warmest month seems to be one of the most important limiting factors of hunter- gatherer density in Europe (Table 1, Figure 4). It is therefore interesting that its impact on maximum hunter- gatherer density is negative between 22kyBP and 8kyBP (figure 5B). The figure 1 shows that between 10 and 15 C the 90th quantile of MWM is decreasing, because of couple of outlier points. These kind of "edge effects" are a known problem in GAMs and therefore there are different kinds of constrained GAMs available.  
+
+It is nevertheless quite unrealistic to assume that increase of MWM would have had negative impact on forager density from the LGM to Mid Holocene and I therefore suggest that you either try to use more conservative smoothing parameter value to get rid of wiggles or switch to constrained GAM, although I dont know if there are quantile versions available for such techniques. The negative (but unrealistic) effect of MWM is at least one of the reasons why MWM appears to increase its importance as a limiting factor over time in Europe.  
+
+Thanks for your point regarding the patterns in this variable. This is one of the points we have been discussing in our revision. Given the issues highlighted in this comment and the points raised by Reviewer- 1 (the fact that there is a large non- analogy for this variable, especially in the late Pleistocene), we have decided to remove this variable from our analyses. As we discussed in R1c1, we do not consider this a significant problem for our work. Our reasoning is that because our focus is mainly on the "environmental mechanisms" by which climate imposes a limitation to population density (captured usually by two to three variables in our dataset) and not the effect of an individual variable.  
+
+R2c9. On page 5, you describe your results so that during the LGM the northern limit of human range would have been in central France and southern Germany. However, my reading of figure 4 is that the whole of France would have been within the human range. You use one individual/100km2 as threshold for human occupancy, which is pretty high given that lowest densities in ethnographic data are 0.2- 0.25 individuals/100km2. But even with your threshold, the occupied area seems to be clearly bigger than you describe in the text. Why this discrepancy? I would suggest that you bravely stand behind your results and describe them as they are.
+
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+This text section is now modified (L188- 192) to reflect a more detailed discussion of the observed pattern.  
+
+R3c1. One is that while I agree that environmental changes seem to have been the main driving force behind evident demographic patterns in the case of human populations and various other species, as the authors indicate, there are also adaptive capacities of humans to buffer and manage at least to some extent, environmental changes and corresponding resource fluctuations. The cited paper by Filho et al. 2021, documents how several African communities deferentially adapted to climate changes. If we assume that at least some of the human groups, during the Last Glacial Maximum and post- LGM period had similar adaptive capacities, it follows that their population sizes, densities an even settlement patterns, will not only reflect a 'passive' causal relationship with a specific climatic limiting factor, but also a unique human capacity to buffer and perhaps even overcome some limitation. Some examples include shelters, fire, and projectile technology. Moreover, one of the main mechanisms is mobility and mainly dispersals to refugia with better resources and climatic conditions.  
+
+The reviewer points to one of the main points we wanted to showcase with this study, but perhaps it was not clear - that climate sets a stage for human adaptation to "act" (L289- 297). You could see this as climate determining a baseline "limit", where human- populations active interaction with the environment, via behaviour and tools, would result in a deviation from this limit. This is a point we make explicit in our text (L293- 297), indicating that deviations from our estimates can be used to signpost which populations had buffering strategies and generate hypotheses as to which could these strategies be.  
+
+R3c2. A second issue is the reliance on ethnographic data. The authors cite the article by Bird a& Codding 2021, about the Promise and peril of ecological and evolutionary modelling using cross- cultural datasets. While the authors of this paper claim that the promise outweighs the peril. It will be useful for the authors to mention in more details, the potential caveats of drawing the analogy between present day and Upper Palaeolithic hunter- gatherers, since various papers argued that the former are not really a good proxy for the latter.  
+
+A paragraph on the inferential limits of the available ethnographic datasets has been added (L148- 152). However, we do consider a very detailed discussion of these issues outside of the scope of this particular study, not least because it has been discussed directly in the recent literature, e.g.: Hamilton, M.J., Tallavaara, M., 2022. Statistical inference, scale and noise in comparative anthropology. Nature Ecology & Evolution 6, 122- 122. https://doi.org/10.1038/s41559- 021- 01637- 3  
+
+R3c3. A third issue is that on page 6, Figure 3, they refer to the date of recolonization of Europe to be 17 kyBP. This is no longer regarded, on the basis of archaeological data, as being the date of onset of the process, as new results indicate that it started
+
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+around 19 kyBP- see the paper by Maier et al. 2020: https://doi.org/10.1007/s41982- 019- 00045- 1 This text section has been amended (L201- 203) and the appropriate reference added.  
+
+R3c4. In sum, the paper is informative and balanced but the above- mentioned points are raised as the way some of the text is worded, it seems that the underlying approach is that human demography is not only affected by environmental shifts, and more specifically climatic changes, but is directly caused only by these. In which case, the assessment of which specific limiting factor exerted the most impact on a given human populations at a given location and time is indeed informative and interesting. But it should be made clearer that the paper does not test the specific role of human cultural capacities, to buffer and even overcome some limiting factors. Moreover, the spatiotemporal variations are expected to be a reflection of the fact that indeed limiting factors varied and that hunter- gatherers needed to overcome different adaptive challenges, but they cannot shed light on how they actually adapted, or alternatively failed to adapt, to these changes.  
+
+Thank you for the thoughtful summary of our ideas in our study. We have now added text to ensure the points the reviewer so correctly highlights are even more evident in the text. Notably, the ideas of environmental conditions as factors affecting and determining human demography in the evaluated period (L42- 44) determine how technology or behaviour resulted in particular populations overcoming the limitations imposed by the rapid clitic changes of the late- Pleistocene (L74- 78).  
+
+R3c5. Some of the figures need to be improved in terms of colors and legends.: We have done a substation change in the figures color and legend to clarify their message, and fully explain what the objective of these is.  
+
+R3c6. Figure 1. What are the abscissa? It is not clear from the figure legend. Figure 1 now show what is the variable in the Abscissa (the same as the title)  
+
+R3c7. Figure 3, Change color for Maximum temperature of the Warmest Month. In figure- 1, we have now plated all the used variables and used a colour scheme that facilitates the readability of the variables.  
+
+R3c8. Figure 4, side panel legend, should be Population density and not population size. It is also difficult to understand the panels, what is the difference between each side and the colors are difficult to detect at this scale.  
+
+Figure one has been redrawn, and the density and limiting factors maps have been speared to clarify and enhance the message of each plot.  
+
+R3c9. Figure 5, is it assessing population size or population density? We now clarify that the top panel shows the changes in the evaluated period (21kyBP to 8kyBP) in the proportion of ice- free cells where a viable is considered the limiting
+
+<--- Page Split --->
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+factor (predicts the min population density). The bottom panel shows the estimated population density based on the average climatic condition across Europe for each evaluated variable.
+
+<--- Page Split --->
+
+Reviewers' Comments:  
+
+Reviewer #1:  
+
+Remarks to the Author:  
+
+The authors have addressed my major comments by 1) removing the MwM variable in assessing the limiting climate factors so that the serious non- analogy problem can be bypassed; 2) testing NPP as a potential limiting factor in the analysis; and 3) extending the discussion regarding the significance of the results in archaeological perspective. Overall I'm satisfied with these revisions. But there are some points to be clarified in the revised manuscript:  
+
+For the variable temperature seasonality, why are the values so large, \(\sim 2000^{\circ}C\) (Figure 1F and Figure 2F)? How is it calculated? And for precipitation seasonality, is it the standard deviation of the monthly precipitation?  
+
+Table 1: why are the metrics substantially lower than that in the previous version? Is it because now you have used h- block cross validation to address spatial auto- correlation? Besides, why do the deviance explained and \(R^2\) differ so much for some variables like PWM and TAP?  
+
+Line 165: what is the threshold of population density to define an occupied grid cell?  
+
+Figure 4: It would be more informative if you could overlay the localities of the archaeological sites that correspond to each time interval on the predicted population density maps. It can serve as a qualitative comparison. Besides, the current color legend looks weird - the ticks are not at the boundaries of each color segment.  
+
+In addition, there are still quite a few careless errors and inconsistencies in the revised manuscript. Below are some examples.  
+
+Line 110: "most of environmental variable produced models that explaining over \(50\%\) of the population density variation" - according to Table 1, the explanatory power of the variables are mostly below \(50\%\) .  
+
+Line 230: "PET" here should be TAP?  
+
+Line 234: "PET and TAP were the main limiting factors" - according to Fig. 6, it should be TS and TAP. Line 254- 255: MwM is no longer used in the prediction.  
+
+Table 1: "TSeson" and "PREC" - inconsistent with those in the text. And check the footnote of Table 1.  
+
+Figure 1: The precipitation has been log- transformed, right? Need to specify it.  
+
+Figure 3 lower panel: why Precip. Dryiest Month is higher than Precip. Wettest Month?  
+
+Figure 5 caption: what is "F- J"?  
+
+It is the authors' job to closely check every sentence, figures and tables to avoid any inconsistency or contradiction!  
+
+Reviewer #2:  
+
+Remarks to the Author:  
+
+Authors have successfully revised their manuscript. I have just one follow- up comment because authors might have misunderstood my earlier comment about models having one predictor at a time. My intention was not to suggest to add multiple predictors to achieve better predictive ability, but to take into account the fact that the effect of a predictor can change when one takes into account the effect(s) of other potential predictor(s).  
+
+For example, ET and MCM both appear to be important limiting factors and also representing different kinds of limiting factors, ET relating to energy availability and MCM to annual limits. However, these variables are also highly correlated, which already indicates that it will be difficult to tell apart their
+
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+individual effects. When you include both variables as predictors in the same model it actually turns out that the effect of ET is not statistically significant, response of population density to ET being more or less flat. Similarly, if you add e.g. NPP, ET and TS to the same model their effect (response shapes) are different from their effect when each is the only predictor in the model.  
+
+To me, all this suggests that the real limiting effects of climate variables can be different from those you get when you include these variables separately as predictors. However, I don't know how severe issue this truly is, but I would like to know your thoughts on that. If it really is an issue, one might use PCA to create uncorrelated climate variables and use these as predictors in the models.  
+
+Best wishes, Miikka Tallavaara  
+
+Reviewer #3: Remarks to the Author: I am fully satisfied with the revised version and with the revised manuscript and the changes.
+
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+## REVIEWER COMMENTS  
+
+## Reviewer #1  
+
+R12Co. The authors have addressed my major comments by 1) removing the MWM variable in assessing the limiting climate factors so that the serious non- analogy problem can be bypassed; 2) testing NPP as a potential limiting factor in the analysis; and 3) extending the discussion regarding the significance of the results in archaeological perspective. Overall, I'm satisfied with these revisions. But there are some points to be clarified in the revised manuscript:  
+
+We appreciate your assessment regarding our revision.  
+
+R1C1. For the variable temperature seasonality, why are the values so large, \(\sim 2000^{\circ}\mathrm{C}\) (Figure 1F and Figure 2F)? How is it calculated? Thanks for bringing this to our attention. Temperature Seasonality (TS) is estimated as the SD of mean annual temperatures X 100. For Clarity, we have now done two things. First, we now clarify that TS is measured as the SD of mean annual temperatures (so that values are in the same order of magnitude as other temperature variables). Second, we specify how (TS) is calculated in Table 1.  
+
+R1C2. And for precipitation seasonality, is it the standard deviation of the monthly precipitation?  
+
+As for TS, we now explain in table 1 how precipitation seasonality (PS) is estimated. In short, yes, it is calculated as the variation in monthly precipitation. However, instead of the SD in monthly precipitation, we use the Coefficient of Variation (CV) as this is the standard when estimating bioclimatic variables. We also clarify this in the "variable" and "units" columns of Table 1.  
+
+R1C3. Table 1: why are the metrics substantially lower than that in the previous version? Is it because now you have used h- block cross validation to address spatial autocorrelation? Besides, why do the deviance explained and \(\mathrm{R}^2\) differ so much for some variables like PWM and TAP?  
+
+Yes, the values are lower due to using an h- block cross- validation approach to define the random samples. Furthermore, two points explain the lower deviance- explained when compared to the R2 values. First, adding the variable does not add more explanatory power to the model compared to an intercept- only model (hence the low deviance explained and likely low unadjusted R2. Second, we can interpret the higher R2 as the models built on the training dataset can accurately describe the test dataset, which ensures the idea of model transferability. To ensure these points are clear, we add these points of clarification to table 1 legend.  
+
+R1C4. Line 165: what is the threshold of population density to define an occupied grid cell?  
+
+Ecoinformatics and Biodiversity Aarhus University Ny Munkegade 116 DK- 8000 Aarhus C Denmark  
+
+Tel.: +45 8715 0000 Fax: +45 8715 4326 E- mail: nat@au.dk Web: bio.au.dk/en
+
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+A cell was defined as occupied if our model predicted population densities above 0.2 individuals per 100km2 (the lowest densities in the ethnographic dataset). This point is added to the main text (L162- 163) and the methods (L405- 406).  
+
+R1C5. Figure 4: It would be more informative if you could overlay the localities of the archaeological sites that correspond to each time interval on the predicted population density maps. It can serve as a qualitative comparison. Besides, the current color legend looks weird – the ticks are not at the boundaries of each color segment.  
+
+We explored adding the localities of the archaeological sites to figure 4 but decided not to include these as these create an unnecessary layer of complexity for the figure. We also address the point raised by the reviewer regarding the figure colour legend.  
+
+R1C6. Line 110: "most of environmental variable produced models that explaining over 50% of the population density variation" – according to Table 1, the explanatory power of the variables are mostly below 50%.  
+
+We appreciate the reviewer catching this inconsistency coming from a legacy text from the first version. We now changed the sentence, so it does not specify a cut- off value (50%) but the range of mean deviance across the 1000 different models (L110).  
+
+R1C7. Line 230: "PET" here should be TAP?  
+
+We consider that here the variable to include is PET, as we are building from the idea of a relationship between productivity and Evapotranspiration. This is the case as the second point relates to energy availability, not climate variability. To further justify this link, we add a reference (L232).  
+
+R1C8. Line 234: "PET and TAP were the main limiting factors" – according to Fig. 6, it should be TS and TAP.  
+
+We appreciate the reviewer catching this inconsistency coming from a legacy text from the original submission. We now changed the sentence accordingly.  
+
+R1C9. Line 254- 255: MWM is no longer used in the prediction. We appreciate the reviewer catching this legacy text from the first submission. We have rephrased this point (L256).  
+
+R1C10. Table 1: "TSeson" and "PREC" – inconsistent with those in the text. And check the footnote of Table 1.  
+
+The acronym was changed as suggested in the table for consistency with the text.  
+
+R1C11. Figure 1: The precipitation has been log- transformed, right? Need to specify it. We have added this clarification to the corresponding axes in figure 1 and table 1.  
+
+R1C12. Figure 3 lower panel: why Precip. Dryiest Month is higher than Precip. Wettest Month?
+
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+
+We appreciate the reviewer catching this inconsistency. This was a problem in the code calling the different variables after we removed the Temperature of the Warmest Month, which caused a mismatch between the names and the data plotted in the bottom panels of figure 3. The figure now has corrected this.  
+
+R1C13. Figure 5 caption: what is "F- J"? We appreciate the reviewer catching this legacy text from the first submission. This text was removed.  
+
+## Reviewer #2.  
+
+R2C1. Authors have successfully revised their manuscript. I have just one follow- up comment because authors might have misunderstood my earlier comment about models having one predictor at a time.  
+
+My intention was not to suggest to add multiple predictors to achieve better predictive ability, but to take into account the fact that the effect of a predictor can change when one takes into account the effect(s) of other potential predictor(s).  
+
+For example, ET and MCM both appear to be important limiting factors and also representing different kinds of limiting factors, ET relating to energy availability and MCM to annual limits. However, these variables are also highly correlated, which already indicates that it will be difficult to tell apart their individual effects. When you include both variables as predictors in the same model it actually turns out that the effect of ET is not statistically significant, response of population density to ET being more or less flat. Similarly, if you add e.g. NPP, ET and TS to the same model their effect (response shapes) are different from their effect when each is the only predictor in the model.  
+
+To me, all this suggests that the real limiting effects of climate variables can be different from those you get when you include these variables separately as predictors. However, I don't know how severe issue this truly is, but I would like to know your thoughts on that. If it really is an issue, one might use PCA to create uncorrelated climate variables and use these as predictors in the models.  
+
+We thank the reviewer for his positive feedback on our revision and the clarification of his original point. As we now understand the reviewer's point, the issue is that significant absolute effects from univariate models would not translate into relative effects determined by multivariate models.  
+
+While we agree with the point, we consider that the multiple regression approach does not translate to the idea of limiting factors we are evaluating here. We argue that multiple regression coefficients indicate effects in the context of other variables (hence contingent on which variables are included or omitted in a model). Therefore, these determine how much each variable contributes to the change in population density. To define which variables set a lower boundary, we require a measure of absolute effects provided by univariate approaches. Focusing on the relative effects would not allow us
+
+<--- Page Split --->
+
+to define limiting factors but which variable(s) contribute the most to changes in population density from the "regional" mean.  
+
+Suppose we could build models for change in population density for each evaluated time bin. In that case, we could define the variable that contributes the most to population density at each time bin. Still, this is not a limiting factor but the variable that contributes the most to changes in population density form the regional average (i.e., the model intercept). Last, there is the issue of translating these relative effects into space, which our approach based on univariate models can do.  
+
+Furthermore, while PCA, or other ordination approaches, could be used here to determine "groups of variables" and the variable most representative of such "group", we will still be looking at relative effects when using the two or three most important axes. There also be questions about how suitable it is to use the eigenvectors generated by the ordination under current conditions to "reorganize" past climatic surfaces where the correlations between variables change.  
+
+In summary, we consider that using univariate models, while far from perfect, is a practical approach to assessing the absolute effects of each variable and comparing these between variables over time. Also, it allows us to link our models to a process. All these points are now made in the text (L72- 80) and the methods (L364- 368).  
+
+## Reviewer #3.  
+
+I am fully satisfied with the revised version and with the revised manuscript and the changes.  
+
+Thanks for your positive assessment.
+
+<--- Page Split --->
+
+Reviewers' Comments:  
+
+Reviewer #1:  Remarks to the Author:  I'm satisfied with the revisions and have no further comments.  
+
+Reviewer #2:  Remarks to the Author:  
+
+While I still slightly disagree with you about the effects of univariate vs multivariate models on the results, I'm happy to do so. It is good that you now explain in the manuscript your choices regarding the matter, so I'm fully satisfied with this revised manuscript.  
+
+Best,  Miikka Tallavaara
+
+<--- Page Split --->
+
+## REVIEWER COMMENTS  
+
+Reviewer #2 (Remarks to the Author):  
+
+While I still slightly disagree with you about the effects of univariate vs multivariate models on the results, I'm happy to do so. It is good that you now explain in the manuscript your choices regarding the matter, so I'm fully satisfied with this revised manuscript.  
+
+We appreciate your assessment regarding our revision and your willingness to agree to disagree regarding the effects of univariate vs multivariate models on the results.
+
+<--- Page Split --->

peer_reviews/supplementary_0_Peer Review File__0226534d35058c645a89e4e44377c8b2e4f25f5dddefbf9c78c868b60659db7b/supplementary_0_Peer Review File__0226534d35058c645a89e4e44377c8b2e4f25f5dddefbf9c78c868b60659db7b_det.mmd

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+<|ref|>title<|/ref|><|det|>[[99, 40, 507, 90]]<|/det|>
+# nature portfolio  
+
+<|ref|>text<|/ref|><|det|>[[106, 110, 373, 139]]<|/det|>
+Peer Review File  
+
+<|ref|>text<|/ref|><|det|>[[108, 154, 880, 211]]<|/det|>
+Changes in limiting factors for forager population dynamics in Europe across the Last Glacial- Interglacial Transition  
+
+<|ref|>image<|/ref|><|det|>[[94, 732, 262, 780]]<|/det|>  
+
+<|ref|>text<|/ref|><|det|>[[270, 732, 880, 784]]<|/det|>
+Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 90, 286, 103]]<|/det|>
+Reviewers' Comments:  
+
+<|ref|>text<|/ref|><|det|>[[116, 120, 217, 133]]<|/det|>
+Reviewer #1:  
+
+<|ref|>text<|/ref|><|det|>[[116, 135, 291, 148]]<|/det|>
+Remarks to the Author:  
+
+<|ref|>text<|/ref|><|det|>[[115, 150, 879, 315]]<|/det|>
+This study aims to identify the limiting climate factors of hunter- gatherer population density in Europe from the LGM to early Holocene, which is a tempting research question that, if answered convincingly, could provide clues in resilience and adaptation strategies of ancient human societies. The authors built statistical models (quantile Generalised Additive Models) of population density versus each of the 18 climate variables based on contemporary hunter- gatherer dataset, and hindcasted population density using paleoclimate outputs from a climate model. This workflow and statistical techniques are not new (e.g. Tallavaara et al. 2015 pnas), but selecting the factor that predicts the lowest population density across space and time, based on the concept of limiting factor, provides a fresh angle of view. However, I have some major concerns about the robustness of the analysis and significance of current results, as outlined below. Based on these, I cannot recommend its publication, at least not in its current form.  
+
+<|ref|>text<|/ref|><|det|>[[116, 330, 279, 343]]<|/det|>
+Robustness of results:  
+
+<|ref|>text<|/ref|><|det|>[[115, 344, 874, 433]]<|/det|>
+Mean temperature of the Warmest Month (MwM) is identified a major limiting factor during all critical periods (Fig. 4), but MwM is the variable that has the most severe non- analogy problem comparing present- day climate space and past climates (Fig. 2 and Supplementary material S2). Although mentioned at Lines 126- 128, considering the strong relevance to the main findings, the risk of an unreliable extrapolation out of the range of data used to fit the statistical model is higher than acknowledged here.  
+
+<|ref|>text<|/ref|><|det|>[[115, 433, 878, 540]]<|/det|>
+In addition, it is not clear to me why the authors chose 90th percentile of population density to do the hindcast. First, the results, in principle, would not be comparable to previous estimates in literature. Second, does the resulted limiting factors change dependent on the choice of the percentile? Although the general shape of the population density versus climate relationship looks similar across different percentiles for each individual climate factor (Lines 311- 312), it is the relative magnitudes between all predicted densities by these factors that ultimately selects the limiting factor. Thus, it is not straightforward whether your results are sensitive to the choice of percentiles.  
+
+<|ref|>text<|/ref|><|det|>[[115, 539, 866, 597]]<|/det|>
+Regarding the comparison between hindcasted population density and the archeological population proxy (Fig. 3), I would not say they are "in line with" each other (Lines 139- 140). The black curve in Fig. 3 starts to increase already since 18 ka, which is relatively flat in the red curve; the red curve increases significantly during GI1 and GS1, whereas it is stale in the black curve.  
+
+<|ref|>text<|/ref|><|det|>[[115, 597, 844, 627]]<|/det|>
+By the way, at Lines 101- 103, why do you separate higher and lower predictive accuracy by a threshold of "explained deviances \(< 0.79\) " According to Table 1, these predictors are all so close.  
+
+<|ref|>text<|/ref|><|det|>[[116, 642, 317, 655]]<|/det|>
+About mechanism insights:  
+
+<|ref|>text<|/ref|><|det|>[[115, 656, 875, 805]]<|/det|>
+From the results it is hard to infer mechanisms regarding how the identified limiting factor has constrained population density. This is limited by the fact that only temperature and precipitation and their variants were used as predictors, without direct information about productivity; whereas climate impacts population density via indirect effects on ecosystem attributes like NPP (e.g. Freeman et al. 2020, doi:10.1016/j.jas.2020.105168). Throughout the text the authors have tried to relate some of the factors to environmental productivity, but it was highly speculative. Let me take Lines 185- 192 as an example. During 14.7ka to 11.7ka, the importance of ET decreases while importance of MwM and temperature seasonality increases. But all three variables are linked to NPP (and possibly other aspects of the ecosystem). From these changes one still cannot judge what process is taking effect in the end.  
+
+<|ref|>text<|/ref|><|det|>[[116, 806, 869, 850]]<|/det|>
+Given this, why not use NPP as a predictor in the first place? Data availability for the hindcast should not be a problem since simulated NPP for the past 21,000 years are publicly accessible from some climate models already.  
+
+<|ref|>text<|/ref|><|det|>[[115, 864, 825, 895]]<|/det|>
+Significance of the results in archaeological perspective: I commend the authors' effort to put the (more of ecology- oriented) results into archaeological
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 89, 866, 150]]<|/det|>
+context, but currently it is still limited in qualitative descriptions scattered in the text. If the authors could achieve a more systematic compilation of archaeological records regarding how these societies have tackled with the limiting climate factors and find a consistency with your hindcasted results in space and time, it would add much merit to this study, with broader significance and impacts.  
+
+<|ref|>text<|/ref|><|det|>[[115, 163, 860, 239]]<|/det|>
+Aside from the above concerns, the organization of Results and discussion needs to be improved. Adding sub- headings would help. Besides, descriptions of the results in the text should be more careful. Currently they are sometimes inconsistent with the table or figures. For example, at Lines 104- 110, it says seasonal temperature variables are among the lowest explained deviance, which is not the case as listed in Table 1.  
+
+<|ref|>text<|/ref|><|det|>[[115, 253, 872, 283]]<|/det|>
+A minor point is that the uncertainties/biases in the paleoclimate outputs of the CCSM3 climate model should be discussed.  
+
+<|ref|>text<|/ref|><|det|>[[115, 297, 870, 328]]<|/det|>
+Code availability: though not mandatory, it is strongly encouraged to make the code readily available so as to enable reproduction of the results.  
+
+<|ref|>text<|/ref|><|det|>[[115, 342, 880, 373]]<|/det|>
+Table 1: A conceptual confusion: MCM is not "extreme events". Same for MWM, PDM, and PWM. Extreme events are events that occur with low frequency, not the regular seasonal maxima or minima.  
+
+<|ref|>text<|/ref|><|det|>[[115, 387, 587, 402]]<|/det|>
+There are a few careless errors in the manuscript, for example:  
+
+<|ref|>text<|/ref|><|det|>[[115, 402, 675, 416]]<|/det|>
+Line 266: "16 climatic predictors": there are 18 climate variables in Table 1.  
+
+<|ref|>text<|/ref|><|det|>[[115, 417, 825, 446]]<|/det|>
+Line 289: it says "We use a subsample of 159 hunter- gatherers populations..." in the Reporting summary, while here it says "127 populations".  
+
+<|ref|>text<|/ref|><|det|>[[115, 447, 733, 462]]<|/det|>
+Table 1: Acronym of "Precipitation of the Wettest Month" should be PWM, not PDM.  
+
+<|ref|>text<|/ref|><|det|>[[115, 462, 825, 492]]<|/det|>
+Figure caption of Fig. 3: "Minimum temperature of the Coldest Month" should be "Mean...", and "Maximum temperature of the Warmest Month" should be "Mean..."  
+
+<|ref|>text<|/ref|><|det|>[[115, 493, 875, 523]]<|/det|>
+Figure caption of Supplementary material S1: it is not "the six most important environmental factors". Please check carefully throughout the paper.  
+
+<|ref|>text<|/ref|><|det|>[[115, 567, 216, 580]]<|/det|>
+Reviewer #2:  
+
+<|ref|>text<|/ref|><|det|>[[115, 582, 291, 595]]<|/det|>
+Remarks to the Author:  
+
+<|ref|>text<|/ref|><|det|>[[115, 596, 877, 656]]<|/det|>
+This is truly thought- provoking, highly interesting and novel contribution to the hunter- gatherer ecology. I very much like the approach of applying the analysis of ecological limiting factors, for the first time, to prehistoric hunter- gatherers. Below I have highlighted few issues that you could consider to revise to further improve the paper.  
+
+<|ref|>text<|/ref|><|det|>[[115, 671, 245, 700]]<|/det|>
+Best regards, Miikka Tallavaara  
+
+<|ref|>text<|/ref|><|det|>[[115, 716, 130, 728]]<|/det|>
+1.  
+
+<|ref|>text<|/ref|><|det|>[[115, 730, 878, 805]]<|/det|>
+Given that many of the climate predictors are highly correlated (as you also write in the manuscript), they will convey partly the same information. This can potentially make it difficult to differentiate the importance between different climatic variables as limiting factors. I would suggest that you add some more justification for using large number correlated variables in the analysis. Or, alternatively, consider reducing the dimensionality of the data.  
+
+<|ref|>text<|/ref|><|det|>[[115, 821, 130, 833]]<|/det|>
+2.  
+
+<|ref|>text<|/ref|><|det|>[[115, 835, 880, 894]]<|/det|>
+Partly related to the item 1, you could provide justification for using univariate instead of multivariate approach. The effect of a predictor variable can change (sometimes dramatically) when controlled for the effects of other variables by adding them to the model. Therefore, you should explain why you rely on univariate approach, or, alternatively, try to add the best predictor candidates in the same model
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 90, 404, 104]]<|/det|>
+and see how the results would change.  
+
+<|ref|>text<|/ref|><|det|>[[116, 120, 130, 131]]<|/det|>
+3.  
+
+<|ref|>text<|/ref|><|det|>[[115, 133, 868, 258]]<|/det|>
+Binford's data is notorious for spatial auto- correlation, especially because in particular areas, he has basically split closely living ecologically, demographically and culturally similar groups into smaller units even though one might consider many of those belonging to the same ethnic group. This can lead to inflated performance metrics in traditional cross- validation schemes. The idea of cross- validation is to test the model with data that the model has not seen before, so in the presence of spatial auto- correlation, test data can be "too" similar to training data. Therefore, your performance metrics are quite likely "too good" and I suggest that you could use some kind of spatial block cross- validation scheme, such as h- block cross validation. See, e.g.  
+
+<|ref|>text<|/ref|><|det|>[[115, 256, 840, 280]]<|/det|>
+Salonen, J.S., et al, 2016. Calibrating aquatic microfossil proxies with regression- tree ensembles:  
+
+<|ref|>text<|/ref|><|det|>[[115, 280, 435, 293]]<|/det|>
+Cross- validation with modern chironomid and diatom data. The Holocene 26, 1040- 1048.  
+
+<|ref|>text<|/ref|><|det|>[[115, 294, 830, 323]]<|/det|>
+https://doi.org/10.1177/0959683616632881 https://quantpalaeo.wordpress.com/2013/12/15/h- block- cross- validation- of- transfer- functions/ https://cran.r- project.org/web/packages/blockCV/vignettes/BlockCV_for_SDM.html  
+
+<|ref|>text<|/ref|><|det|>[[115, 345, 130, 356]]<|/det|>
+4.  
+
+<|ref|>text<|/ref|><|det|>[[115, 358, 875, 448]]<|/det|>
+Related to the above issue, on page 3 you write that no single environmental variable explained more than \(81\%\) of the population density variation among ethnographic foraging societies. It might be because of my ignorance of quantile regression, but I'm not sure if you can really say that quantile regression model can explain some percentage of the variation in a response variable. So clarify this and explain what the explained deviance is measuring in your quantile regressions, is it the goodness of fit of the 90th quantile or what?  
+
+<|ref|>text<|/ref|><|det|>[[115, 462, 879, 537]]<|/det|>
+I'm also not sure if one can directly compare your performance metrics (explained deviance) to e.g. our metrics (R2) (Tallavaara et al. 2015). Besides, our R2 e.g. for multivariate GAM is clearly smaller (0.6), not marginally better, than any your values. After a lot of exploration with Binford's data, I also think that it is really difficult to push the R2 of (multivariate) population density models well above 0.7 unless you really overfit the model.  
+
+<|ref|>text<|/ref|><|det|>[[115, 552, 130, 564]]<|/det|>
+5.  
+
+<|ref|>text<|/ref|><|det|>[[115, 566, 871, 640]]<|/det|>
+On page 5, you provide the modelled population size estimates for Europe, which seems to be pretty high. The LGM estimate is twice as large as our previous estimate (which has been argued to be way too large by some) despite we having larger geographical area. However, am I right that your estimates are actually maximum estimates based on the modelled 90th quantile? Whatever the case, this needs to be stated clearly in the text and in the relevant figure captions.  
+
+<|ref|>text<|/ref|><|det|>[[115, 655, 130, 667]]<|/det|>
+6.  
+
+<|ref|>text<|/ref|><|det|>[[115, 670, 872, 730]]<|/det|>
+I might have missed it somehow, but which of the many univariate models you are using when estimating the population size or average density (including figures 3 and 4a- e)? Or is it ensemble of all models? This is nevertheless important information and if it is missing, you should clearly provide the information in the text and also to relevant figure captions.  
+
+<|ref|>text<|/ref|><|det|>[[115, 745, 130, 757]]<|/det|>
+7.  
+
+<|ref|>text<|/ref|><|det|>[[115, 760, 874, 853]]<|/det|>
+Mean temperature of the warmest month seems to be one of the most important limiting factors of hunter- gatherer density in Europe (Table 1, Figure 4). It is therefore interesting that its impact on maximum hunter- gatherer density is negative between 22kyBP and 8kyBP (figure 5B). The figure 1 shows that between 10 and 15 C the 90th quantile of MWM is decreasing, because of couple of outlier points. These kind of "edge effects" are a known problem in GAMs and therefore there are different kinds of constrained GAMs available:  
+
+<|ref|>text<|/ref|><|det|>[[115, 853, 800, 881]]<|/det|>
+https://www.researchgate.net/publication/271740857_Shape_constrained_additive_models https://arxiv.org/pdf/1812.07696. pdf
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 89, 872, 180]]<|/det|>
+It is nevertheless quite unrealistic to assume that increase of MwM would have had negative impact on forager density from the LGM to Mid Holocene and I therefore suggest that you either try to use more conservative smoothing parameter value to get rid of wiggles or switch to constrained GAM, although I dont know if there are quantile versions available for such techniques. The negative (but unrealistic) effect of MwM is at least one of the reasons why MwM appears to increase its importance as a limiting factor over time in Europe.  
+
+<|ref|>text<|/ref|><|det|>[[115, 195, 131, 207]]<|/det|>
+8.  
+
+<|ref|>text<|/ref|><|det|>[[115, 208, 878, 314]]<|/det|>
+On page 5, you describe your results so that during the LGM the northern limit of human range would have been in central France and southern Germany. However, my reading of figure 4 is that the whole of France would have been within the human range. You use one individual/100km2 as threshold for human occupancy, which is pretty high given that lowest densities in ethnographic data are 0.2- 0.25 individuals/100km2. But even with your threshold, the occupied area seems to be clearly bigger than you describe in the text. Why this discrepancy? I would suggest that you bravely stand behind your results and describe them as they are :-)  
+
+<|ref|>text<|/ref|><|det|>[[115, 358, 216, 371]]<|/det|>
+Reviewer #3:  
+
+<|ref|>text<|/ref|><|det|>[[115, 373, 291, 386]]<|/det|>
+Remarks to the Author:  
+
+<|ref|>text<|/ref|><|det|>[[112, 386, 883, 583]]<|/det|>
+The manuscript focuses on the relation between the environmental factors explored here and population density. One central assumption that is adopted in this paper is that for foragers, demographic and environmental changes correlate strongly. And that there are causal relations between different environmental variables and human responses through time and Space. They then focus on limiting environmental factor which are defined as the variable predicting the lowest population density at a given place and time and assume that one of these limiting factors, or a combination of several, limited the scarcest recourse, and in turn regulate population sizes and densities. They then identify the dominant climatic constraints for hunter- gatherer population densities and then hindcast their changing dynamics in Europe for the period between 20kyBP to 8kyBP. They detect spaciotemporal variations in these factors in relation to the assessed demographic data for human groups which suggests that European Upper Palaeolithic hunter- gatherers at various regions and periods needed to overcome very different adaptive challenges.  
+
+<|ref|>text<|/ref|><|det|>[[115, 611, 879, 655]]<|/det|>
+The paper is overall well written and the introduction and Results and Discussion are detailed, and cite a lot of relevant and up to date sources. Moreover, the main caveats associated with their data sources and analyses are mentioned and discussed  
+
+<|ref|>text<|/ref|><|det|>[[115, 670, 774, 685]]<|/det|>
+I would like to raise three issues which I think can be handled in the revised manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[115, 700, 875, 865]]<|/det|>
+One is that while I agree that environmental changes seem to have been the main driving force behind evident demographic patterns in the case of human populations and various other species, as the authors indicate, there are also adaptive capacities of humans to buffer and manage at least to some extent, environmental changes and corresponding resource fluctuations. The cited paper by Filho et al. 2021, documents how several African communities deferentially adapted to climate changes. If we assume that at least some of the human groups, during the Last Glacial Maximum and post- LGM period had similar adaptive capacities, it follows that their population sizes, densities an even settlement patterns, will not only reflect a 'passive' causal relationship with a specific climatic limiting factor, but also a unique human capacity to buffer and perhaps even overcome some limitation. Some examples include shelters, fire, and projectile technology. Moreover, one of the main mechanisms is mobility and mainly dispersals to refugia with better resources and climatic conditions.  
+
+<|ref|>text<|/ref|><|det|>[[115, 878, 857, 894]]<|/det|>
+A second issue is the reliance on ethnographic data. The authors cite the article by Bird a& Codding
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 89, 872, 165]]<|/det|>
+2021, about the Promise and peril of ecological and evolutionary modelling using cross- cultural datasets. While the authors of this paper claim that the promise outweighs the peril. It will be useful for the authors to mention in more details, the potential caveats of drawing the analogy between present day and Upper Palaeolithic hunter- gatherers, since various papers argued that the former are not really a good proxy for the latter.  
+
+<|ref|>text<|/ref|><|det|>[[115, 179, 878, 239]]<|/det|>
+A third issue is that on page 6, Figure 3, they refer to the date of recolonization of Europe to be 17 kyBP. This is no longer regarded, on the basis of archaeological data, as being the date of onset of the process, as new results indicate that it started around 19 kyBP- see the paper by Maier et al. 2020: https://doi.org/10.1007/s41982- 019- 00045- 1  
+
+<|ref|>text<|/ref|><|det|>[[115, 253, 883, 401]]<|/det|>
+In sum, the paper is informative and balanced but the above- mentioned points are raised as the way some of the text is worded, it seems that the underlying approach is that human demography is not only affected by environmental shifts, and more specifically climatic changes, but is directly caused only by these. In which case, the assessment of which specific limiting factor exerted the most impact on a given human populations at a given location and time is indeed informative and interesting. But it should be made clearer that the paper does not test the specific role of human cultural capacities, to buffer and even overcome some limiting factors. Moreover, the spatiotemporal variations are expected to be a reflection of the fact that indeed limiting factors varied and that hunter- gatherers needed to overcome different adaptive challenges, but they cannot shed light on how they actually adapted, or alternatively failed to adapt, to these changes.  
+
+<|ref|>sub_title<|/ref|><|det|>[[115, 432, 241, 446]]<|/det|>
+## Minor comments  
+
+<|ref|>text<|/ref|><|det|>[[115, 447, 661, 462]]<|/det|>
+Some of the figures need to be improved in terms of colors and legends.:  
+
+<|ref|>text<|/ref|><|det|>[[115, 477, 639, 492]]<|/det|>
+Figure 1. What are the abscissa? It is not clear from the figure legend.  
+
+<|ref|>text<|/ref|><|det|>[[115, 507, 659, 522]]<|/det|>
+Figure 3, Change color for Maximum temperature of the Warmest Month.  
+
+<|ref|>text<|/ref|><|det|>[[115, 536, 856, 580]]<|/det|>
+Figure 4, side panel legend, should be Population density and not population size It is also difficult to understand the panels, what is the difference between each side and the colors are difficult to detect at this scale.  
+
+<|ref|>text<|/ref|><|det|>[[115, 595, 577, 610]]<|/det|>
+Figure 5, is it assessing population size or population density?
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[93, 169, 237, 184]]<|/det|>
+## Final responses:  
+
+<|ref|>text<|/ref|><|det|>[[92, 185, 740, 300]]<|/det|>
+R1c1: Mean temperature of the Warmest Month (MWM) is identified a major limiting factor during all critical periods (Fig. 4), but MWM is the variable that has the most severe non- analogy problem comparing present- day climate space and past climates (Fig. 2 and Supplementary material S2). Although mentioned at Lines 126- 128, considering the strong relevance to the main findings, the risk of an unreliable extrapolation out of the range of data used to fit the statistical model is higher than acknowledged here.  
+
+<|ref|>text<|/ref|><|det|>[[92, 301, 740, 433]]<|/det|>
+To acknowledge the points made by the reviewer regarding the non- analogy problem with the variable, Mean temperature of the Warmest Month, we remove this variable from the pool of factors used to assess population densities and limiting factors. We explain this in the text in L137- 140 and L401- 403. We do not consider that removing this variable is a significant problem for our analyses. We reason that our objective is not to determine how a specific variable determines population densities but on the possible processes (as we specify in Table 1) by which climate can determine population densities.  
+
+<|ref|>text<|/ref|><|det|>[[92, 449, 740, 582]]<|/det|>
+R1c2: It is not clear why the authors chose 9oth percentile of population density to do the hindcast. First, the results, in principle, would not be comparable to previous estimates in literature. Second, does the resulted limiting factors change dependent on the choice of the percentile? Although the general shape of the population density versus climate relationship looks similar across different percentiles for each individual climate factor (Lines 311- 312), it is the relative magnitudes between all predicted densities by these factors that ultimately selects the limiting factor. Thus, it is not straightforward whether your results are sensitive to the choice of percentiles.  
+
+<|ref|>text<|/ref|><|det|>[[92, 583, 740, 732]]<|/det|>
+The reviewer's point regarding the need to justify why we chose the 9oth percentile in our analyses is welcomed. We have done all the analyses using the 10th, 50th, and 9oth percentile in this revision. As we now clarify in the text (L62- 63; L365- 359; L407- 412), our goal is not to quantify the population size on each evaluated grid but to indicate what are the potential climatic limiting factors and which could be the expected values (maxi- mum/average/minimum) given this climatic limit. Furthermore, our results and discussion focus on how observed deviations from these estimates can be used to generate hypotheses to how different societies have (or not) tackled these climatic limits, allowing them to have larger population sizes.  
+
+<|ref|>text<|/ref|><|det|>[[92, 749, 740, 830]]<|/det|>
+R1c3: Regarding the comparison between hindcasted population density and the archaeological population proxy (Fig. 3), I would not say they are "in line with" each other (Lines 139- 140). The black curve in Fig. 3 starts to increase already since 18 ka, which is relatively flat in the red curve; the red curve increases significantly during GI1 and GS1, whereas it is stale in the black curve.  
+
+<|ref|>text<|/ref|><|det|>[[92, 832, 740, 881]]<|/det|>
+While we now acknowledge the nuanced description of the trends by the reviewer in the text (L172- 176), we consider that a perfect match on the timing of events cannot be expected as these are variables representing trends at two different resolutions. Having
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[92, 167, 739, 234]]<|/det|>
+said that, the archaeological population proxy and our population density estimates show a strong correlation (rho = - 0.7) when aggregated at the same temporal resolution as the archaeological population proxy. We now make this point explicit in our text (L172- 176).  
+
+<|ref|>text<|/ref|><|det|>[[92, 250, 739, 300]]<|/det|>
+R1c4: By the way, at Lines 101- 103, why do you separate higher and lower predictive accuracy by a threshold of "explained deviances < 0.79"? According to Table 1, these predictors are all so close.  
+
+<|ref|>text<|/ref|><|det|>[[92, 301, 739, 350]]<|/det|>
+We do make this distinction anymore. Now we acknowledge that there are differences in the predictive accuracy between variables, but that accuracy amongst predictors is somewhat similar (L122- 124).  
+
+<|ref|>text<|/ref|><|det|>[[92, 366, 740, 598]]<|/det|>
+R1c5: From the results it is hard to infer mechanisms regarding how the identified limiting factor has constrained population density. This is limited by the fact that only temperature and precipitation and their variants were used as predictors, without direct information about productivity; whereas climate impacts population density via indirect effects on ecosystem attributes like NPP (e.g. Freeman et al. 2020, doi:10.1016/j.jas.2020.105168). Throughout the text the authors have tried to relate some of the factors to environmental productivity, but it was highly speculative. Let me take Lines 185- 192 as an example. During 14.7ka to 11.7ka, the importance of ET decreases while importance of MWM and temperature seasonality increases. But all three variables are linked to NPP (and possibly other aspects of the ecosystem). From these changes one still cannot judge what process is taking effect in the end. Given this, why not use NPP as a predictor in the first place? Data availability for the hindcast should not be a problem since simulated NPP for the past 21,000 years are publicly accessible from some climate models already.  
+
+<|ref|>text<|/ref|><|det|>[[93, 600, 473, 615]]<|/det|>
+To address the comment, we have done two things:  
+
+<|ref|>text<|/ref|><|det|>[[92, 616, 740, 749]]<|/det|>
+First, we no use Net Primary Productivity (NPP) in our work as a predictor. Using the Miami model, we calculate this variable (Lieth, 1972, as described in Table 1). We use this modelling approach instead of other possible NPP products as we want to reduce the potential biases that could come from using environmental datasets from alternative sources. As we do this, NPP as a predictor shows that it is not a significant factor. Second, we now refer to Effective Temperature and Potential Evapotranspiration as factors determining energy availability in a broad context (Table 1). NPP relates only to a variable indicating the energy available to hunter- gatherers from primary producers.  
+
+<|ref|>text<|/ref|><|det|>[[92, 765, 740, 864]]<|/det|>
+R1c6: I commend the authors' effort to put the (more of ecology- oriented) results into archaeological context, but currently it is still limited in qualitative descriptions scattered in the text. If the authors could achieve a more systematic compilation of archaeological records regarding how these societies have tackled with the limiting climate factors and find a consistency with your hindcasted results in space and time, it would add much merit to this study, with broader significance and impacts.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[92, 167, 740, 250]]<|/det|>
+Thank you for this comment – naturally, we love to expand on this particular issue. We now provide an extended discussion of how the archaeological record explicitly links to the identified limiting factors and how different forager groups overcame these. We also provide additional references relating to pyrotechnology, shelter, energy capture, etc. (e.g. L223- 226 and L262- 271).  
+
+<|ref|>text<|/ref|><|det|>[[92, 251, 740, 333]]<|/det|>
+R1c7: Aside from the above concerns, the organization of Results and discussion needs to be improved. Adding sub- headings would help. Besides, descriptions of the results in the text should be more careful. Currently they are sometimes inconsistent with the table or figures. For example, at Lines 104- 110, it says seasonal temperature variables are among the lowest explained deviance, which is not the case as listed in Table 1.  
+
+<|ref|>text<|/ref|><|det|>[[92, 334, 740, 383]]<|/det|>
+As suggested, we have added subheadings to the Results and discussion section to provide a clear outline of our study results and their implication and relevance. We have now addressed all inconsistencies between the tables, figures and text.  
+
+<|ref|>text<|/ref|><|det|>[[92, 400, 740, 432]]<|/det|>
+R1c8: A minor point is that the uncertainties/biases in the paleoclimate outputs of the CCSM3 climate model should be discussed.  
+
+<|ref|>text<|/ref|><|det|>[[92, 434, 740, 516]]<|/det|>
+We would like to evaluate and discuss how CCSM3 SynTrace paleoclimate simulations uncertainties propagate to our population density models and definition of limiting factors. However, the used downscaled and debiased paleoclimatic simulations do not contain uncertainty estimates, and this is a point we acknowledge in our manuscript methods (L394- 397).  
+
+<|ref|>text<|/ref|><|det|>[[92, 532, 740, 565]]<|/det|>
+R1c9: Code availability: though not mandatory, it is strongly encouraged to make the code readily available so as to enable reproduction of the results.  
+
+<|ref|>text<|/ref|><|det|>[[92, 567, 740, 615]]<|/det|>
+We have now made the code and data used in this study available through a project GitHub site: https://github.com/AlejoOrdonez/PaleoPopDen. This is now part of the data availability statement.  
+
+<|ref|>text<|/ref|><|det|>[[92, 632, 740, 681]]<|/det|>
+R1c10: Table 1: A conceptual confusion: MCM is not "extreme events". Same for MWM, PDM, and PWM. Extreme events are events that occur with low frequency, not the regular seasonal maxima or minima.  
+
+<|ref|>text<|/ref|><|det|>[[92, 683, 433, 698]]<|/det|>
+We have now renamed these as annual limits.  
+
+<|ref|>text<|/ref|><|det|>[[92, 716, 707, 748]]<|/det|>
+R1c11: Line 266: "16 climatic predictors": there are 18 climate variables in Table 1. We have now change these to the current number of predictors.  
+
+<|ref|>text<|/ref|><|det|>[[92, 765, 740, 797]]<|/det|>
+R1c12: Line 289: it says "We use a subsample of 159 hunter- gatherers populations..." in the Reporting summary, while here it says "127 populations".  
+
+<|ref|>text<|/ref|><|det|>[[92, 799, 740, 814]]<|/det|>
+We have corrected this to there is consistency with the Reporting summary.  
+
+<|ref|>text<|/ref|><|det|>[[92, 831, 740, 863]]<|/det|>
+R1c13: Table 1: Acronym of "Precipitation of the Wettest Month" should be PWM, not PDM.  
+
+<|ref|>text<|/ref|><|det|>[[92, 866, 365, 880]]<|/det|>
+We have corrected this as suggested.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[92, 183, 740, 250]]<|/det|>
+R1c14: Figure caption of Fig. 3: "Minimum temperature of the Coldest Month" should be "Mean...", and "Maximum temperature of the Warmest Month" should be "Mean..." We have changed the figure layout to include all used variables and ensure the titles and legend match Table 1  
+
+<|ref|>text<|/ref|><|det|>[[92, 266, 740, 299]]<|/det|>
+R1c15: Figure caption of Supplementary material S1: it is not "the six most important environmental factors".  
+
+<|ref|>text<|/ref|><|det|>[[92, 300, 678, 316]]<|/det|>
+We have removed this figure as all regressions are now shown in the main text.  
+
+<|ref|>text<|/ref|><|det|>[[92, 332, 740, 432]]<|/det|>
+R2c1. Given that many of the climate predictors are highly correlated (as you also write in the manuscript), they will convey partly the same information. This can potentially make it difficult to differentiate the importance between different climatic variables as limiting factors. I would suggest that you add some more justification for using large number correlated variables in the analysis. Or, alternatively, consider reducing the dimensionality of the data.  
+
+<|ref|>text<|/ref|><|det|>[[92, 433, 740, 516]]<|/det|>
+While we acknowledge in our study that the level of correlation between predictors is high, this level of relationship amongst predictors allows us to "[justify] our grouping of individual variables within groups of possible explanatory mechanisms (as listed in Table 1)" (L69- 74; L124- 129; and L326- 327). We also provide further justifications for this in our response to the reviewer's flowing point.  
+
+<|ref|>text<|/ref|><|det|>[[92, 532, 740, 632]]<|/det|>
+R2c2. Partly related to the item 1, you could provide justification for using univariate instead of multivariate approach. The effect of a predictor variable can change (sometimes dramatically) when controlled for the effects of other variables by adding them to the model. Therefore, you should explain why you rely on univariate approach, or, alternatively, try to add the best predictor candidates in the same model and see how the results would change.  
+
+<|ref|>text<|/ref|><|det|>[[92, 633, 740, 700]]<|/det|>
+As we now explicitly state in our text, "we are not aiming at determining the best combination of variables to predict population density, but rather at determining the limiting effect of a given environmental driver" (L69- 74). This perspective aligns with the core idea of limiting factors behind the current study.  
+
+<|ref|>text<|/ref|><|det|>[[92, 715, 740, 864]]<|/det|>
+R2c3. Binford's data is notorious for spatial auto- correlation, especially because in particular areas, he has basically split closely living ecologically, demographically and culturally similar groups into smaller units even though one might consider many of those belonging to the same ethnic group. This can lead to inflated performance metrics in traditional cross- validation schemes. The idea of cross- validation is to test the model with data that the model has not seen before, so in the presence of spatial auto- correlation, test data can be "too" similar to training data. Therefore, your performance metrics are quite likely "too good" and I suggest that you could use some kind of spatial block cross- validation scheme, such as h- block cross validation.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[92, 167, 739, 234]]<|/det|>
+As suggested, we have used an h- block cross- validation approach (L68; L377- 383; L398- 401) to determine, for each qGAM model, its' performance and use these multiple models to control for model specification variability in our estimates of Population Density.  
+
+<|ref|>text<|/ref|><|det|>[[92, 250, 740, 365]]<|/det|>
+R2c4. Related to the above issue, on page 3 you write that no single environmental variable explained more than \(81\%\) of the population density variation among ethnographic foraging societies. It might be because of my ignorance of quantile regression, but I'm not sure if you can really say that quantile regression model can explain some percentage of the variation in a response variable. So clarify this and explain what the explained deviance is measuring in your quantile regressions, is it the goodness of fit of the 90th quantile or what?  
+
+<|ref|>text<|/ref|><|det|>[[92, 366, 739, 466]]<|/det|>
+You are right in your assessment that quantile GAMs cannot provide an estimate of the "percentage of the variation in a response variable" (i.e., R2). Our values here refer to the 50th percentile qGAM (or a traditional GAM), a point that was not clear in the original submission. For these, it is possible to determine an R2 value. This revision ensures that the point is explicitly made in the text (L374- 377). In both the main text and the method section (L110- 120; Table 1), we now also describe the model deviance.  
+
+<|ref|>text<|/ref|><|det|>[[92, 483, 739, 582]]<|/det|>
+R2c5. I'm also not sure if one can directly compare your performance metrics (explained deviance) to e.g. our metrics (R2) (Tallavaara et al. 2015). Besides, our R2 e.g. for multivariate GAM is clearly smaller (0.6), not marginally better, than any your values. After a lot of exploration with Binford's data, I also think that it is really difficult to push the R2 of (multivariate) population density models well above 0.7 unless you really overfit the model.  
+
+<|ref|>text<|/ref|><|det|>[[92, 584, 739, 632]]<|/det|>
+We agree that a proper comparison between the performance of our models and those in other publications is not so straightforward. Therefore decided to omit this statement in the revised text.  
+
+<|ref|>text<|/ref|><|det|>[[92, 649, 739, 748]]<|/det|>
+R2c6. On page 5, you provide the modelled population size estimates for Europe, which seems to be pretty high. The LGM estimate is twice as large as our previous estimate (which has been argued to be way too large by some) despite we having larger geographical area. However, am I right that your estimates are actually maximum estimates based on the modelled 90th quantile? Whatever the case, this needs to be stated clearly in the text and in the relevant figure captions.  
+
+<|ref|>text<|/ref|><|det|>[[92, 750, 739, 881]]<|/det|>
+We are aware of this, and it is a result of us using the 90th percentile model when describing these trends - as accurately pointed out in the comments. This revision states that "Taken at face value, these figures are gross overestimations of actual sustained and demographically viable human land- use across this timeframe" (L169- 170). Furthermore, we state in the text that our goal is NOT to predict population density but rather to show the limiting effects of climate on this important variable (L74- 76). Therefore, it makes sense to consider maximum (90th- percentile), average (50th- percentile), and minimum (10th- percentile) values as descriptors of these possible
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[92, 166, 738, 200]]<|/det|>
+limits. These are clarifications we also make when describing our population size/density estimates (L154- 169; L177- 180).  
+
+<|ref|>text<|/ref|><|det|>[[92, 216, 740, 300]]<|/det|>
+R2c7. I might have missed it somehow, but which of the many univariate models you are using when estimating the population size or average density (including figures 3 and 4a- e)? Or is it ensemble of all models? This is nevertheless important information and if it is missing, you should clearly provide the information in the text and also to relevant figure captions.  
+
+<|ref|>text<|/ref|><|det|>[[92, 301, 738, 334]]<|/det|>
+This information was only in the methods in the original submission (L386- 397), and now it is part of the main text (L84- 89) and the relevant legends.  
+
+<|ref|>text<|/ref|><|det|>[[92, 350, 740, 468]]<|/det|>
+R2c8. Mean temperature of the warmest month seems to be one of the most important limiting factors of hunter- gatherer density in Europe (Table 1, Figure 4). It is therefore interesting that its impact on maximum hunter- gatherer density is negative between 22kyBP and 8kyBP (figure 5B). The figure 1 shows that between 10 and 15 C the 90th quantile of MWM is decreasing, because of couple of outlier points. These kind of "edge effects" are a known problem in GAMs and therefore there are different kinds of constrained GAMs available.  
+
+<|ref|>text<|/ref|><|det|>[[92, 468, 740, 583]]<|/det|>
+It is nevertheless quite unrealistic to assume that increase of MWM would have had negative impact on forager density from the LGM to Mid Holocene and I therefore suggest that you either try to use more conservative smoothing parameter value to get rid of wiggles or switch to constrained GAM, although I dont know if there are quantile versions available for such techniques. The negative (but unrealistic) effect of MWM is at least one of the reasons why MWM appears to increase its importance as a limiting factor over time in Europe.  
+
+<|ref|>text<|/ref|><|det|>[[92, 584, 740, 732]]<|/det|>
+Thanks for your point regarding the patterns in this variable. This is one of the points we have been discussing in our revision. Given the issues highlighted in this comment and the points raised by Reviewer- 1 (the fact that there is a large non- analogy for this variable, especially in the late Pleistocene), we have decided to remove this variable from our analyses. As we discussed in R1c1, we do not consider this a significant problem for our work. Our reasoning is that because our focus is mainly on the "environmental mechanisms" by which climate imposes a limitation to population density (captured usually by two to three variables in our dataset) and not the effect of an individual variable.  
+
+<|ref|>text<|/ref|><|det|>[[92, 749, 740, 881]]<|/det|>
+R2c9. On page 5, you describe your results so that during the LGM the northern limit of human range would have been in central France and southern Germany. However, my reading of figure 4 is that the whole of France would have been within the human range. You use one individual/100km2 as threshold for human occupancy, which is pretty high given that lowest densities in ethnographic data are 0.2- 0.25 individuals/100km2. But even with your threshold, the occupied area seems to be clearly bigger than you describe in the text. Why this discrepancy? I would suggest that you bravely stand behind your results and describe them as they are.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[93, 167, 739, 201]]<|/det|>
+This text section is now modified (L188- 192) to reflect a more detailed discussion of the observed pattern.  
+
+<|ref|>text<|/ref|><|det|>[[92, 217, 740, 433]]<|/det|>
+R3c1. One is that while I agree that environmental changes seem to have been the main driving force behind evident demographic patterns in the case of human populations and various other species, as the authors indicate, there are also adaptive capacities of humans to buffer and manage at least to some extent, environmental changes and corresponding resource fluctuations. The cited paper by Filho et al. 2021, documents how several African communities deferentially adapted to climate changes. If we assume that at least some of the human groups, during the Last Glacial Maximum and post- LGM period had similar adaptive capacities, it follows that their population sizes, densities an even settlement patterns, will not only reflect a 'passive' causal relationship with a specific climatic limiting factor, but also a unique human capacity to buffer and perhaps even overcome some limitation. Some examples include shelters, fire, and projectile technology. Moreover, one of the main mechanisms is mobility and mainly dispersals to refugia with better resources and climatic conditions.  
+
+<|ref|>text<|/ref|><|det|>[[92, 450, 730, 583]]<|/det|>
+The reviewer points to one of the main points we wanted to showcase with this study, but perhaps it was not clear - that climate sets a stage for human adaptation to "act" (L289- 297). You could see this as climate determining a baseline "limit", where human- populations active interaction with the environment, via behaviour and tools, would result in a deviation from this limit. This is a point we make explicit in our text (L293- 297), indicating that deviations from our estimates can be used to signpost which populations had buffering strategies and generate hypotheses as to which could these strategies be.  
+
+<|ref|>text<|/ref|><|det|>[[92, 599, 736, 715]]<|/det|>
+R3c2. A second issue is the reliance on ethnographic data. The authors cite the article by Bird a& Codding 2021, about the Promise and peril of ecological and evolutionary modelling using cross- cultural datasets. While the authors of this paper claim that the promise outweighs the peril. It will be useful for the authors to mention in more details, the potential caveats of drawing the analogy between present day and Upper Palaeolithic hunter- gatherers, since various papers argued that the former are not really a good proxy for the latter.  
+
+<|ref|>text<|/ref|><|det|>[[92, 716, 733, 817]]<|/det|>
+A paragraph on the inferential limits of the available ethnographic datasets has been added (L148- 152). However, we do consider a very detailed discussion of these issues outside of the scope of this particular study, not least because it has been discussed directly in the recent literature, e.g.: Hamilton, M.J., Tallavaara, M., 2022. Statistical inference, scale and noise in comparative anthropology. Nature Ecology & Evolution 6, 122- 122. https://doi.org/10.1038/s41559- 021- 01637- 3  
+
+<|ref|>text<|/ref|><|det|>[[92, 833, 730, 882]]<|/det|>
+R3c3. A third issue is that on page 6, Figure 3, they refer to the date of recolonization of Europe to be 17 kyBP. This is no longer regarded, on the basis of archaeological data, as being the date of onset of the process, as new results indicate that it started
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[92, 167, 737, 216]]<|/det|>
+around 19 kyBP- see the paper by Maier et al. 2020: https://doi.org/10.1007/s41982- 019- 00045- 1 This text section has been amended (L201- 203) and the appropriate reference added.  
+
+<|ref|>text<|/ref|><|det|>[[92, 234, 735, 433]]<|/det|>
+R3c4. In sum, the paper is informative and balanced but the above- mentioned points are raised as the way some of the text is worded, it seems that the underlying approach is that human demography is not only affected by environmental shifts, and more specifically climatic changes, but is directly caused only by these. In which case, the assessment of which specific limiting factor exerted the most impact on a given human populations at a given location and time is indeed informative and interesting. But it should be made clearer that the paper does not test the specific role of human cultural capacities, to buffer and even overcome some limiting factors. Moreover, the spatiotemporal variations are expected to be a reflection of the fact that indeed limiting factors varied and that hunter- gatherers needed to overcome different adaptive challenges, but they cannot shed light on how they actually adapted, or alternatively failed to adapt, to these changes.  
+
+<|ref|>text<|/ref|><|det|>[[92, 434, 730, 533]]<|/det|>
+Thank you for the thoughtful summary of our ideas in our study. We have now added text to ensure the points the reviewer so correctly highlights are even more evident in the text. Notably, the ideas of environmental conditions as factors affecting and determining human demography in the evaluated period (L42- 44) determine how technology or behaviour resulted in particular populations overcoming the limitations imposed by the rapid clitic changes of the late- Pleistocene (L74- 78).  
+
+<|ref|>text<|/ref|><|det|>[[92, 550, 727, 599]]<|/det|>
+R3c5. Some of the figures need to be improved in terms of colors and legends.: We have done a substation change in the figures color and legend to clarify their message, and fully explain what the objective of these is.  
+
+<|ref|>text<|/ref|><|det|>[[92, 616, 660, 649]]<|/det|>
+R3c6. Figure 1. What are the abscissa? It is not clear from the figure legend. Figure 1 now show what is the variable in the Abscissa (the same as the title)  
+
+<|ref|>text<|/ref|><|det|>[[92, 666, 715, 714]]<|/det|>
+R3c7. Figure 3, Change color for Maximum temperature of the Warmest Month. In figure- 1, we have now plated all the used variables and used a colour scheme that facilitates the readability of the variables.  
+
+<|ref|>text<|/ref|><|det|>[[92, 732, 717, 780]]<|/det|>
+R3c8. Figure 4, side panel legend, should be Population density and not population size. It is also difficult to understand the panels, what is the difference between each side and the colors are difficult to detect at this scale.  
+
+<|ref|>text<|/ref|><|det|>[[92, 783, 704, 815]]<|/det|>
+Figure one has been redrawn, and the density and limiting factors maps have been speared to clarify and enhance the message of each plot.  
+
+<|ref|>text<|/ref|><|det|>[[92, 832, 720, 881]]<|/det|>
+R3c9. Figure 5, is it assessing population size or population density? We now clarify that the top panel shows the changes in the evaluated period (21kyBP to 8kyBP) in the proportion of ice- free cells where a viable is considered the limiting
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[92, 168, 714, 216]]<|/det|>
+factor (predicts the min population density). The bottom panel shows the estimated population density based on the average climatic condition across Europe for each evaluated variable.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[116, 90, 286, 104]]<|/det|>
+Reviewers' Comments:  
+
+<|ref|>text<|/ref|><|det|>[[116, 120, 217, 133]]<|/det|>
+Reviewer #1:  
+
+<|ref|>text<|/ref|><|det|>[[116, 135, 291, 148]]<|/det|>
+Remarks to the Author:  
+
+<|ref|>text<|/ref|><|det|>[[115, 149, 880, 224]]<|/det|>
+The authors have addressed my major comments by 1) removing the MwM variable in assessing the limiting climate factors so that the serious non- analogy problem can be bypassed; 2) testing NPP as a potential limiting factor in the analysis; and 3) extending the discussion regarding the significance of the results in archaeological perspective. Overall I'm satisfied with these revisions. But there are some points to be clarified in the revised manuscript:  
+
+<|ref|>text<|/ref|><|det|>[[115, 238, 881, 283]]<|/det|>
+For the variable temperature seasonality, why are the values so large, \(\sim 2000^{\circ}C\) (Figure 1F and Figure 2F)? How is it calculated? And for precipitation seasonality, is it the standard deviation of the monthly precipitation?  
+
+<|ref|>text<|/ref|><|det|>[[115, 298, 868, 343]]<|/det|>
+Table 1: why are the metrics substantially lower than that in the previous version? Is it because now you have used h- block cross validation to address spatial auto- correlation? Besides, why do the deviance explained and \(R^2\) differ so much for some variables like PWM and TAP?  
+
+<|ref|>text<|/ref|><|det|>[[115, 357, 744, 372]]<|/det|>
+Line 165: what is the threshold of population density to define an occupied grid cell?  
+
+<|ref|>text<|/ref|><|det|>[[115, 386, 855, 446]]<|/det|>
+Figure 4: It would be more informative if you could overlay the localities of the archaeological sites that correspond to each time interval on the predicted population density maps. It can serve as a qualitative comparison. Besides, the current color legend looks weird - the ticks are not at the boundaries of each color segment.  
+
+<|ref|>text<|/ref|><|det|>[[115, 476, 856, 505]]<|/det|>
+In addition, there are still quite a few careless errors and inconsistencies in the revised manuscript. Below are some examples.  
+
+<|ref|>text<|/ref|><|det|>[[115, 505, 880, 550]]<|/det|>
+Line 110: "most of environmental variable produced models that explaining over \(50\%\) of the population density variation" - according to Table 1, the explanatory power of the variables are mostly below \(50\%\) .  
+
+<|ref|>text<|/ref|><|det|>[[116, 551, 390, 565]]<|/det|>
+Line 230: "PET" here should be TAP?  
+
+<|ref|>text<|/ref|><|det|>[[115, 566, 875, 595]]<|/det|>
+Line 234: "PET and TAP were the main limiting factors" - according to Fig. 6, it should be TS and TAP. Line 254- 255: MwM is no longer used in the prediction.  
+
+<|ref|>text<|/ref|><|det|>[[115, 596, 875, 610]]<|/det|>
+Table 1: "TSeson" and "PREC" - inconsistent with those in the text. And check the footnote of Table 1.  
+
+<|ref|>text<|/ref|><|det|>[[115, 611, 712, 625]]<|/det|>
+Figure 1: The precipitation has been log- transformed, right? Need to specify it.  
+
+<|ref|>text<|/ref|><|det|>[[115, 626, 757, 640]]<|/det|>
+Figure 3 lower panel: why Precip. Dryiest Month is higher than Precip. Wettest Month?  
+
+<|ref|>text<|/ref|><|det|>[[115, 641, 350, 655]]<|/det|>
+Figure 5 caption: what is "F- J"?  
+
+<|ref|>text<|/ref|><|det|>[[115, 670, 872, 700]]<|/det|>
+It is the authors' job to closely check every sentence, figures and tables to avoid any inconsistency or contradiction!  
+
+<|ref|>text<|/ref|><|det|>[[115, 744, 216, 758]]<|/det|>
+Reviewer #2:  
+
+<|ref|>text<|/ref|><|det|>[[115, 760, 291, 774]]<|/det|>
+Remarks to the Author:  
+
+<|ref|>text<|/ref|><|det|>[[115, 775, 872, 850]]<|/det|>
+Authors have successfully revised their manuscript. I have just one follow- up comment because authors might have misunderstood my earlier comment about models having one predictor at a time. My intention was not to suggest to add multiple predictors to achieve better predictive ability, but to take into account the fact that the effect of a predictor can change when one takes into account the effect(s) of other potential predictor(s).  
+
+<|ref|>text<|/ref|><|det|>[[115, 850, 875, 894]]<|/det|>
+For example, ET and MCM both appear to be important limiting factors and also representing different kinds of limiting factors, ET relating to energy availability and MCM to annual limits. However, these variables are also highly correlated, which already indicates that it will be difficult to tell apart their
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 88, 879, 150]]<|/det|>
+individual effects. When you include both variables as predictors in the same model it actually turns out that the effect of ET is not statistically significant, response of population density to ET being more or less flat. Similarly, if you add e.g. NPP, ET and TS to the same model their effect (response shapes) are different from their effect when each is the only predictor in the model.  
+
+<|ref|>text<|/ref|><|det|>[[115, 163, 881, 224]]<|/det|>
+To me, all this suggests that the real limiting effects of climate variables can be different from those you get when you include these variables separately as predictors. However, I don't know how severe issue this truly is, but I would like to know your thoughts on that. If it really is an issue, one might use PCA to create uncorrelated climate variables and use these as predictors in the models.  
+
+<|ref|>text<|/ref|><|det|>[[115, 238, 245, 268]]<|/det|>
+Best wishes, Miikka Tallavaara  
+
+<|ref|>text<|/ref|><|det|>[[115, 312, 815, 358]]<|/det|>
+Reviewer #3: Remarks to the Author: I am fully satisfied with the revised version and with the revised manuscript and the changes.
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[94, 148, 310, 164]]<|/det|>
+## REVIEWER COMMENTS  
+
+<|ref|>sub_title<|/ref|><|det|>[[94, 182, 201, 197]]<|/det|>
+## Reviewer #1  
+
+<|ref|>text<|/ref|><|det|>[[92, 214, 734, 314]]<|/det|>
+R12Co. The authors have addressed my major comments by 1) removing the MWM variable in assessing the limiting climate factors so that the serious non- analogy problem can be bypassed; 2) testing NPP as a potential limiting factor in the analysis; and 3) extending the discussion regarding the significance of the results in archaeological perspective. Overall, I'm satisfied with these revisions. But there are some points to be clarified in the revised manuscript:  
+
+<|ref|>text<|/ref|><|det|>[[94, 315, 500, 330]]<|/det|>
+We appreciate your assessment regarding our revision.  
+
+<|ref|>text<|/ref|><|det|>[[92, 347, 736, 464]]<|/det|>
+R1C1. For the variable temperature seasonality, why are the values so large, \(\sim 2000^{\circ}\mathrm{C}\) (Figure 1F and Figure 2F)? How is it calculated? Thanks for bringing this to our attention. Temperature Seasonality (TS) is estimated as the SD of mean annual temperatures X 100. For Clarity, we have now done two things. First, we now clarify that TS is measured as the SD of mean annual temperatures (so that values are in the same order of magnitude as other temperature variables). Second, we specify how (TS) is calculated in Table 1.  
+
+<|ref|>text<|/ref|><|det|>[[92, 480, 720, 512]]<|/det|>
+R1C2. And for precipitation seasonality, is it the standard deviation of the monthly precipitation?  
+
+<|ref|>text<|/ref|><|det|>[[92, 514, 730, 595]]<|/det|>
+As for TS, we now explain in table 1 how precipitation seasonality (PS) is estimated. In short, yes, it is calculated as the variation in monthly precipitation. However, instead of the SD in monthly precipitation, we use the Coefficient of Variation (CV) as this is the standard when estimating bioclimatic variables. We also clarify this in the "variable" and "units" columns of Table 1.  
+
+<|ref|>text<|/ref|><|det|>[[92, 612, 730, 678]]<|/det|>
+R1C3. Table 1: why are the metrics substantially lower than that in the previous version? Is it because now you have used h- block cross validation to address spatial autocorrelation? Besides, why do the deviance explained and \(\mathrm{R}^2\) differ so much for some variables like PWM and TAP?  
+
+<|ref|>text<|/ref|><|det|>[[92, 680, 734, 812]]<|/det|>
+Yes, the values are lower due to using an h- block cross- validation approach to define the random samples. Furthermore, two points explain the lower deviance- explained when compared to the R2 values. First, adding the variable does not add more explanatory power to the model compared to an intercept- only model (hence the low deviance explained and likely low unadjusted R2. Second, we can interpret the higher R2 as the models built on the training dataset can accurately describe the test dataset, which ensures the idea of model transferability. To ensure these points are clear, we add these points of clarification to table 1 legend.  
+
+<|ref|>text<|/ref|><|det|>[[92, 829, 734, 860]]<|/det|>
+R1C4. Line 165: what is the threshold of population density to define an occupied grid cell?  
+
+<|ref|>text<|/ref|><|det|>[[300, 911, 492, 965]]<|/det|>
+Ecoinformatics and Biodiversity Aarhus University Ny Munkegade 116 DK- 8000 Aarhus C Denmark  
+
+<|ref|>text<|/ref|><|det|>[[606, 920, 730, 965]]<|/det|>
+Tel.: +45 8715 0000 Fax: +45 8715 4326 E- mail: nat@au.dk Web: bio.au.dk/en
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[92, 147, 725, 197]]<|/det|>
+A cell was defined as occupied if our model predicted population densities above 0.2 individuals per 100km2 (the lowest densities in the ethnographic dataset). This point is added to the main text (L162- 163) and the methods (L405- 406).  
+
+<|ref|>text<|/ref|><|det|>[[92, 214, 733, 278]]<|/det|>
+R1C5. Figure 4: It would be more informative if you could overlay the localities of the archaeological sites that correspond to each time interval on the predicted population density maps. It can serve as a qualitative comparison. Besides, the current color legend looks weird – the ticks are not at the boundaries of each color segment.  
+
+<|ref|>text<|/ref|><|det|>[[92, 279, 735, 329]]<|/det|>
+We explored adding the localities of the archaeological sites to figure 4 but decided not to include these as these create an unnecessary layer of complexity for the figure. We also address the point raised by the reviewer regarding the figure colour legend.  
+
+<|ref|>text<|/ref|><|det|>[[92, 346, 725, 395]]<|/det|>
+R1C6. Line 110: "most of environmental variable produced models that explaining over 50% of the population density variation" – according to Table 1, the explanatory power of the variables are mostly below 50%.  
+
+<|ref|>text<|/ref|><|det|>[[92, 397, 733, 446]]<|/det|>
+We appreciate the reviewer catching this inconsistency coming from a legacy text from the first version. We now changed the sentence, so it does not specify a cut- off value (50%) but the range of mean deviance across the 1000 different models (L110).  
+
+<|ref|>text<|/ref|><|det|>[[92, 463, 427, 479]]<|/det|>
+R1C7. Line 230: "PET" here should be TAP?  
+
+<|ref|>text<|/ref|><|det|>[[92, 480, 727, 545]]<|/det|>
+We consider that here the variable to include is PET, as we are building from the idea of a relationship between productivity and Evapotranspiration. This is the case as the second point relates to energy availability, not climate variability. To further justify this link, we add a reference (L232).  
+
+<|ref|>text<|/ref|><|det|>[[92, 562, 728, 595]]<|/det|>
+R1C8. Line 234: "PET and TAP were the main limiting factors" – according to Fig. 6, it should be TS and TAP.  
+
+<|ref|>text<|/ref|><|det|>[[92, 597, 733, 629]]<|/det|>
+We appreciate the reviewer catching this inconsistency coming from a legacy text from the original submission. We now changed the sentence accordingly.  
+
+<|ref|>text<|/ref|><|det|>[[92, 645, 704, 694]]<|/det|>
+R1C9. Line 254- 255: MWM is no longer used in the prediction. We appreciate the reviewer catching this legacy text from the first submission. We have rephrased this point (L256).  
+
+<|ref|>text<|/ref|><|det|>[[92, 711, 737, 743]]<|/det|>
+R1C10. Table 1: "TSeson" and "PREC" – inconsistent with those in the text. And check the footnote of Table 1.  
+
+<|ref|>text<|/ref|><|det|>[[92, 745, 692, 761]]<|/det|>
+The acronym was changed as suggested in the table for consistency with the text.  
+
+<|ref|>text<|/ref|><|det|>[[92, 778, 733, 811]]<|/det|>
+R1C11. Figure 1: The precipitation has been log- transformed, right? Need to specify it. We have added this clarification to the corresponding axes in figure 1 and table 1.  
+
+<|ref|>text<|/ref|><|det|>[[92, 828, 720, 860]]<|/det|>
+R1C12. Figure 3 lower panel: why Precip. Dryiest Month is higher than Precip. Wettest Month?
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[92, 147, 738, 213]]<|/det|>
+We appreciate the reviewer catching this inconsistency. This was a problem in the code calling the different variables after we removed the Temperature of the Warmest Month, which caused a mismatch between the names and the data plotted in the bottom panels of figure 3. The figure now has corrected this.  
+
+<|ref|>text<|/ref|><|det|>[[92, 230, 712, 280]]<|/det|>
+R1C13. Figure 5 caption: what is "F- J"? We appreciate the reviewer catching this legacy text from the first submission. This text was removed.  
+
+<|ref|>sub_title<|/ref|><|det|>[[93, 298, 206, 312]]<|/det|>
+## Reviewer #2.  
+
+<|ref|>text<|/ref|><|det|>[[92, 330, 736, 380]]<|/det|>
+R2C1. Authors have successfully revised their manuscript. I have just one follow- up comment because authors might have misunderstood my earlier comment about models having one predictor at a time.  
+
+<|ref|>text<|/ref|><|det|>[[92, 381, 733, 431]]<|/det|>
+My intention was not to suggest to add multiple predictors to achieve better predictive ability, but to take into account the fact that the effect of a predictor can change when one takes into account the effect(s) of other potential predictor(s).  
+
+<|ref|>text<|/ref|><|det|>[[92, 431, 734, 579]]<|/det|>
+For example, ET and MCM both appear to be important limiting factors and also representing different kinds of limiting factors, ET relating to energy availability and MCM to annual limits. However, these variables are also highly correlated, which already indicates that it will be difficult to tell apart their individual effects. When you include both variables as predictors in the same model it actually turns out that the effect of ET is not statistically significant, response of population density to ET being more or less flat. Similarly, if you add e.g. NPP, ET and TS to the same model their effect (response shapes) are different from their effect when each is the only predictor in the model.  
+
+<|ref|>text<|/ref|><|det|>[[92, 580, 727, 661]]<|/det|>
+To me, all this suggests that the real limiting effects of climate variables can be different from those you get when you include these variables separately as predictors. However, I don't know how severe issue this truly is, but I would like to know your thoughts on that. If it really is an issue, one might use PCA to create uncorrelated climate variables and use these as predictors in the models.  
+
+<|ref|>text<|/ref|><|det|>[[92, 679, 734, 745]]<|/det|>
+We thank the reviewer for his positive feedback on our revision and the clarification of his original point. As we now understand the reviewer's point, the issue is that significant absolute effects from univariate models would not translate into relative effects determined by multivariate models.  
+
+<|ref|>text<|/ref|><|det|>[[92, 747, 736, 863]]<|/det|>
+While we agree with the point, we consider that the multiple regression approach does not translate to the idea of limiting factors we are evaluating here. We argue that multiple regression coefficients indicate effects in the context of other variables (hence contingent on which variables are included or omitted in a model). Therefore, these determine how much each variable contributes to the change in population density. To define which variables set a lower boundary, we require a measure of absolute effects provided by univariate approaches. Focusing on the relative effects would not allow us
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[92, 147, 730, 180]]<|/det|>
+to define limiting factors but which variable(s) contribute the most to changes in population density from the "regional" mean.  
+
+<|ref|>text<|/ref|><|det|>[[92, 181, 730, 280]]<|/det|>
+Suppose we could build models for change in population density for each evaluated time bin. In that case, we could define the variable that contributes the most to population density at each time bin. Still, this is not a limiting factor but the variable that contributes the most to changes in population density form the regional average (i.e., the model intercept). Last, there is the issue of translating these relative effects into space, which our approach based on univariate models can do.  
+
+<|ref|>text<|/ref|><|det|>[[92, 281, 735, 380]]<|/det|>
+Furthermore, while PCA, or other ordination approaches, could be used here to determine "groups of variables" and the variable most representative of such "group", we will still be looking at relative effects when using the two or three most important axes. There also be questions about how suitable it is to use the eigenvectors generated by the ordination under current conditions to "reorganize" past climatic surfaces where the correlations between variables change.  
+
+<|ref|>text<|/ref|><|det|>[[92, 381, 737, 446]]<|/det|>
+In summary, we consider that using univariate models, while far from perfect, is a practical approach to assessing the absolute effects of each variable and comparing these between variables over time. Also, it allows us to link our models to a process. All these points are now made in the text (L72- 80) and the methods (L364- 368).  
+
+<|ref|>sub_title<|/ref|><|det|>[[92, 464, 207, 478]]<|/det|>
+## Reviewer #3.  
+
+<|ref|>text<|/ref|><|det|>[[92, 480, 717, 512]]<|/det|>
+I am fully satisfied with the revised version and with the revised manuscript and the changes.  
+
+<|ref|>text<|/ref|><|det|>[[92, 514, 368, 529]]<|/det|>
+Thanks for your positive assessment.
+
+<--- Page Split --->
+<|ref|>text<|/ref|><|det|>[[115, 90, 286, 104]]<|/det|>
+Reviewers' Comments:  
+
+<|ref|>text<|/ref|><|det|>[[115, 120, 584, 165]]<|/det|>
+Reviewer #1:  Remarks to the Author:  I'm satisfied with the revisions and have no further comments.  
+
+<|ref|>text<|/ref|><|det|>[[115, 209, 288, 238]]<|/det|>
+Reviewer #2:  Remarks to the Author:  
+
+<|ref|>text<|/ref|><|det|>[[115, 240, 867, 283]]<|/det|>
+While I still slightly disagree with you about the effects of univariate vs multivariate models on the results, I'm happy to do so. It is good that you now explain in the manuscript your choices regarding the matter, so I'm fully satisfied with this revised manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[115, 300, 245, 328]]<|/det|>
+Best,  Miikka Tallavaara
+
+<--- Page Split --->
+<|ref|>sub_title<|/ref|><|det|>[[93, 147, 310, 163]]<|/det|>
+## REVIEWER COMMENTS  
+
+<|ref|>text<|/ref|><|det|>[[93, 181, 379, 196]]<|/det|>
+Reviewer #2 (Remarks to the Author):  
+
+<|ref|>text<|/ref|><|det|>[[92, 214, 718, 280]]<|/det|>
+While I still slightly disagree with you about the effects of univariate vs multivariate models on the results, I'm happy to do so. It is good that you now explain in the manuscript your choices regarding the matter, so I'm fully satisfied with this revised manuscript.  
+
+<|ref|>text<|/ref|><|det|>[[92, 297, 736, 329]]<|/det|>
+We appreciate your assessment regarding our revision and your willingness to agree to disagree regarding the effects of univariate vs multivariate models on the results.
+
+<--- Page Split --->

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