Análise pertinente demonstrando que o baixo consumo de alimentos ultraprocessados não indicaria, necessariamente, uma alimentação saudável, tendo em vista o baixo consumo de frutas, verduras, legumes e variedade alimentar na mesma população.

Ponto relevante, que merece destaque, pois incentiva os demais municípios brasileiros quanto à adoção de leis municipais para que a alimentação fornecida pelas cantinas escolares sejam mais nutritivas.

Annotation!

for - The projected timing of climate departure from recent variability - Camilo Mora et al. - 6th mass extinction - biodiversity loss - question - 2024 - Sept 13 - how do we reconcile climate departure with quantification of earth system boundary biodiversity safe and just limit? - to - climate departure map - map of major cities - 2013 - to - researchgate paper - The projected timing of climate departure from recent variability - 2013 - Camilo Mora et al

paper details - title: The projected timing of climate departure from recent variability - author: - Camilo Mora, - Abby G. Frazier, - Ryan J. Longman, - Rachel S. Dacks, - Maya M. Walton, - Eric J. Tong, - Joseph J. Sanchez, - Lauren R. Kaiser, - Yuko O. Stender, - James M. Anderson, - Christine M. Ambrosino, - Iria Fernandez-Silva, - Louise M. Giuseffi, - Thomas W. Giambelluca - date - 9 October, 2013 - publication Nature 502, 183-187 (2013) - https://doi.org/10.1038/nature12540 - https://www.nature.com/articles/nature12540

to - https://hyp.is/0BdCglsHEe-2CteEQbOBfw/www.researchgate.net/publication/257598710_The_projected_timing_of_climate_departure_from_recent_variability

Summary - This is an extremely important paper with a startling conclusion of the magnitude of the social and economic impacts of the biodiversity disruption coming down the pipeline - It is likely that very few governments are prepared to adapt to these levels of ecosystemic disruption - Climate departure is defined as an index of the year when: - The projected mean climate of a given location moves to a state that is - continuously outside the bounds of historical variability - Climate departure is projected to happen regardless of how aggressive our climate mitigation pathway - The business-as-usual (BAU) scenario in the study is RCP85 and leads to a global climate departure mean of 2047 (+/- 14 years s.d.) while - The more aggressive RCP45 scenario (which we are currently far from) leads to a global climate departure mean of 2069 (+/- 18 years s.d.) - So regardless of how aggressive we mitigate, we cannot avoid climate departure. - What consequences will this have on economies around the world? How will we adapt? - The world is not prepared for the vast ecosystem changes, which will reshape our entire economy all around the globe.

question - 2024 - Sept 13 - how do we reconcile climate departure with quantification of earth system boundary biodiversity safe and just limit? - Annotating the Sept 11, 2024 published Earth Commission paper in Lancet, the question arises: - How do we reconcile climate departure dates with the earth system boundary quantification of safe limits for biodiversity? - There, it is claimed that: - 50 to 60 % of intact nature is required<br /> - https://hyp.is/Mt8ocnIEEe-C0dNSJFTjyQ/www.thelancet.com/journals/lanplh/article/PIIS2542-5196(24)00042-1/fulltext - a minimum of 20 to 25% of human modified ecosystems is required - https://hyp.is/AKwa4nIHEe-U1oNQDdFqlA/www.thelancet.com/journals/lanplh/article/PIIS2542-5196(24)00042-1/fulltext - in order to mitigate major species extinction and social disruption crisis - And yet, Mora et al.'s research and subsequent climate departure map shows climate departure is likely to take place everywhere on the globe, with - aggressive RCP decarbonization pathway only delaying climate departure from - Business-As-Usual RCP pathway - by a few decades at most - And this was a 2011 result. 13 years later in 2024, I expect climate departure dates have likely gotten worse and moved closer to the present

from - Gupta, Joyeeta et al.(2024). A just world on a safe planet: a Lancet Planetary Health–Earth Commission report on Earth-system boundaries, translations, and transformations. The Lancet Planetary Health, Volume 0, Issue 0 - https://hyp.is/go?url=https%3A%2F%2Fwww.thelancet.com%2Fjournals%2Flanplh%2Farticle%2FPIIS2542-5196(24)00042-1%2Ffulltext&group=world

to - climate departure map - of major cities of the world - 2013 - https://hyp.is/tV1UOFsKEe-HFQ-jL-6-cw/www.hawaii.edu/news/2013/10/09/study-in-nature-reveals-urgent-new-time-frame-for-climate-change/ - full research paper - researchgate

nó là quyền thiên liêng hoặc quyền thừa kế, mà là quyền chung được phân bố ở cấp độ cá nhân trong một cộng động chính trị

REVISAR EEN HIPERPLASIA SUPRARREENAL CONGENITA

lowercase o: Methodology overview

Author response:

The following is the authors’ response to the original reviews.

Public Reviews: 

Reviewer #1 (Public Review): 

In this manuscript, Ferhat and colleagues describe their study aimed at developing a blood-brain barrier (BBB) penetrant agent that could induce hypothermia and provide neuroprotection from the sequelae of status epilepticus (SE) in mice. Hypothermia is used clinically in an attempt to reduce neurological sequelae of injury and disease. Hypothermia can be effective, but physical means used to reduce core body temperature are associated with untoward effects. Pharmacological means to induce hypothermia could be as effective with fewer untoward complications. Intracerebroventricularly applied neurotensin can cause hypothermia; however, neurotensin applied peripherally is degraded and does not cross the BBB. Here the authors develop and characterize a neurotensin conjugate that can reach the brain, induce hypothermia, and reduce seizures, cognitive changes, and inflammatory changes associated with status epilepticus. 

Strengths: <br /> (1) In general, the study is well-reasoned, well-designed, and seemingly well-executed. 

(2) Strong dose-response assessment of multiple neurotensin conjugates in mice. 

(3) Solid assessment of binding affinity, in vitro stability in blood, and brain uptake of the conjugate. 

(4) Appropriate inclusion of controls for SE and for drug injections. However, perhaps a vehicle control could have been employed. 

Sham animals received saline 0.9% which is the vehicle control considering it was used to dilute the water-soluble VH-N412 molecule.

(5) Multifaceted assessment of neurodegeneration, inflammation, and mossy fiber sprouting in the different groups. 

(6) Inclusion of behavioral assessments. 

(7) Evaluates NSTR1 receptor distribution in multiple ways; however, does not evaluate changes in receptor distribution or ping wo/w SE and/or various drugs. 

(8) Demonstrates that this conjugate can induce hypothermia and have positive effects on the sequelae of SE. Could have a great impact on the application of pharmacologically-induced hypothermia as a neuroprotective measure in patients. 

Weaknesses: 

(1) The authors make the claim, repeatedly, that the hypothermia caused by the neurotensin conjugate is responsible for the effects they see; however, what they really show is that the conjugate causes hypothermia AND has favorable effects on the sequelae of SE. They need to discuss that they did not administer the conjugate without allowing the pharmacological hypothermia (e.g., by warming the animal, etc.). 

We agree with Reviewer 1. We indeed hypothesize that it is principally the hypothermia induced by the NT conjugate that is responsible for the effects we observe. However, we do not exclude the possibility that the conjugate itself can have direct effects on the sequelae of SE. We tried to address this question with the in vitro experiments. Our results suggest that indeed, in the absence of hypothermia, the conjugate showed intrinsic neuroprotection of cultured hippocampal neurons challenged with excitotoxic agents such as NMDA or KA. Besides the description of these results in the “Results Section”, end of page 19 of the original manuscript, we had discussed them at the end of the “Discussion Section”, top of page 43 of the original manuscript.

In order to separate the hypothermia component from the potential direct neuroprotective effects of the NT conjugate, we did consider abolishing hypothermia in animals that were injected with the NT conjugate by warming them up. However, it is particularly difficult to increase in a well- controlled manner the body temperature of mice, in particular undergoing seizures, in a closed temperature-controlled chamber. In response to Reviewer 1 demand, we added a few sentences in the “Discussion Section”, page 45 of the revised version.

(2) In the status epilepticus studies, it is unclear how or whether they monitored animals for the development of spontaneous seizures. Can the authors please describe this?

The KA model we used was originally discovered more than 30 years ago, developed and very well characterized and mastered in our laboratory by Ben-Ari (Ben-Ari et al., 1979). Most of KA-treated mice that developed SE after KA injection developed spontaneous seizures subsequent to a latent period of about 1 week as described in Figure 3A, Results Section page 11 and in the reference we had mentioned in the Materials and Methods Section, page 27 (Schauwecker and Steward, 1997).

We agree that information regarding the development of spontaneous seizures is missing. We added 2 references, Gröticke et al., 2008; Wu et al., 2021 in the Materials and Methods Section, page 28 of the revised version, that describe the occurrence of spontaneous seizures after KA administration in mice. We also now added the following information in the Materials and Methods Section, end of page 29: In order to study mice in the chronic stage of epilepsy with spontaneous seizures, they were observed daily (at least 3 hours per day) for general behavior and occurrence of SRS. These are highly reproducible in the mouse KA model, allowing for visual monitoring and scoring of epileptic activity. After 3 weeks, most animals exhibited SRS, with 2 to 3 seizures per day, similar to previous observations (Wu et al., 2021). The detection of at least one spontaneous seizure per day was used as criterion indicating the animals had reached chronic phase that can ultimately be confirmed by mossy fiber sprouting (see Figure 7).

(3) They do not evaluate changes in receptor distribution or ping wo/w SE and/or various drugs. 

It is not clear to us what changes in receptor distribution need evaluation. We suppose the question concerns NTSR1 receptor. It would indeed be very interesting to compare NTSR1 in brain regions and different brain cells wo/w SE and/or various drugs, to assess receptor distribution or re-distribution, if any. However, addressing such a question is a project in itself that could not be addressed in the present study. Reviewer 1 also evokes ping wo/w SE and/or various drugs and if our understanding is correct, Reviewer 1 alludes to PING, Pyramidal Interneuronal Network γ (Dugladze et al., 2013, see reference below). Although we did not assess PING per se, we used multi-electrode arrays (MEA) on hippocampal brain slices stimulated wo/w KA to assess whether the VH-N412 conjugate could modulate pyramidal neuron activity. In order to respond to Reviewer 1 concern we added these data as Figure S2 with corresponding modifications in the Material and Methods Section (pages 34-35), in the Results Section (page 19) and in the Discussion Section page 43 of the revised version of our manuscript.

Dugladze T, Maziashvili N, Börgers C, Gurgenidze S, Häussler U, Winkelmann A, Haas CA, Meier JC, Vida I, Kopell NJ, Gloveli T. GABA(B) autoreceptor-mediated cell type-specific reduction of inhibition in epileptic mice. Proc Natl Acad Sci U S A. 2013 Sep 10;110(37):15073-8. doi: 10.1073/pnas.1313505110. Epub 2013 Aug 26. PMID: 23980149; PMCID: PMC3773756.

Bas du formulaire

(4) It is not clear why several different mouse strains were employed. 

We used 2 mouse strains in our work as mentioned in the Materials and Methods Section, page 21. The conjugates we developed and hypothermia evaluation were initially tested on adult Swiss CD-1 males. For the KA model and for behavioral tests, adult male FVB/N mice were used because they are considered as reliable and well described mouse models of epilepsy, where seizures are associated with cell death (Schauwecker, 2003). This not the case for a number of mouse strains that demonstrate very heterogeneous behavior in SE and heterogeneous neuronal death, sprouting and neuroinflammation. The FVB/N are also well suited for behavioral tests.

In response to the Reviewer 1 demand, the following sentence has been introduced in the Results Section, page 11 and in the Materials and Methods Section, page 21 of the revised manuscript: We assessed our conjugates in a model of KA-induced seizures using adult male FVB/N mice. This mouse strain was selected as a reliable and well described mouse model of epilepsy, where seizures are associated with cell death and neuroinflammation (Schauwecker, 2003; Wu et al., 2021).

Reviewer #2 (Public Review): 

Summary: 

The authors generated analogs consisting of modified neurotensin (NT) peptides capable of binding to low-density lipoprotein (LDL) and NT receptors. Their lead analog was further evaluated for additional validation as a novel therapeutic. The putative mechanism of action for NT in its antiseizure activity is hypothermia, and as therapeutic hypothermia has been demonstrated in epilepsy, NT analogs may confer antiseizure activity and avoid the negative effects of induced hypothermia. 

Strengths: 

The authors demonstrate an innovative approach, i.e. using LDLR as a means of transport into the brain, that may extend to other compounds. They systematically validate their approach and its potential through binding, brain penetration, in vivo antiseizure efficacy, and neuroprotection studies. 

Weaknesses: 

Tolerability studies are warranted, given the mechanism of action and the potential narrow therapeutic index. In vivo studies were used to assess the efficacy of the peptide conjugate analogs in the mouse KA model. However, it would be beneficial to have shown tolerability in naïve animals to better understand the therapeutic potential of this approach. 

Tolerability studies were performed, but the results were not presented in the first version of the manuscript. In order to comply with Reviewer 2 demand, we have added the following text in the Results section, page 11 of the revised version to describe our tolerability results.

Finally, tolerability studies were performed with the administration up to 20 and 40 mg/kg Eq. NT (i.e. 25.8 and 51.6 mg/kg of VH-N412) with n=3 for these doses. The rectal temperature of the animals did not fall below 32.5 to 33.2°C, similar to the temperature induced with the 4 mg/kg Eq. NT dose. We observed no mortality or notable clinical signs other than those associated with the rapid HT effect such as a decrease in locomotor activity. We thus report a very interesting therapeutic index since the maximal tolerated dose (MTD) was > 40 mg/kg Eq. NT, while the maximum effect is observed at a 10x lower dose of 4 mg/kg Eq. NT and an ED50 established at 0.69 mg/kg as shown in Figure 1G.

Mice may be particularly sensitive to hypothermia. It would be beneficial to show similar effects in a rat model. 

We have tested our conjugate in mice, rats, and pigs, with in all cases nice dose response curves. We added a few words in the Discussion Section, page 38 of the revised version to mention that we can elicit hypothermia with our conjugates in the above-mentioned species.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

(1) In Figures 4, 5, 6, 8, and 9, scale bars are needed on all panels. 

We have looked carefully at the Figures. Scale bars are present on all Figures, as mentioned in the Legends of all Figures, but not necessarily on all panel pictures at the same magnification.

(2) The supplemental would seemingly be better moved into the main body of the manuscript. 

In agreement with Reviewer 1 demand, we moved the Supplemental Figures into the main body of the manuscript, except for Figure S1, previously Figure S3, and the new Figure S2. Tables S1 to S5 remain as Supplemental files.

Reviewer #2 (Recommendations For The Authors): 

Activation of LDLRs can have widespread effects in the CNS and peripherally. The authors should further discuss any beneficial or untoward effects of binding to LDL and activating LDLRs. 

As mentioned in the Introduction and in a number of references where we describe the development of our family of LDLR peptide ligands (see below), we only selected peptide ligands that do not compete with LDL, one of the major ligands of the LDLR. We indeed showed that while LDL binds the ligand-binding domain of the LDLR, the peptide ligands we developed bind to the EGF-precursor homology domain of the receptor (See Malcor et al., 2008 below).

We have studied our peptide ligands in vitro and in vivo for more than 15 years and we have not observed beneficial or adverse effects. Actually, one of the members of our LDLR peptide family has been validated as a theragnostic agent and is in Phase 1 clinical trials for brain glioblastoma and pancreatic cancer. Hence, to our knowledge, the peptide ligand we describe in the present study shows no beneficial or untoward effects on LDL binding and activation of the LDLR. In response to Reviewer 2 recommendation, we added the following information and references in the Introduction Section, page 6 of the revised version of our manuscript: These peptides bind the EGF precursor homology domain of the LDLR and thus do not compete with LDL binding on the ligand-binding domain. To our knowledge, they have no beneficial or untoward effects on LDL binding and LDLR activity (Malcor et al., 2012; Jacquot et al., 2016; David et al., 2018; Varini et al., 2019; Acier et al., 2021, Yang et al., 2023; Broc et al., 2024).

Broc B, Varini K, Sonnette R, Pecqueux B, Benoist F, Masse M, Mechioukhi Y, Ferracci G, Temsamani J, Khrestchatisky M, Jacquot G, Lécorché P. LDLR-Mediated Targeting and Productive Uptake of siRNA-Peptide Ligand Conjugates In Vitro and In Vivo. Pharmaceutics. 2024 Apr 17;16(4):548. doi: 10.3390/pharmaceutics16040548. PMID: 38675209; PMCID: PMC11054735.

Yang X, Varini K, Godard M, Gassiot F, Sonnette R, Ferracci G, Pecqueux B, Monnier V, Charles L, Maria S, Hardy M, Ouari O, Khrestchatisky M, Lécorché P, Jacquot G, Bardelang D. Preparation and In Vitro Validation of a Cucurbit[7]uril-Peptide Conjugate Targeting the LDL Receptor. J Med Chem. 2023 Jul 13;66(13):8844-8857. doi: 10.1021/acs.jmedchem.3c00423. Epub 2023 Jun 20. PMID: 37339060. 

Acier A, Godard M, Gassiot F, Finetti P, Rubis M, Nowak J, Bertucci F, Iovanna JL, Tomasini R, Lécorché P, Jacquot G, Khrestchatisky M, Temsamani J, Malicet C, Vasseur S, Guillaumond F. LDL receptor-peptide conjugate as in vivo tool for specific targeting of pancreatic ductal adenocarcinoma. Commun Biol. 2021 Aug 19;4(1):987. doi: 10.1038/s42003-021-02508-0. PMID: 34413441; PMCID: PMC8377056.

Varini K, Lécorché P, Sonnette R, Gassiot F, Broc B, Godard M, David M, Faucon A, Abouzid K, Ferracci G, Temsamani J, Khrestchatisky M, Jacquot G. Target engagement and intracellular delivery of mono- and bivalent LDL receptor- binding peptide-cargo conjugates: Implications for the rational design of new targeted drug therapies. J Control Release. 2019 Nov 28;314:141-161. doi: 10.1016/j.jconrel.2019.10.033. Epub 2019 Oct 20. PMID: 31644939.

David M, Lécorché P, Masse M, Faucon A, Abouzid K, Gaudin N, Varini K, Gassiot F, Ferracci G, Jacquot G, Vlieghe P, Khrestchatisky M. Identification and characterization of highly versatile peptide-vectors that bind non- competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos. PLoS One. 2018 Feb 27;13(2):e0191052. doi: 10.1371/journal.pone.0191052. PMID: 29485998; PMCID: PMC5828360.

Molino Y, David M, Varini K, Jabès F, Gaudin N, Fortoul A, Bakloul K, Masse M, Bernard A, Drobecq L, Lécorché P, Temsamani J, Jacquot G, Khrestchatisky M. Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier. FASEB J. 2017 May;31(5):1807-1827. doi: 10.1096/fj.201600827R. Epub 2017 Jan 20. PMID: 28108572.

Jacquot G, Lécorché P, Malcor JD, Laurencin M, Smirnova M, Varini K, Malicet C, Gassiot F, Abouzid K, Faucon A, David M, Gaudin N, Masse M, Ferracci G, Dive V, Cisternino S, Khrestchatisky M. Optimization and in Vivo Validation of Peptide Vectors Targeting the LDL Receptor. Mol Pharm. 2016 Dec 5;13(12):4094-4105. doi: 10.1021/acs.molpharmaceut.6b00687. Epub 2016 Oct 11. PMID: 27656777.

Malcor JD, Payrot N, David M, Faucon A, Abouzid K, Jacquot G, Floquet N, Debarbieux F, Rougon G, Martinez J, Khrestchatisky M, Vlieghe P, Lisowski V. Chemical optimization of new ligands of the low-density lipoprotein receptor as potential vectors for central nervous system targeting. J Med Chem. 2012 Mar 8;55(5):2227-41. doi: 10.1021/jm2014919. Epub 2012 Feb 14. PMID: 22257077.

As described above, the authors should also comment on the tolerability of these analogs. 

Tolerability studies were performed, but the results were not presented in the first version of the manuscript. In order to comply with Reviewer 2 demand, we have added the following text in the Results section, page 11 of the revised version to describe our tolerability results.

Finally, tolerability studies were performed with the administration up to 20 and 40 mg/kg Eq. NT (i.e. 25.8 and 51.6 mg/kg of VH-N412) with n=3 for these doses. The rectal temperature of the animals did not fall below 32.5 to 33.2°C, similar to the temperature induced with the 4 mg/kg Eq. NT dose. We observed no mortality or notable clinical signs other than those associated with the rapid HT effect such as a decrease in locomotor activity. We thus report a very interesting therapeutic index since the maximal tolerated dose (MTD) was > 40 mg/kg Eq. NT, while the maximum effect is observed at a 10x lower dose of 4 mg/kg Eq. NT and an ED50 established at 0.69 mg/kg as shown in Figure 1G.

Author response:

The following is the authors’ response to the original reviews.

We are deeply appreciative of the reviewers' insightful comments and constructive feedback on our manuscript. In response, we have implemented substantial revisions to enhance the clarity and impact of our work. Key changes include: 

Reframing: We have shifted our focus from cognitive control to attention and memory processes, aligning more closely with our experimental design. This reframing is reflected throughout the manuscript, including additional citations highlighting the triple network model's involvement in memory processing. To reflect this change, we have updated the title to "Causal dynamics of salience, default mode, and frontoparietal networks during episodic memory formation and recall: A multi-experiment iEEG replication".

Control analyses using resting-state epochs: We have conducted new analyses comparing task periods to resting baseline epochs. These results demonstrate enhanced directed information flow from the anterior insula to both the default mode and frontoparietal networks during encoding and recall periods compared to resting state across all four experiments. This finding underscores the anterior insula's critical role in memory and attention processing.

Control analysis using the inferior frontal gyrus: To address specificity concerns, we performed control analyses using the inferior frontal gyrus as a comparison region. This analysis confirms that the observed directed information flow to the default mode and frontoparietal networks is specific to the anterior insula, rather than a general property of task-engaged brain regions.

These revisions, combined with our rigorous methodologies and comprehensive analyses, provide compelling support for the central claims of our manuscript. We believe these changes significantly enhance the scientific contribution of our work.

Our point-by-point responses to the reviewers' comments are provided below.

Reviewer 1:

-  The authors present results from an impressively sized iEEG sample. For reader context, this type of invasive human data is difficult and time-consuming to collect and many similar studies in high-level journals include 5-20 participants, typically not all of whom have electrodes in all regions of interest. It is excellent that they have been able to leverage open-source data in this way. 

-  Preprocessing of iEEG data also seems sensible and appropriate based on field standards. 

-  The authors tackle the replication issues inherent in much of the literature by replicating findings across task contexts, demonstrating that the principles of network communication evidenced by their results generalize in multiple task memory contexts. Again, the number of iEEG patients who have multiple tasks' worth of data is impressive. 

We thank the reviewer for the encouraging comments and appreciate the positive feedback.  

(1.1) The motivation for investigating the tripartite network during memory tasks is not currently well-elaborated. Though the authors mention, for example, that "the formation of episodic memories relies on the intricate interplay between large-scale brain networks (p. 4)", there are no citations provided for this statement, and the reader is unable to evaluate whether the nodes and networks evidenced to support these processes are the same as networks measured here. 

Recommendation: Detail with citations the motivation for assessing the tripartite network in these tasks. Include work referencing network-level and local effects during encoding and recall.

We appreciate the reviewer's feedback and suggestions for improving our framing. We have substantially expanded and revised the Introduction to elaborate on the motivation for investigating the tripartite network during memory tasks, supported by relevant citations.

We now provide a stronger rationale for examining these networks in the context of episodic memory, emphasizing that while the tripartite network has been extensively studied in cognitive control tasks, growing evidence suggests its relevance to episodic memory as a domain-general network. We cite several key studies that demonstrate the involvement of the salience, default mode, and frontoparietal networks in memory processes, including work by Sestieri et al. (2014) and Vatansever et al. (2021), which show the consistent engagement of these networks during memory tasks. We have also included references to studies examining network-level and local effects during encoding and recall, such as the work by Xie et al. (2012) on disrupted intrinsic connectivity in amnestic mild cognitive impairment, and Le Berre et al. (2017) on the role of insula connectivity in memory awareness (pages 4-5).

Furthermore, we have clarified how our study aims to address gaps in the current understanding by investigating the electrophysiological basis of these network interactions during memory formation and retrieval, which has not been explored in previous research. This expanded framing provides a clearer motivation for our investigation and places our study within the broader context of memory and network neuroscience research (pages 3-6).  

(1.2) In addition, though the tripartite network has been proposed to support cognitive control processes, and the neural basis of cognitive control is the framed focus of this work, the authors do not demonstrate that they have measured cognitive control in addition to simple memory encoding and retrieval processes. Tasks that have investigated cognitive control over memory (such as those cited on p. 13 - Badre et al., 2005; Badre & Wagner, 2007; Wagner et al., 2001; Wagner et al., 2005) generally do not simply include encoding, delay, and recall (as the tasks used here), but tend to include some manipulation that requires participants to engage control processes over memory retrieval, such as task rules governing what choice should be made at recall (e.g., from Badre et al., 2005 Fig. 1: congruency of match, associative strength, number of choices, semantic similarity). Moreover, though there are task-responsive signatures in the nodes of the tripartite networks, concluding that cognitive control is present because cognitive control networks are active would be a reverse inference.

Recommendation: If present, highlight components of the tasks that are known to elicit cognitive control processes and cite relevant literature. If the tasks cannot be argued to elicit cognitive control, reframe the motivation to focus on task-related attention or memory processes. If the latter, reframe the motivation for investigating the tripartite network in this context absent control.

We appreciate the reviewer's insightful comment and recommendation. We acknowledge that our tasks do not include specific manipulations designed to elicit cognitive control processes over memory retrieval. In light of this, we have reframed our motivation and discussion to focus on the role of the tripartite network in attention and memory processes more broadly, rather than cognitive control specifically (pages 3-6).

As noted in Response 1.1, we have revised the Introduction to emphasize the domain-general nature of these networks and their involvement in various cognitive processes, including memory. We also highlight how the salience, default mode, and frontoparietal networks contribute to different aspects of memory formation and retrieval, drawing on relevant literature.

Our revised framing examines the salience network's role in detecting behaviorally relevant stimuli and orienting attention during encoding, the default mode network's involvement in internally-driven processes during recall, and the frontoparietal network's contribution to maintaining and manipulating information in working memory. We now present our study as an investigation into how these networks interact during different phases of memory processing, rather than focusing specifically on cognitive control. This approach aligns better with our experimental design and allows us to explore the broader applicability of the tripartite network model to memory processes. 

This revised reframing provides a more accurate representation of our study's scope and contribution to understanding the role of large-scale brain networks in memory formation and retrieval (pages 3-6). 

(1.3) It is currently unclear if the directed information flow from AI to DMN and FPN nodes truly arises from task-related processes such as cognitive control or if it is a function of static brain network characteristics constrained by anatomy (such as white matter connection patterns, etc.). This is a concern because the authors did not find that influences of AI on DMN or FPN are increased relative to a resting baseline (collected during the task) or that directed information flow differs in successful compared to unsuccessful retrieval. I doubt that this AI influence is 1) supporting a switch between the DMN and FPN via the SN or 2) relevant for behavior if it doesn't differ from baseline-active task or across accuracy conditions. An additional comparison that may help investigate whether this is reflective of static connectivity characteristics would be a baseline comparison during non-task rest or sleep periods.  

Recommendation: As described in the task of the concern, analyze the PTE across the same contacts during sleep or task-free rest periods (if present in the dataset). 

We thank the reviewer for this suggestion. We have now carried out additional analyses using resting-state baseline epochs. We found that directed information flow from the AI to both the DMN and FPN were enhanced during the encoding and recall periods compared to resting-state baseline in all four experiments. These new results have now been included in the revised Results (page 12):    

“Enhanced information flow from the AI to the DMN and FPN during episodic memory processing, compared to resting-state baseline  

We next examined whether directed information flow from the AI to the DMN and FPN nodes during the memory tasks differed from the resting-state baseline. Resting-state baselines were extracted immediately before the start of the task sessions and the duration of task and rest epochs were matched to ensure that differences in network dynamics could not be explained by differences in duration of the epochs. Directed information flow from the AI to both the DMN and FPN were higher during both the memory encoding and recall phases and across the four experiments, compared to baseline in all but two cases (Figures S6, S7). These findings provide strong evidence for enhanced role of AI directed information flow to the DMN and FPN during memory processing compared to the resting state.” 

(1.4) Related to the above concern, it is also questionable how directed information flow from AI facilitates switching between FPN and DMN during both encoding and recall if high gamma activity does not significantly differ in AI versus PCC or mPFC during recall as it does during encoding. It seems erroneous to conclude that the network-level communication is happening or happening with the same effect during both task time points when these effects are decoupled in such a way from the power findings.  

We appreciate the reviewer's insightful observation regarding the apparent discrepancy between our directed information flow findings and the high-gamma activity results. This comment highlights an important distinction in interpreting our results, and we thank the reviewer for the opportunity to address this point.

Our findings demonstrate that directed information flow from the AI to the DMN and FPN persists during both encoding and recall, despite differences in local high-gamma activity patterns. This dissociation suggests that the network-level communication facilitated by the AI may operate independently of local activation levels in individual nodes. It is important to note that our directed connectivity analysis (using phase transfer entropy) was conducted on broadband signals (0.5-80 Hz), while the power analysis focused specifically on the high-gamma band (80-160 Hz). These different frequency ranges may capture distinct aspects of neural processing. The broadband connectivity might reflect more general, sustained network interactions, while high-gamma activity may be more sensitive to specific task demands or cognitive processes.

The phase transfer entropy analysis captures directed interactions over extended time periods, while the high-gamma power analysis provides a more temporally precise measure of local neural activity. The persistent directed connectivity from AI during recall, despite changes in local activity, might reflect the AI's ongoing role in coordinating network interactions, even when its local activation is not significantly different from other regions.

Rather than facilitating "switching" between FPN and DMN, as we may have previously overstated, our results suggest that the AI maintains a consistent pattern of influence on both networks across task phases. This influence might serve different functions during encoding (e.g., orienting attention to external stimuli) and recall (e.g., monitoring and evaluating retrieved information), even if local activation patterns differ.

It is crucial to note that in the three verbal tasks, our analysis of memory recall is time-locked to word production onset. However, the precise timing of the internal recall process initiation is unknown. This limitation may affect our ability to capture the full dynamics of network interactions during recall, particularly in the early stages of memory retrieval. Interestingly, in the spatial memory task WMSM, the PCC/precuneus exhibited an earlier onset and enhanced activity compared to the AI. This task may provide a clearer window into recall processes:

findings align with the view that DMN nodes may play a crucial role in triggering internal recall processes. However, the precise timing of internal retrieval initiation remains a challenge in the three verbal tasks, potentially limiting our ability to capture the full dynamics of regional activity, and its replicability, during early stages of recall.

These observations highlight the need for more detailed investigation of the temporal dynamics of network interactions during memory recall. To further elucidate the relationship between directed connectivity and local activity, future studies could employ time-resolved connectivity analyses and investigate coupling between different frequency bands. This could provide a more precise understanding of how network-level communication relates to local neural dynamics across different task phases.

We have revised the manuscript to more accurately reflect these points and avoid overstating the implications of our findings (pages 15-19). We thank the reviewer for prompting this important clarification, which we believe strengthens the interpretation and discussion of our results.

(1.5) Missing information about the methods used for time-frequency conversion for power calculation and the power normalization/baseline-correction procedure bars a thorough evaluation of power calculation methods and results. 

Recommendation: Include more information about how power was calculated. For example, how were time-series data converted to time-frequency (with complex wavelets, filter-hilbert, etc.)? What settings were used (frequency steps, wavelet length)? How were power values checked for outliers and normalized (decibels, Z-transform)? How was baseline correction applied (subtraction, division)?

We have now included detailed information related to our power calculation and normalization steps as we note on page 28: “We first filtered the signals in the high-gamma (80160 Hz) frequency band (Canolty et al., 2006; Helfrich & Knight, 2016; Kai J. Miller, Weaver, & Ojemann, 2009) using sequential band-pass filters in increments of 10 Hz (i.e., 80–90 Hz, 90– 100 Hz, etc.), using a fourth order two-way zero phase lag Butterworth filter. We used these narrowband filtering processing steps to correct for the 1/f decay of power. We then calculated the amplitude (envelope) of each narrow band signal by taking the absolute value of the analytic signal obtained from the Hilbert transform (Foster, Rangarajan, Shirer, & Parvizi, 2015). Each narrow band amplitude time series was then normalized to its own mean amplitude, expressed as a percentage of the mean. Finally, we calculated the mean of the normalized narrow band amplitude time series, producing a single amplitude time series. Signals were then smoothed using 0.2s windows with 90% overlap (Kwon et al., 2021) and normalized with respect to 0.2s pre-stimulus periods by subtracting the pre-stimulus baseline from the post-stimulus signal.” 

(1.6) If revisions to the manuscript can address concerns about directed information flow possibly being due to anatomical constraints - such as by indicating that directed information flow is not present during non-task rest or sleep - this work may convey important information about the structure and order of communication between these networks during attention to tasks in general. However, the ability of the findings to address cognitive control-specific communication and the nature of neurophysiological mechanisms of this communication - as opposed to the temporal order and structure of recruited networks - may be limited.

We appreciate the reviewer's insightful feedback, which has led to significant improvements in our manuscript. In response, we have made the following key changes. We have shifted our focus from cognitive control to the broader roles of the tripartite network in attention and memory processes. This reframing aligns more closely with our experimental design and the nature of our tasks. We have revised the Introduction, Results, and Discussion sections to reflect this perspective, providing a more accurate representation of our study's scope and contribution. Additionally, to strengthen our findings, we have conducted new analyses comparing task periods to resting-state baselines. These analyses revealed that directed information flow from the anterior insula to both the DMN and FPN was significantly enhanced during memory encoding and recall periods compared to resting-state across all four experiments. This finding provides robust evidence for the specific involvement of these network interactions in memory processing. Please also see Response 1.2 above. 

(1.7) Because phase-transfer entropy is presented as a "causal" analysis in this investigation (PTE), I also believe it is important to highlight for readers recent discussions surrounding the description of "causal mechanisms" in neuroscience (see "Confusion about causation" section from Ross and Bassett, 2024, Nature Neuroscience). A large proportion of neuroscientists (admittedly, myself included) use "causal" only to refer to a mechanism whose modulation or removal (with direct manipulation, such as by lesion or stimulation) is known to change or control a given outcome (such as a successful behavior). As Ross and Bassett highlight, it is debatable whether such mechanistic causality is captured by Granger "causality" (a.k.a. Granger prediction) or the parametric PTE, and the imprecise use of "causation" may be confusing. The authors could consider amending language regarding this analysis if they are concerned about bridging these definitions of causality across a wide audience. 

We thank the reviewer for this suggestion. We would like to clarify here that we define causality in our manuscript as follows: a brain region has a causal influence on a target if knowing the past history of temporal signals in both regions improves the ability to predict the target's signal in comparison to knowing only the target's past, as defined in earlier studies (Granger, 1969; Lobier, Siebenhühner, Palva, & Matias, 2014). We have now included this clarification in the Introduction section (page 6).  

We also agree with the reviewer that to more mechanistically establish a causal link between the neural dynamics and behavior, lesion or brain stimulation studies are necessary. We have now acknowledged this in the revised Discussion as we note: “Although our computational methods suggest causal influences, direct causal manipulations, such as targeted brain stimulation during memory tasks, are needed to establish definitive causal relationships between network nodes.” (page 19). 

Minor additional information that would be helpful to the reader to include: 

(1.8) How exactly was line noise (p. 24) removed? (For example, if notch filtered, how were slight offsets of the line noise from exactly 60.0Hz and harmonics identified and handled?). 

We would like to clarify here that to filter line noise and its harmonics, we used bandstop filters at 57-63 Hz, 117-123 Hz, and 177-183 Hz. To create a band-stop filter, we used a fourth order two-way zero phase lag Butterworth filter. This information has now been included in the revised Methods (page 26). 

(1.9) Why were the alpha and beta bands collapsed for narrowband filtering?

Please note that we did not combine the alpha (8-12 Hz) and beta (12-30 Hz) bands for narrowband filtering, rather these two frequency bands were analyzed separately. However, we combined the delta (0.5-4 Hz) and theta (4-8 Hz) frequency bands into a combined delta-theta (0.5-8 Hz) frequency band for our analysis since previous human electrophysiology studies have not settled on a specific band of frequency (delta or theta) for memory processing. Previous human iEEG (Ekstrom et al., 2005; Ekstrom & Watrous, 2014; Engel & Fries, 2010; Gonzalez et al., 2015; Watrous, Tandon, Conner, Pieters, & Ekstrom, 2013) as well as scalp EEG and MEG studies, have shown that both the delta and theta frequency band oscillations play a prominent role for human memory encoding as well as retrieval (Backus, Schoffelen, Szebényi, Hanslmayr, & Doeller, 2016; Clouter, Shapiro, & Hanslmayr, 2017; Griffiths, Martín-Buro, Staresina, & Hanslmayr, 2021; Guderian & Düzel, 2005; Guderian, Schott, Richardson-Klavehn, & Düzel, 2009).  

Reviewer 2:

In this study, the authors leverage a large public dataset of intracranial EEG (the University of Pennsylvania RAM repository) to examine electrophysiologic network dynamics involving the participation of salience, frontoparietal, and default mode networks in the completion of several episodic memory tasks. They do this through a focus on the anterior insula (AI; salience network), which they hypothesize may help switch engagement between the DMN and FPN in concert with task demands. By analyzing high-gamma spectral power and phase transfer entropy (PTE; a putative measure of information "flow"), they show that the AI shows higher directed PTE towards nodes of both the DMN and FPN, during encoding and recall, across multiple tasks. They further demonstrate that high-gamma power in the PCC/precuneus is decreased relative to the AI during memory encoding. They interpret these results as evidence of "triple-network" control processes in memory tasks, governed by a key role of the AI. 

I commend the authors on leveraging this large public dataset to help contextualize network models of brain function with electrophysiological mechanisms - a key problem in much of the fMRI literature. I also appreciate that the authors emphasized replicability across multiple memory tasks, in an effort to demonstrate conserved or fundamental mechanisms that support a diversity of cognitive processes. However, I believe that their strong claims regarding causal influences within circumscribed brain networks cannot be supported by the evidence as presented. In my efforts to clearly communicate these inadequacies, I will suggest several potential analyses for the authors to consider that might better link the data to their central hypotheses.

We thank the reviewer for the encouraging comments and suggestions for improving the manuscript. Please see our detailed responses and clarifications below. 

(2.1) As a general principle, the effects that the authors show - both in regards to their highgamma power analysis and PTE analysis - do not offer sufficient specificity for a reader to understand whether these are general effects that may be repeated throughout the brain, or whether they reflect unique activity to the networks/regions that are laid out in the Introduction's hypothesis. This lack of specificity manifests in several ways, and is best communicated through examples of control analyses. 

We appreciate the reviewer's insightful comment regarding the specificity of our findings. We agree that additional analyses could provide valuable context for interpreting our results. In response, we have conducted the following additional analyses and made corresponding revisions to the manuscript:

Following the reviewer's suggestion, we have selected the inferior frontal gyrus (IFG, BA 44) as a control region. The IFG serves as an ideal control region due to its anatomical adjacency to the AI, its involvement in a wide range of cognitive control functions including response inhibition (Cai, Ryali, Chen, Li, & Menon, 2014), and its frequent co-activation with the AI in fMRI studies. Furthermore, the IFG has been associated with controlled retrieval of memory (Badre et al., 2005; Badre & Wagner, 2007; Wagner et al., 2001), making it a compelling region for comparison. We repeated our PTE analysis using the IFG as the source region, comparing its directed influence on the DMN and FPN nodes to that of the AI.  

Our analysis revealed a striking contrast between the AI and IFG in their patterns of directed information flow. While the AI exhibited strong causal influences on both the DMN and FPN, the IFG showed the opposite pattern. Specifically, both the DMN and FPN demonstrated higher influence on the IFG than the reverse, during both encoding and recall periods, and across all four memory experiments (Figures S4, S5). 

These findings highlight the unique role of the AI in orchestrating large-scale network dynamics during memory processes. The AI's pattern of directed information flow stands in contrast to that of the IFG, despite their anatomical proximity and shared involvement in cognitive control processes. This dissociation underscores the specificity of the AI's function in coordinating network interactions during memory formation and retrieval. These results have now been included in our revised Results on page 11.  

(2.2) First, the PTE analysis is focused solely on the AI's interactions with nodes of the DMN and FPN; while it makes sense to focus on this putative "switch" region, the fact that the authors report significant PTE from the AI to nodes of both networks, in encoding and retrieval, across all tasks and (crucially) also at baseline, raises questions about the meaningfulness of this statistic. One way to address this concern would be to select a control region that would be expected to have little/no directed causal influence on these networks and repeat the analysis. Alternatively (or additionally), the authors could examine the time course of PTE as it evolves throughout an encoding/retrieval interval, and relate that to the timing of behavioral events or changes in high-gamma power. This would directly address an important idea raised in their own Discussion, "the AI is wellpositioned to dynamically engage and disengage with other brain areas."  

Please see Response 2.1 above for additional analyses related to control region.  

We also appreciate the reviewer's suggestion regarding time-resolved PTE analysis. However, it's important to note that our current methodology does not allow for such fine-grained temporal analysis. This is due to the fact that PTE, which is an information theoretic measure and relies on constructing histograms of occurrences of singles, pairs, or triplets of instantaneous phase estimates from the phase time-series (Hillebrand et al., 2016) (Methods), requires sufficient number of cycles in the phase time-series for its reliable estimation (Lobier et al., 2014). PTE is based on estimating the time-delayed directed influences from one time-series to the other and its estimate is the most accurate when a large number of time-points (cycles) are available (Lobier et al., 2014). Since our encoding and recall epochs in the verbal recall tasks were only 1.6 seconds long, which corresponds to only 800 time-points with a 500 Hz sampling rate, we used the entire encoding and recall epochs for the most efficient estimate of PTE, rather than estimating PTE in a time-resolved manner. Please note that this is consistent with previous literature which have used ~ 225000 time-points (3 minutes of resting-state data with 1250 Hz sampling rate) for estimating PTE, for example, see (Hillebrand et al., 2016). 

This limitation prevents us from examining how directed connectivity evolves throughout the encoding and retrieval intervals on a moment-to-moment basis. Future studies employing longer task epochs or alternative methods for time-resolved connectivity analysis could provide valuable insights into the dynamic engagement and disengagement of the AI with other brain areas based on task demands. Such analyses could potentially reveal task-specific temporal patterns in the AI's influence on DMN and FPN nodes during different phases of memory processing.

Finally, it is crucial to note that in the three verbal tasks, our analysis of memory recall is timelocked to word production onset. However, the precise timing of the internal retrieval process initiation is unknown. This limitation may affect our ability to capture the full dynamics of network interactions during recall, particularly in the early stages of memory retrieval. Interestingly, in the spatial memory task, where this timing issue is less problematic due to the nature of the task, we observe that the PCC/precuneus shows an earlier onset of activity compared to the AI. This process is aligned with the view that DMN nodes may trigger internal recall processes, the full extent and replication of which across verbal and spatial tasks could not be examined in this study.  

We have added a discussion of these limitations and future directions to the manuscript to provide a more nuanced interpretation of our findings and to highlight important areas for further investigation (page 19). 

(2.3) Second, the authors state that high-gamma suppression in the PCC/precuneus relative to the AI is an anatomically specific signature that is not present in the FPN. This claim does not seem to be supported by their own evidence as presented in the Supplemental Data (Figures S2 and S3), which to my eye show clear evidence of relative suppression in the MFG and dPPC (e.g. S2a and S3a, most notably) which are notated as "significant" with green bars. I appreciate that the magnitude of this effect may be greater in the PCC/precuneus, but if this is the claim it should be supported by appropriate statistics and interpretation.  

We thank the reviewer for raising this point. We have now directly compared the high-gamma power of the PCC/precuneus with the dPPC and MFG nodes of the FPN and we note that the suppression effects of the PCC/precuneus are stronger compared to those of the dPPC and MFG during memory encoding (Figures S8, S9). 

(2.4) I commend the authors on emphasizing replicability, but I found their Bayes Factor (BF) analysis to be difficult to interpret and qualitatively inconsistent with the results that they show. For example, the authors state that BF analysis demonstrates "high replicability" of the gamma suppression effect in Figure 3a with that of 3c and 3d. While it does appear that significant effects exist across all three tasks, the temporal structure of high gamma signals appears markedly different between the two in ways that may be biologically meaningful. Moreover, it appears that the BF analysis did not support replicability between VFR and CATVFR, which is very surprising; these are essentially the same tasks (merely differing in the presence of word categories) and would be expected to have the highest degree of concordance, not the lowest. I would suggest the authors try to analytically or conceptually reconcile this surprising finding. 

We appreciate the reviewer's commendation on our emphasis on replicability and thank the reviewer for the opportunity to provide clarification.

First, we would like to clarify the nature of our BF analysis. Bayes factors are calculated as the ratio of the marginal likelihood of the replication data, given the posterior distribution estimated from the original data, and the marginal likelihood for the replication data under the null hypothesis of no effect (Ly, Etz, Marsman, & Wagenmakers, 2019). Specifically, BFs use the posterior distribution from the first experiment as a prior distribution for the replication test of the second experiment to constitute a joint multivariate distribution (i.e., the additional evidence for the alternative hypothesis given what was already observed in the original study) and this joint distribution is dependent on the similarity between the two experiments (Ly et al., 2019).  This analysis revealed that PCC/precuneus suppression, in comparison to the AI during memory encoding, observed in the VFR during memory encoding was detected in two other tasks, PALVCR, and WMSM with high BFs. In the CATVFR task, although there were short time periods of PCC/precuneus suppression (Figure 3), the effects were not strong enough like the three other tasks.  

Regarding the high-gamma suppression effect, our BF analysis indeed supports replicability across the VFR, PALVCR, and WMSM tasks. While we agree with the reviewer that the temporal structure of high-gamma signals appears different across tasks, the BF analysis focuses on the overall presence of the suppression effect rather than its precise temporal profile. The high BFs indicate that the core finding - PCC/precuneus suppression relative to the AI during memory encoding - is replicated across these tasks, despite differences in the timing of this suppression. Moreover, at no time point did responses in the PCC/precuneus exceed that of the AI in any of the four memory encoding tasks. 

The reason for differences in temporal profiles is not clear. While VFR and CATVFR are similar, the addition of categorical structure in CATVFR may have introduced cognitive processes that alter the temporal dynamics of regional responses. Moreover, differences in electrode placements across participants in each experiment may also have contributed to variability in the observed effects. Further studies using within-subjects experimental designs are needed to address this. 

We have updated our Results and Discussion sections to reflect these points and to provide a more nuanced interpretation of the replicability across tasks.  

(2.5) To aid in interpretability, it would be extremely helpful for the authors to assess acrosstask similarity in high-gamma power on a within-subject basis, which they are wellpowered to do. For example, could they report the correlation coefficient between HGP timecourses in paired-associates versus free-recall tasks, to better establish whether these effects are consistent on a within-subject basis? This idea could similarly be extended to the PTE analysis. Across-subject correlations would also be a welcome analysis that may provide readers with better-contextualized effect sizes than the output of a Bayes Factor analysis.  

We thank the reviewer for this suggestion. However, a within-subject analysis was not possible because very few participants participated in multiple memory tasks. 

For example, for the AI-PCC/Pr analysis, only 1 individual participated in both the VFR and PALVCR tasks (Tables S2a, S2c). Similarly, for AI-mPFC analysis, only 3 subjects participated in both the VFR and PALVCR tasks (Tables S2a, S2c).  

Due to these small sample sizes, it was not feasible for us to assess replicability across tasks on a within-subject basis in our dataset. Therefore, for all our analysis, we have pooled electrode pairs across subjects and then subjected these to a linear mixed effects modeling framework for assessing significance and then subsequently assessing replicability of these effects using the Bayes factor (BF) framework.    

Recommendations For The Authors: 

(2.6) I would emphasize manuscript organization in a potential rewrite; it was very difficult to follow which analyses were attempting to show a contrast between effects versus a similarity between effects. Results were grouped by the underlying experimental conditions (e.g. encoding/recall, network identity, etc.) but may be better grouped according to the actual effects that were found. 

We thank the reviewer for this suggestion. We considered this possibility, but we feel that the Results section is best organized in the order of the hypotheses we set out to test, starting from analyzing local brain activity using high-gamma power analysis, and then results related to analyzing brain connectivity using PTE. All these results are systematically ordered by presenting results related to encoding first and then the recall periods as they appear sequentially in our task-design, presenting the results related to the VFR task first and then demonstrating replicability of the results in the three other experiments. Results are furthermore arranged by nodes, where we first discuss results related to the DMN nodes, and then the same for the FPN nodes. This is to ensure systematic, unbiased organization of all our results for the readers to clearly follow the hypotheses, statistical analyses, and the brain regions considered. Therefore, for transparency and ethical reasons, we would respectfully like to present our results as they appear in our current manuscript, rather than presenting the results based on effect sizes. 

However, please note that we indeed have ordered our results in the Discussion section based on actual effects, as suggested by the reviewer.  

(2.7) The absence of a PTE effect when analyzing through the lens of successful vs. unsuccessful memory is an important limitation of the current study and a significant departure from the wealth of subsequent memory effects reported in the literature (which the authors have already done a good job citing, e.g. Burke et al. 2014 Neuroimage). I'm glad that the authors raised this in their Discussion, but it is important that the results of such an analysis actually be shown in the manuscript. 

We thank the reviewer for this suggestion. We have now included the results related to PTE dynamics for successful vs. unsuccessful memory trials in the revised Results section as we note on page 12: 

“Differential information flow from the AI to the DMN and FPN for successfully recalled and forgotten memory trials 

We examined memory effects by comparing PTE between successfully recalled and forgotten memory trials. However, this analysis did not reveal differences in directed influence from the AI on the DMN and FPN or the reverse, between successfully recalled and forgotten memory trials during the encoding as well as recall periods in any of the memory experiments (all ps>0.05).”

(2.8) I believe the claims of causality through the use of the PTE are overstated throughout the manuscript and may contribute to further confusion in the literature regarding how causality in the brain can actually be understood. See Mehler and Kording, 2018 arXiv for an excellent discussion on the topic (https://arxiv.org/abs/1812.03363). My recommendation would be to significantly tone down claims that PTE reflects causal interactions in the brain. 

We thank the reviewer for this suggestion. We would like to clarify here that we define causality in our manuscript as follows: a brain region has a causal influence on a target if knowing the past history of temporal signals in both regions improves the ability to predict the target's signal in comparison to knowing only the target's past, as defined in earlier studies (Granger, 1969; Lobier et al., 2014). We have now included this clarification in the Introduction section (page 6).  

We also agree with the reviewer that to more mechanistically establish a causal link between the neural dynamics and behavior, lesion or brain stimulation studies are necessary. We have now acknowledged this in the revised Discussion as we note: “Although our computational methods suggest causal influences, direct causal manipulations, such as targeted brain stimulation during memory tasks, are needed to establish definitive causal relationships between network nodes.” (page 19). 

Finally, we have now significantly toned down our claims that PTE reflects causal interactions in the brain, in the Introduction, Results, and Discussion sections of our revised manuscript.  

(2.9) Relatedly, it may be useful for the authors to consider a supplemental analysis that uses classic measures of inter-regional synchronization, e.g. the PLV, and compare to their PTE findings. They cite literature to suggest a metric like the PTE may be useful, but this hardly rules out the potential utility of investigating narrowband phase synchronization. 

We thank the reviewer for this suggestion. We have now run new analyses based on PLV to examine phase synchronization between the AI and the DMN and FPN. However, we did not find a significant PLV for the interactions between the AI and DMN and FPN nodes for the different task periods compared to the resting baselines, as we note on page 13: 

“Narrowband phase synchronization between the AI and the DMN and FPN during encoding and recall compared to resting baseline  

We next directly compared the phase locking values (PLVs) (see Methods for details) between the AI and the PCC/precuneus and mPFC nodes of the DMN and also the dPPC and MFG nodes of the FPN for the encoding and the recall periods compared to resting baseline. However, narrowband PLV values did not significantly differ between the encoding/recall vs. rest periods, in any of the delta-theta (0.5-8 Hz), alpha (8-12 Hz), beta (12-30 Hz), gamma (30-80 Hz), and high-gamma (80-160 Hz) frequency bands. These results indicate that PTE, rather than phase synchronization, more robustly captures the AI dynamic interactions with the DMN and the FPN.” 

Please note that phase locking measures such as the PLV or coherence do not probe directed causal influences and cannot address how one region drives another. Instead, our study examined the direction of information flow between the AI and the DMN and FPN using robust estimators of the direction of information flow. PTE assesses with the ability of one time-series to predict future values of other time-series, thus estimating the time-delayed causal influences between the two time-series, whereas PLV or coherence can only estimate “instantaneous” phase synchronization, but not predict the future time-series. 

Additionally, please note that the directed information flow from the AI to both the DMN and FPN were enhanced during the encoding and recall periods compared to resting state across all four experiments, in a new set of analyses that we have carried out in our revised manuscript. Specifically, we have now carried out our task versus rest comparison by using resting baseline epochs before the start of the entire session of the task periods, rather than our previously used rest epochs which were in between the task periods. These new results have now been included in the revised Results as we note on page 12:  

“Enhanced information flow from the AI to the DMN and FPN during episodic memory processing, compared to resting-state baseline

We next examined whether directed information flow from the AI to the DMN and FPN nodes during the memory tasks differed from the resting-state baseline. Resting-state baselines were extracted immediately before the start of the task sessions and the duration of task and rest epochs were matched to ensure that differences in network dynamics could not be explained by differences in duration of the epochs. Directed information flow from the AI to both the DMN and FPN were higher during both the memory encoding and recall phases and across the four experiments, compared to baseline in all but two cases (Figures S6, S7). These findings provide strong evidence for enhanced role of AI directed information flow to the DMN and FPN during memory processing compared to the resting state.”  

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Cap. 1 Quando o santo

the law does not take guilt consciences into account.

Inserire sotto immagini di leonardo www.leonefoundation.it

Author response:

The following is the authors’ response to the previous reviews.

Public reviews

This study describes a group of CRH-releasing neurons, located in the paraventricular nucleus of the hypothalamus, which, in mice, affects both the state of sevoflurane anesthesia and a grooming behavior observed after it. PVHCRH neurons showed elevated calcium activity during the post-anesthesia period. Optogenetic activation of these PVHCRH neurons during sevoflurane anesthesia shifts the EEG from burst-suppression to a seemingly activated state (an apparent arousal effect), although without a behavioral correlate. Chemogenetic activation of the PVHCRH neurons delays sevoflurane-induced loss of righting reflex (another apparent arousal effect). On the other hand, chemogenetic inhibition of PVHCRH neurons delays recovery of righting reflex and decreases sevoflurane-induced stress (an apparent decrease in the arousal effect). The authors conclude that PVHCRH neurons "integrate" sevoflurane-induced anesthesia and stress. The authors also claim that their findings show that sevoflurane itself produces a post-anesthesia stress response that is independent of any surgical trauma, such as an incision. In its revised form, the article does not achieve its intended goal and will not have impact on the clinical practice of anesthesiology nor on anesthesiology research.

Thanks for the reviews. Please see our responses to the following comments.

Weaknesses:

The most significant weaknesses remain:

a) overinterpretation of the significance of their findings

b) the failure to use another anesthetic as a control,

c) a failure to compellingly link their post-sevoflurane measures in mice to anything measured in humans, and

d) limitations in the novelty of the findings. These weaknesses are related to the primary concerns described below:

Concerns about the primary conclusion that PVHCRH neurons integrate the anesthetic effects and post-anesthesia stress response of sevoflurane GA

(1) After revision, their remain multiple places where it is claimed that PVHCRH neurons mediate the anesthetic effects of sevoflurane (impact statement: we explain "how sevoflurane-induced general anesthesia works..."; introduction: "the neuronal mechanisms that mediate the anesthetic effects...of sevoflurane GA remain poorly understood" and "PVHCRH neurons may act as a crucial node integrating the anesthetic effect and stress response of sevoflurane").The manuscript simply does not support these statements. The authors show that a short duration exposure to sevoflurane inhibits PVHCRH neurons, but this is followed by hyperexcitability of these neurons for a short period after anesthesia is terminated. They show that the induction and recovery from sevoflurane anesthesia can be modulated by PVHCRH neuronal activity, most likely through changes in brain state (measured by EEG). They also show that PVHCRH neuronal activity modulates corticosterone levels and grooming behavior observed post-anesthesia (which the authors argue are two stress responses).These two things (effects during anesthesia and effects post-anesthesia)may be mechanistically unrelated to each other. None of these observations relate to the primary mechanism of action for sevoflurane. All claims relating to "anesthetic effects" should be removed. Even the term "integration" seems wrong-it implies the PVH is combining information about the anesthetic effect and post-anesthesia stress responses.

As requested, we have removed all claims related to ‘anesthetic effects’ or ‘integration’. Please see the revised manuscript.

(2) lt is important to compare the effects of sevoflurane with at least one other inhaled ether anesthetic as one step towards elevating the impact of this paper to the level required for a journal such as eLife. Isoflurane, desflurane, and enflurane are ether anesthetics that are very similar to each other, as well as being similar to sevoflurane. For example, one study cited by the authors (Marana et al.2013) concludes that there is weak evidence for differences in stress-related hormones between sevoflurane and desflurane, with lower levels of cortisol and ACTH observed during the desflurane intraoperative period. It is important to determine whether desflurane activates PVHCRH neurons in the post-anesthesia period, and whether this is accompanied by excess grooming in the mice, because this will distinguish whether the effects of sevoflurane generalize to other inhaled anesthestics, or, alternatively, relate to unique idiosyncratic properties of this gas that may not be a part of its anesthetic properties.

Thanks for your insightful comments and suggestions. Regarding your request for additional experiments, we acknowledge the value they could add to our study. However, investigating whether the effects of sevoflurane generalize to other inhaled anesthetics is beyond the scope of our current study. There is evidence indicating the prevalence of anesthetic stress caused by inhaled ether anesthetics1,2. The post-anesthesia stress-related behaviors caused by sevoflurane administration are reminiscent of delirium observed clinically. Notably, studies have shown that the use of desflurane for maintenance of anesthesia did not significantly affect the incidence or duration of delirium compared to sevoflurane administration3. This suggests that our observations likely represent a generalized response to inhaled ether anesthetic rather than being specific to sevoflurane.

Concerns about the clinical relevance of the experiments

In anesthesiology practice, perioperative stress observed in patients is more commonly related to the trauma of the surgical intervention, with inadequate levels of antinociception or unconsciousness intraoperatively and/or poor post-operative pain control. The authors seem to be suggesting that the sevoflurane itself is causing stress because their mice receive sevoflurane but no invasive procedures, but there is no evidence of sevoflurane inducing stress in human patients. It is important to know whether sevoflurane effectively produces behavioral stress in the recovery room in patients that could be related to the putative stress response (excess grooming) observed in mice. For example, in surgeries or procedures which required only a brief period of unconsciousness that could be achieved by administering sevoflurane alone (comparable to the 30 min administered to the mice), is there clinical evidence of post-operative stress? It is also important to describe a rationale for using a 30 min sevoflurane exposure. What proportion of human surgeries using sevoflurane use exposure times that are comparable to this?

It is also the case that there are explicit published findings showing that mild and moderate surgical procedures in children receiving sevoflurane (which might be the closest human proxy to the brief 30 minutes sevoflurane exposure used here) do not have elevated cortisol (Taylor et al, J Clin Endocrinol Metab, 2013). This again raises the question of whether the enhanced grooming or elevated corticosterone observed in the mice here has any relevance to humans.

Thanks for the comments. Most ear, nose, and throat surgeries in children involve a short period of anesthesia with sevoflurane alone4-6, which is similar to the 30-minute exposure in our mouse study. In clinical settings, emergence delirium and agitation are common in young children undergoing sevoflurane anesthesia7, often accompanied by troublesome excitation phenomena during induction and awakening8. These clinical observations align with the post-operative stress response (e.g., excessive grooming) we identified in our study.

It is the experience of one of the reviewers that human patients who receive sevoflurane as the primary anesthetic do not wake up more stressed than if they had had one of the other GABAergic anesthetics. If there were signs of stress upon emergence (increased heart rate, blood pressure, thrashing movements) from general anesthesia, this would be treated immediately. The most likely cause of post-operative stress behaviors in humans is probably inadequate anti-nociception during the procedure, which translates into inadequate post-op analgesia and likely delirium. It is the case that children receiving sevoflurane do have a higher likelihood of post-operative delirium. Perhaps the authors' studies address a mechanism for delirium associated with sevoflurane, but this is barely mentioned. Delirium seems likely to be the closest clinical phenomenon to what was studied. As noted by the Besnier et al (2017) article cited by the authors, surgery can elevate postoperative glucocorticoidstress hormones, but it generally correlates with the intensity of the surgical procedure. Besnier et al also note the elevation of glucocorticoids is generally considered to be adaptive. Thus, reducing glucocorticoids during surgery with sevoflurane may hamper recovery, especially as it relates to tissue damage, which was not measured or considered here. This paper only considers glucocorticoid release as a negative factor, which causes "immunosuppression", "proteolysis", and "delays postoperative recovery and leads to increased morbidity".

Thanks for the comments. We agree that the post-anesthetic stress behaviors mentioned in our manuscript are similar to the clinical phenomenon of delirium, which were defined in Cheng Li’s study as ‘sevoflurane-induced post-operative delirium’9. Therefore, we conducted additional behavioral tests for cognitive function, including the Y-maze and novel object recognition test, in mice administrated 30-minute sevoflurane anesthesia. The results demonstrate that chemogenetic inhibition of PVHCRH neurons ameliorated the short-term memory impairment in mice exposed to 30-minute sevoflurane GA (Figure 7-figure supplement 9), suggesting PVHCRH neurons may involve in modulating sevoflurane-induced postoperative delirium.

Concerns about the novelty of the findings:

The key finding here is that CRH neurons mediate measures of arousal, and arousal modulates sevoflurane anesthesia induction and recovery. However, CRH is associated with arousal in numerous studies. In fact, the authors' own work, published in eLife in 2021, showed that stimulating the hypothalamic CRH cells lead to arousal and their inhibition promoted hypersomnia. In both papers the authors use fos expression in CRH cells during a specific event to implicate the cells, then manipulate them and measure EEG responses. In the previous work, the cells were active during wakefulness; here- they were active in the awake state the follows anesthesia (Figure1). Thus, the findings in the current work are incremental and not particularly impactful. Claims like "Here, a core hypothalamic ensemble, corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus, is discovered" are overstated. PVHCRH cell populations were discovered in the 1980s. Suggesting that it is novel to identify that hypothalamic CRH cells regulate post-anesthesia stress is unfounded as well: this PVH population has been shown over four decades to regulate a plethora of different responses to stress. Anesthesia stress is no different. Their role in arousal is not being discovered in this paper. Even their role in grooming is not discovered in this paper.

Thanks for the comments. As requested, we have revised our manuscript by removing overstated sentences. Please see the revised manuscript. In terms of novelty, our study reveals that PVHCRH neurons are implicated not only in the induction and emergence of sevoflurane general anesthesia but also in sevoflurane-induced post-operative delirium. This finding represents a novel contribution to the field, as it has not been previously reported by other studies.

The activation of CRH cells in PVH has already been shown to result in grooming by Jaideep Bains (a paper cited by the authors). Thus, the involvement of these cells in this behavior is not surprising. The authors perform elaborate manipulations of CRH cells and numerous analyses of grooming and related behaviors. For example, they compare grooming and paw licking after anesthesia with those after other stressors such as forced swim, spraying mice with water, physical attack and restraint. The authors have identified a behavioral phenomenon in a rodent model that does not have a clear correlation with a behavior state observed in humans during the use of sevoflurane as part of an anesthetic regimen. The grooming behaviors are not a model of the emergence delirium or the cognitive dysfunction observed commonly in patients receiving sevoflurane for general anesthesia. Emergence delirium is commonly seen in children after sevoflurane is used as part of general anesthesia and cognitive dysfunction is commonly observed in adults-particularly the elderly-- following general anesthesia. No features of delirium or cognitive dysfunction are measured here.

As requested, behavioral tests for cognitive function have been conducted and displayed in Figure 7-figure supplement 9.

Other concerns:

In Figure 2, cFos was measured in the PVH at different points before, during and after sevoflurane. The greatest cFos expression was seen in Post 2, the latest time point after anesthesia. However, this may simply reflect the fact that there is a delay between activity levels and expression of cFos (as noted by the authors, 2-3 hours). Thus, sacrificing mice 30 minutes after the onset of sevoflurane application would be expected to drive minimal cFos expression, and the cFos observed at 30 minutes would not accurately reflect the activity levels during the sevoflurane. Also, the authors state that the hyperactivity, as measured by cFos, lasted "approximately 1 hours before returning to baseline", but there is no data to support this return to baseline.

Thanks for the comments. We apologize that the protocol we used for c-fos staining may not accurately reflect the activity levels, so we have removed Figure 2F. The sentence ‘lasted approximately 1 hours before returning to baseline’ refers to the calcium signal but not c-fos level.

In Figure 7, the number of animals appears to change from panel to panel even though they are supposed to show animals from the same groups. For example, cort was measured in only 3 saline-treated O2 animals (Fig 7E), but cFos and CRH were assessed in 4 (Fig C,D). Similarly, grooming time and time spent in open arms was measured in 6 saline-treated O2 controls (Fig 7F, H) but central distance was measured in 8(Fig 7G). There are other group number discrepancies in this figure--the number of data points in the plots do not match what is reported in the legend for numerous groups. Similarly, Figure 4 has a mismatch between the Ns reported in the legend and the number of points plotted per bar. For example, there were 10 animals in the hM3Di group; all are shown for the LORR and time to emergence plots, but only8 were used for time to induction. The legends reported N=7 for the mCherry group, yet 9 are shown for the time to emergence panel. No reason for exclusions is cited. These figures (and their statistics) should be corrected.

Thanks for the comments. We have rechecked and corrected our figures and illustrations in the revised manuscript.

Recommendations for the authors:

In Figure 6, the BSR pre-stim data points for panels F and H look exactly identical, even though these data are from two different sets of mice. It seems likely that one of these panels is not depicting the correct pre-stim data points. Please check this.

Thanks for the comments. We have corrected this mistake.

General anesthesia is a combination of behavioral and physiological states induced and maintained primarily by pharmacologic agents. The authors do not provide a definition of general anesthesia.

Thanks for the advice. We have added the definition of general anesthesia in the introduction part.

The first sentence of the abstract closely resembles the first sentence of the abstract of Brown，Purdon and Van Dort,Annu. Rev. Neurosci. 2011,34:601-28 yet, there is no citation.

Thanks for the comments. We have revised the first sentence.

ln the Discussion, the authors cite the research on circuitry that is relevant for emergence from general anesthesia. Conspicuously missing from this section of the paper is the large body of work by Solt and colleagues which has demonstrated that dopamine agonists (such as methylphenidate), electrical stimulation of the ventral tegmental area and optogenetic stimulation of the D1 neurons in the ventral tegmental area can hasten emergence from general anesthesia. Also omitted is the work of Kelzand colleagues and a discussion of neural inertia.

Thanks for the suggestions. We have added these citations as requested.

As regards the weaknesses of p-values for reporting the results of scientific studies, l offer the following reference to the authors. Ronald L. Wasserstein & Nicole A.Lazar (2016)The ASA Statement on p-Values: Context, Process, and Purpose, The American Statistician,70:2,129- 133, DOl:10.1080/00031305.2016.1154108

Thanks for the suggestions. We have revised the manuscript as requested.

The methods for the CRF antibody are unclear. It was previously suggested that the antibody be validated (for example, show an absence of immunostaining with CRF knockdown) because the concentration of antiserum (1:800) is quite high, suggesting either the antibody is not potent or (more concerning) not specific. The methods also indicated that colchicine was infused ICV prior to perfusion for staining of cFos and CRF, but no surgical methods are described that would enable ICV infusion, and it is not clear why colchicine was used. Please clarify.

The anti-CRF antibody is validated by other studies11,12. F For CRF immunostaining, animals' brains were pre-treated with intraventricular injections of colchicine (20 μg in 500 nL saline) 24 hours before perfusion to inhibit fast axonal transport13,14. Additional details regarding these methods have been included in the Method section of the revised manuscript.

Editor's note:

Full statistical reporting including exact p-values alongside summary statistics (test statistic and df) and 95% confidence intervals is lacking.

Thanks for the suggestions. We have added full statistical reporting in the revised manuscript as requested.

Reference

(1) Marana, E. et al. Desflurane versus sevoflurane: a comparison on stress response. Minerva Anestesiol 79, 7-14 (2013).

(2) Yang, L., Chen, Z. & Xiang, D. Effects of intravenous anesthesia with sevoflurane combined with propofol on intraoperative hemodynamics, postoperative stress disorder and cognitive function in elderly patients undergoing laparoscopic surgery. Pak J Med Sci 38, 1938-1944, doi:10.12669/pjms.38.7.5763 (2022).

(3) Driscoll, J. N. et al. Comparing incidence of emergence delirium between sevoflurane and desflurane in children following routine otolaryngology procedures. Minerva Anestesiol 83, 383-391, doi:10.23736/s0375-9393.16.11362-8 (2017).

(4) Galinkin, J. L. et al. Use of intranasal fentanyl in children undergoing myringotomy and tube placement during halothane and sevoflurane anesthesia. Anesthesiology 93, 1378-1383, doi:10.1097/00000542-200012000-00006 (2000).

(5) Greenspun, J. C., Hannallah, R. S., Welborn, L. G. & Norden, J. M. Comparison of sevoflurane and halothane anesthesia in children undergoing outpatient ear, nose, and throat surgery. J Clin Anesth 7, 398-402, doi:10.1016/0952-8180(95)00071-o (1995).

(6) Messieha, Z. Prevention of sevoflurane delirium and agitation with propofol. Anesth Prog 60, 67-71, doi:10.2344/0003-3006-60.3.67 (2013).

(7) Shi, M. et al. Dexmedetomidine for the prevention of emergence delirium and postoperative behavioral changes in pediatric patients with sevoflurane anesthesia: a double-blind, randomized trial. Drug Des Devel Ther 13, 897-905, doi:10.2147/dddt.S196075 (2019).

(8) Veyckemans, F. Excitation and delirium during sevoflurane anesthesia in pediatric patients. Minerva Anestesiol 68, 402-405 (2002).

(9) Xu, Y., Gao, G., Sun, X., Liu, Q. & Li, C. ATPase Inhibitory Factor 1 Is Critical for Regulating Sevoflurane-Induced Microglial Inflammatory Responses and Caspase-3 Activation. Front Cell Neurosci 15, 770666, doi:10.3389/fncel.2021.770666 (2021).

(10) Friedman, E. B. et al. A conserved behavioral state barrier impedes transitions between anesthetic-induced unconsciousness and wakefulness: evidence for neural inertia. PLoS One 5, e11903, doi:10.1371/journal.pone.0011903 (2010).

(11) Giardino, W. J. et al. Parallel circuits from the bed nuclei of stria terminalis to the lateral hypothalamus drive opposing emotional states. Nat Neurosci 21, 1084-1095, doi:10.1038/s41593-018-0198-x (2018).

(12) Yeo, S. H., Kyle, V., Blouet, C., Jones, S. & Colledge, W. H. Mapping neuronal inputs to Kiss1 neurons in the arcuate nucleus of the mouse. PLoS One 14, e0213927, doi:10.1371/journal.pone.0213927 (2019).

(13) de Goeij, D. C. et al. Repeated stress-induced activation of corticotropin-releasing factor neurons enhances vasopressin stores and colocalization with corticotropin-releasing factor in the median eminence of rats. Neuroendocrinology 53, 150-159, doi:10.1159/000125712 (1991).

(14) Yuan, Y. et al. Reward Inhibits Paraventricular CRH Neurons to Relieve Stress. Curr Biol 29, 1243-1251.e1244, doi:10.1016/j.cub.2019.02.048 (2019).

A note to sailors journeying across the sea, and a central message/theme for the reader to interpret Potentially a message to communicate a lesson, appreciate the time you spend alive and in your prime/youth because there will only be one opportunity to experience mortality/life

vibration is an interesting word to describe a passionate sensation

BBB rhyme scheme

Quantification theory and logic of relatives

Para ampliar mi punto de vista recomiendo ver los siguientes documentales: 1. Utopia o distopia: https://youtu.be/34Kz-PP_X7c?si=oQeF3HH6_Rv6tTqi 2. El cambio disruptivo. https://youtu.be/34Kz-PP_X7c?si=OU9zvxGwD-2OH2kM 3. El limite ético de las IA: https://youtu.be/sHVwwriaT6k?si=ZOI9PgVqv52u67Yf

Hay dos elementos:

• La empresa es estadounidense, pero el data set es multilingüe. Por tanto, el modelo vectorial de lenguaje puede expresar información en distintos lenguajes, expresando, de todos modos los sesgos en el conjunto de datos que lo alimentan.
• Los modelos de lenguaje capturan relaciones entre palabras que se escapan a la pronunciación o l atraducción literal particular y reflejan relaciones probabilísitcas, por tanto una relación probabilística en un idioma (rey y reina) se puede reflejar en otra (king y queen).

Sobre el examen de abogados, la pregunta es sí esos exámenes miden cosas que van más allá de la manipulación del texto. La idea de que el examen prueba más a la prueba que a la persona que lo presenta y como esta también refleja sesgos de los evaluadores (por ejemplo frete a qué es ser inteligente, para el caso del IQ o qué es ser un abogado, en caso de la barra de abogados).

example of a metaphor

He compares the girl to a new found land, saying that she is the safest when only one man, him, is with her.

Creo que es importante mantener una postura esperanzada pero crítica, no sólo frente a las tecnologías venideras, sino a las que tenemos "naturalizadas" ya. De este modo podemos saber cuándo es mejor la bici o el metro (subterraneo!) que el carro particular o no revisar notificaciones de redes sociales al despertar o saber cuándo es mejor escribir uno mismo a que lo haga una IA.

Acceso.

che significa?

dipende :D Ci possono essere: costi fissi, variabili, solo all'inizio, su ogni rata o meno ...

O UR &c

Dost disney look just like 'my website?' Owe Janet for "apparently making the idiotic deal that destroyed facebook's connection to imgur" which makes it look like my website wasn't kept up as well as I wanted it to be.

I used to have a script that would back up all the imgur.com pictures and store them locally also; after using imgur for the emails because "when you send emails from the same domain over and over again" your throughput decreases.

In any case i stop[ped using that script around when i stopped using Vultr/London where all of the primary development of "the website code" that modified MDBOOK was hand written by "this hacker."

MIT, CALTECH, ANDREW CARNEGIE MELON:

Please find me a psuedo-human that can survive walking through the Langoliers; which might mean you have to be "a princess of something" like Brooke and Kitty and ...

Well I still wanna Fuck Bianca Rose so ... "her mom can call me an asshole as much as she wants" she is one shiny pretty looking thing.

Also i watched her grow up; kinda feel like "i spent some time raising her" and could tell just by the way she skipped up to me and said "hello Adam" that she was .. without a doubt, one of God's favourite little princesses.

In this first stanza of the poem, we see a recurring theme of seasons appear. However, Eliot’s depictions of the seasons divagate from traditional representations of them since the very first line: “April is the cruellest month, breeding / Lilacs out of the dead land” (53). April, the epitome of the expected vitality of spring (a season of rebirth,) is described here with the infertility of the waste land. By personifying April as “cruel”, Eliot plays with an unconventional symbolism of seasons: spring brings discomfort and pain to the land because it forces life up on a spiritually barren world.

When I then moved on to consider the winter and summer imageries in the following lines, I saw a similar use of seasons as a means to reflect inner turmoil as in the Countess Marie Larisch’s book, “My Past.” In the ending paragraphs, the narrator awakens to the earlier dramatic and ominous events of the story with a sense of dread, but commented on the tendency for the anxiety to dissipate in the summertime than in the wintertime (6). In this story, winter is linked to an inescapable sadness, an inability to banish one’s worries. On the other hand, Eliot characterized winter as a season of numbness, covering the earth in “forgetful snow” (53). Winter is a time of stasis and preservation, where pain is deferred until spring disrupts this quietude by forcing life to return. Hence, while both the story and The Waste Land denote winter as a period of emotional or spiritual stasis, it is for different reasons.

Summer, meanwhile, seems disorienting to Eliot—”Summer surprised us”—almost as if warmth and life are unexpected and perhaps even unwanted. Yet, at the end of the stanza, the phrase “go south” finds resonance in the penultimate paragraph of the book: “‘O for the sunshine and warmth of the South’" (6). Summer and the desire to go south symbolize an attempt to escape the present states, but in both cases, this escape is complicated. In the story, the narrator realizes that the idealized south, the Riviera, is “perfectly impossible.” Similarly, in The Waste Land, the speaker’s actions of reading all night and going south in winter feel more like distractions than real solutions.

DOI: 10.1016/j.jbc.2024.107779

Resource: (Abcam Cat# ab81887, RRID:AB_1658411)

Curator: @scibot

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What is this?

DOI: 10.1096/fj.201901751R

Resource: RRID:BDSC_34363

Curator: @scibot

SciCrunch record: RRID:BDSC_34363

What is this?

This right here shows that at home she probably doesn't get the same love or attention from her parents. And maybe she doesn't have food at home, so the food the nurses brought and them changing her sheets even though they looked clean might've been a shock to her.

AIDS: disease, HIV can kill cells in the immune system so it weakens the body and it cannot fight serious infections. The body becomes tripartite: the virus (active, antagonist), the immune system (passive, the victim) and the person who caught it (both the victim and the perpetrator, passive and active)

knew love but did not know himself

Ode is spoken/delivered to a person

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1:

Comment #1: Insufficient Network Analysis for Explainability: The paper does not sufficiently delve into network analysis to determine whether the model's predictions are based on accurately identifying and matching the 18 items of the ROCF or if they rely on global, item-irrelevant features. This gap in analysis limits our understanding of the model's decision-making process and its clinical relevance.

Response #1: Thank you for your comment. We acknowledge the importance of understanding the decision-making process of AI models is crucial for their acceptance and utility in clinical settings. However, we believe that our current approach, which focuses on providing individual scores for each of the 18 items of the Rey-Osterrieth Complex Figure (ROCF), inherently offers a higher level of explainability and practical utility for clinicians than a network analysis could. Our multi-head convolutional neural network is designed with a dedicated output head for each of the 18 items in the ROCF, and thus provides separate scores for each of the 18 items in the ROCF. This architecture helps that the model focuses on individual elements rather than relying on global, item-irrelevant features.

This item-specific approach directly aligns with the traditional clinical assessment method, thereby making the results more interpretable and actionable for clinicians. The individual scores for each item provide detailed insights into a patient's performance. Clinicians can use these scores to identify specific areas of strength and weakness in a patient's visuospatial memory and drawing abilities.

Furthermore, we evaluated the model's performance on each of the 18 items separately, providing detailed metrics that show consistent accuracy across all items. This item-level performance analysis offers clear evidence that the model is not relying on irrelevant global features but is indeed making decisions based on the specific characteristics of each item. We believe that our approach provides a level of explainability that is directly useful and relevant to clinical practitioners.

Comment #2: Generative Model Consideration: The critique suggests exploring generative models to model the joint distribution of images and scores, which could offer deeper insights into the relationship between scores and specific visual-spatial disabilities. The absence of this consideration in the study is seen as a missed opportunity to enhance the model's explainability and clinical utility.

Response #2: Thank you for your thoughtful comment and the suggestion to explore generative models. We appreciate the potential benefits that generative models to model the joint distribution of images and scores. However, we chose not to pursue this approach in our study for several reasons: First, our primary goal was to develop a model that provides accurate and interpretable scores for each of the 18 individual items in the ROCF figure. Second, generative models, while powerful, would add a layer of complexity that might diminish the clarity and immediate clinical applicability of our results. Generative models, (particularly deep learning-based) can be challenging to interpret in terms of how they make decisions or why they produce specific outputs. This lack can be a concern in critical applications involving neurological and psychiatric disorders. Clinicians require tools that provide clear insights without the need for additional layers of analysis. Our current model provides detailed, item-specific scores that clinicians can directly use to assess visuospatial memory and drawing abilities. Initially, we explored using generative models (i.e. GANs) for data augmentation to address the scarcity of low-score images compared to high-score images. Moreover, for the low-score images, the same score can be achieved by numerous combinations of figure elements. However, due to our extensive available dataset, we did not observe any substantial performance improvements in our model. Nevertheless, future studies could explore generative models, such as Variational Autoencoders (VAEs) or Bayesian Networks, and test them on the data from the current prospective study to compare their performance with our results.

In the revised manuscript, we have included additional sentences discussing the potential use of generative models and their implications for future research.

“The data augmentation did not include generative models. Initially, we explored using generative models, specifically GANs, for data augmentation to address the scarcity of low-score images compared to high-score images. However, due to the extensive available dataset, we did not observe any substantial performance improvements in our model. Nevertheless, Future studies could explore generative models, such as Variational Autoencoders (VAEs) or Bayesian Networks, which can then be tested on the data from the current prospective study and compared with our results.”

Comment #3: Lack of Detailed Model Performance Analysis Across Subject Conditions: The study does not provide a detailed analysis of the model's performance across different ages, health conditions, etc. This omission raises questions about the model's applicability to diverse patient populations and whether separate models are needed for different subject types.

Response #3: Thank you for your this important comment. Although the initial version of our manuscript already provided detailed “item-specific” and “across total scores” performance metrics, we recognize the importance of including detailed analyses across different patient demographics to enhance the robustness and applicability of our findings. In response to your comment, we have conducted additional analyses that provide a comprehensive evaluation of model performance across various patient demographics, such as age groups, gender, and different neurological and psychiatric conditions. This additional analysis demonstrates the generalizability and reliability of our model across diverse populations. We have included these analyses in the revised manuscript.

“In addition, we have conducted a comprehensive model performance analysis to evaluate our model's performance across different ROCF conditions (copy and recall), demographics (age, gender), and clinical statuses (healthy individuals and patients) (Figure 4A). These results have been confirmed in the prospective validation study (Supplementary Figure S6). Furthermore, we included an additional analysis focusing on specific diagnoses to assess the model's performance in diverse patient populations (Figure 4B). Our findings demonstrate that the model maintains high accuracy and generalizes well across various demographics and clinical conditions.”

Comment #4: Data Augmentation: While the data augmentation procedure is noted as clever, it does not fully encompass all affine transformations, potentially limiting the model's robustness.

Response #4: We appreciate your feedback on our data augmentation strategy. We acknowledge that while our current approach significantly improves robustness against certain semantic transformations, it may not fully cover all possible affine transformations.

Here, we provide further clarification and justification for our chosen methods and their impact on the model's performance: In our study, we implemented a data augmentation pipeline to enhance the robustness of our model against common and realisitc geometric and semantic-preserving transformations. This pipeline included rotations, perspective changes, and Gaussian blur, which we found to be particularly effective in improving the model's resilience to variations in input data. These transformations are particularly relevant for the present application since users in real-life are likely to take pictures of drawings that might be slightly rotated or with a slightly tilted perspective. With these intuitions in mind, we randomly transformed drawings during training. Each transformation was a combination of Gaussian blur, a random perspective change, and a rotation with angles chosen randomly between -10° and 10°. These transformations are representative of realistic scenarios where images might be slightly tilted or photographed from different angles. We intentionally did not explicitly address all affine transformations, such as shearing or more complex geometric transformations because these transformations could alter the score of individual items of the ROCF and would be disruptive to the model.

As noted in our manuscript and demonstrated in supplementary Figure S1, the data augmentation pipeline significantly improved the model's robustness against rotations and changes in perspective. Importantly, our tablet-based scoring application can further ensure that the photos taken do not exhibit excessive semantic transformations. By leveraging the gyroscope built into the tablet, the application can help users align the images properly, minimizing issues such as excessive rotation or skew. This built-in functionality helps maintain the quality and consistency of the images, reducing the likelihood of significant semantic transformations that could affect model performance.

Comment #5: Additionally, the rationale for using median crowdsourced scores as ground truth, despite evidence of potential bias compared to clinician scores, is not adequately justified.

Response #5: Thank you for this valuable comment. Clarifying the rationale behind using the median score of crowdsourcing as the ground truth is indeed important. To reach high accuracy in predicting individual sample scores of the ROCFs, it is imperative that the scores of the training set are based on a systematic scheme with as little human bias as possible influencing the score. However, our analysis (see results section) and previous work (Canham et al., 2000) suggested that the scoring conducted by clinicians may not be consistent, because the clinicians may be unwittingly influenced by the interaction with the patient/participant or by the clinicians factor (e.g. motivation and fatigue). For this reason and the incomplete availability of clinician scores for all figures (i.e. for 19% of the 20’225 figures), we did not use the clinicians scores as ground truth scores. Instead, we have trained a large pool (5000 workers) of human internet workers (crowdsourcing) to score ROCFs drawings guided by our self-developed interactive web application. Each element of the figure was scored by several human workers (13 workers on average per figure). We have obtained the ground truth for each drawing by computing the median for each item in the figure, and then summed up the medians to get the total score for the drawing in question. To further ensure high-quality data annotation, we identified and excluded crowdsourcing participants that have a high level of disagreement (>20% disagreement) with this rating from trained clinicians, who carefully scored manually a subset of the data in the same interactive web application.

We chose the median score for several reasons: (1) the median score is less influenced by outliers compared to the mean. Given the variability of scoring between different clinicians and human workers (see human MSE and clinician MSE), using the median ensures that the ground truth is not skewed by extreme values, leading to more stable and reliable scores. (2) Crowdsource data do not always follow a normal distribution. In cases where the distribution is skewed or not symmetric, the median can be a more representative measure of the center. (3) The original scoring system involves ordinal scales (0,0.5,1,2). For ordinal scales, the median is often more appropriate than the mean. Finally, by aggregating multiple scores from a large pool of crowdsourced raters, the median provides a consensus that reflects the most common assessment. This approach mitigates the variability introduced by individual rater biases and ensures a more consistent ground truth. In clinical settings, the consensus of multiple expert opinions often serves as the benchmark for assessments. The use of median scores mirrors this practice, providing a ground truth that is representative of collective human judgment.

Canham, R. O., S. L. Smith, and A. M. Tyrrell. 2000. “Automated Scoring of a Neuropsychological Test:

The Rey Osterrieth Complex Figure.” Proceedings of the 26th Euromicro Conference. EUROMICRO 2000. Informatics: Inventing the Future. https://doi.org/10.1109/eurmic.2000.874519.

Reviewer #2:

Comment #1: There is no detail on how the final scoring app can be accessed and whether it is medical device-regulated.

Response #1: We appreciate the opportunity to provide more information about the current status and plans for our scoring application. At this stage, the final scoring app is not publicly accessible as it is currently undergoing rigorous beta testing with a select group of clinicians in real-world settings. The feedback from these clinicians is instrumental in refining the app’s features, interface, and overall functionality to improve its usability and user experience. This ensures that the app meets the high standards required for clinical tools. Following the successful completion of the beta testing phase, we aim to seek FDA approval for the scoring app. Achieving this regulatory milestone will guarantee that the app meets the stringent requirements for medical devices, providing an additional layer of confidence in its safety and efficacy for clinical use. Once FDA approval is obtained, we plan to make the app publicly accessible to clinicians and healthcare institutions worldwide. Detailed instructions on how to access and use the app will be provided at that time on our website (https://www.psychology.uzh.ch/en/areas/nec/plafor/research/rfp.html).

Comment #2: No discussion on the difference in sample sizes between the pre-registration of the prospective study and the results (e.g., aimed for 500 neurological patients but reported data from 288). Demographics for the assessment of the representation of healthy and non-healthy participants were not present.

Response #2: Thank you for your comment. We believe there might have been a misunderstanding regarding our preregistration details. In the preregistration, we planned to prospectively acquire ROCF drawings from 1000 healthy subjects. Each subject should have drawn two ROCF drawings (copy and memory condition). Consequently, 2000 samples should have been collected. In addition, in our pre-registration plan, we aimed to collect 500 drawings from patients (i.e. 250 patients), not 500 patients as the reviewer suggested (https://osf.io/82796). Thus in total, the goal was to obtain 2500 ROCF figures. The final prospective data set, which contained 2498 ROCF images from 961 healthy adults and 288 patients very closely matches the sample size, we aimed for in the the pre-registration. We do not see a necessity to discuss this negligible discrepancy in the main manuscript. The prospective data set remains substantial and sufficient to test our model on the independent prospective data set. Importantly, we want to highlight that the test set in the retrospective data set (4045 figures) was also never seen by the model. Both the retrospective and prospective data sets demonstrate substantial global diversity as the data has been collected in 90 different countries. Please note, that Supplementary Figures S2 & S3 provide detailed demographics of the participants in the prospectively collected data, present their performance in the copy and (immediate) recall condition across the lifespan, and the worldwide distribution of the origin of the data.

Comment #3: Supplementary Figure S1 & S4 is poor quality, please increase resolution.

Response #3: We apologize for the poor quality of Supplementary Figures S1 and S4 in the initial submission. In the revised version of our submission, we have increased the resolution of both Supplementary Figure S1 and Supplementary Figure S4 to ensure that all details are clearly visible and the figures are of high quality.

Comment #4: Regarding medical device regulation; if the app is to be used in clinical practice (as it generates a score and classification of performance), I believe such regulation is necessary - but there are ways around it. This should be detailed.

Response #4: We agree that regulation is essential for any application intended for use in clinical practice, particularly one that generates scores and classifications of performance. As discussed in response #1, the final scoring application is currently undergoing intensive beta testing in real-world settings with a limited group of clinicians and is therefore not publicly accessible at this time. We are fully committed to obtaining the necessary regulatory approvals before the app is made publicly accessible for clinical use. Once the beta testing phase is complete and the app has been refined based on clinician feedback, we will prepare and submit a comprehensive regulatory dossier. This submission will include all necessary data on the app's development, testing, validation, and clinical utility. We are adhering to relevant regulatory standards and guidelines, such as ISO 13485 for medical devices and the FDA's guidance on software as a medical device (SaMD).

Comment #7: Need to clarify that work was already done and pre-printed in 2022 for the main part of this study, and that this paper contributes to an additional prospective study.

Response #7: We would like to clarify that the pre-print the reviewer is referring to is indeed the current paper submitted to ELife. The submitted paper includes both the work that was pre-printed in 2022 and the additional prospective study, as you correctly identified.

Reviewer #3:

Comment #1: The considerable effort and cost to make the model only for an existing neuropsychological test.

Response #1: We acknowledge that significant effort and resources were dedicated to developing our model for the Rey-Osterrieth Complex Figure (ROCF) test. Below, we provide a detailed rationale for this investment and the broader implications of our work. The ROCF test is one of the most widely used neuropsychological assessments worldwide, providing critical insights into visuospatial memory and executive function. While the initial effort and cost are substantial, the long-term benefits of an automated, reliable, objective, fast and widely applicable neuropsychological assessment tool justify the investment. The scoring application will significantly reduce the time for scoring the test and thus provide more efficient use of clinical resources, and the potential for broader applications makes this a worthwhile endeavor. The methods and infrastructure developed for this model can be adapted and scaled to other neuropsychological tests and assessments (e.g. Taylor Figure).

Comment #2: I was truly impressed by the authors' establishment of a system that organizes the methods and fields of diverse specialties in such a remarkable way. I know the primary purpose of ROCFT. However, beyond the score, neuropsychologically, these are observed by specialists while ROCFT and that is attractive of the test: the turn of each stroke (e.g., from right to left, from the main structure to the margin or small structure), the process to total completeness as a figure, e.g., confidential speed and concentration, the boldness of strokes, unnatural fragmentation of strokes, the not deviated place in a paper, turning of the figure itself (before the scanning level), the total size, the level compared with the age, education, and experiences of the patient. Those are reflected by the disease, visuospatial intelligence, executive function, and ability to concentrate. Scores are crucial, but by observing the drawing process, we can obtain diverse facts or parts of symptoms that imply the complications of human behavior.

Response #2: Thank you for your insightful comments and observations regarding our system for organizing diverse specialties within the ROCFT methodology. We agree that beyond the numerical scores, the detailed observation of the drawing process provides invaluable neuropsychological insights. How strokes are executed, from their direction and placement to the overall completion process, offers a nuanced understanding of factors like spatial orientation, concentration, and executive function. In fact, we are working on a ROCF pen tracking application, which enables the patient to draw the ROCF with a digital pen on a tablet. The tablet can 1) assess the sequence order of drawing the items and the number of strokes, 2) record the exact coordinate of each drawn pixel at each time point of the assessment, 3) measure the duration for each pen stroke as well as total drawing time, and 4) assess the pen stroke pressure. Through this, we aim to extract additional information on processing speed, concentration, and other cognitive domains. However, this development is outside the scope of the current manuscript.

Dealing with challenges - slide name needs changing in menu can we not use the pink text and perhaps change the slide layout to one without the big left hand panel? The arrows used are from our old brand can you update o somethat use brand ywllow? https://www.staffnet.manchester.ac.uk/brand/visual-identity/colour/

Reviewer #1 (Public review):

The manuscript by Griesius et al. addresses the dendritic integration of synaptic input in cortical GABAergic interneurons (INs). Dendritic properties, passive and active, of principal cells have been extensively characterized, but much less is known about the dendrites of INs. The limited information is particularly relevant in view of the high morphological and physiological diversity of IN types. The few studies that investigated IN dendrites focused on parvalbumin-expressing INs. In fact, in a previous study, the authors examined dendritic properties of PV INs, and found supralinear dendritic integration in basal, but not in apical dendrites (Cornford et al., 2019 eLife).

In the present study, complementary to the prior work, the authors investigate whether dendrite-targeting IN types, NDNF-expressing neurogliaform cells, and somatostatin(SOM)-expressing O-LM neurons, display similar active integrative properties by combining clustered glutamate-uncaging and pharmacological manipulations with electrophysiological recording and calcium imaging from genetically identified IN types in mouse acute hippocampal slices.

The main findings are that NDNF IN dendrites show strong supralinear summation of spatially- and temporally-clustered EPSPs, which is changed into sublinear behavior by bath application of NMDA receptor antagonists, but not by Na+-channel blockers. L-type calcium channel blockers abolished the supralinear behavior associated calcium transients but had no or only weak effect on EPSP summation. SOM IN dendrites showed similar, albeit weaker NMDA-dependent supralinear summation, but no supralinear calcium transients were detected in these INs. In summary, the study demonstrates that different IN types are endowed with active dendritic integrative mechanisms, but show qualitative and quantitative divergence in these mechanisms.

While the research is conceptionally not novel, it constitutes an important incremental gain in our understanding of the functional diversity of GABAergic INs. In view of the central roles of IN types in network dynamics and information processing in the cortex, results and conclusions are of interest to the broader neuroscience community.

The experiments are well designed, and closely follow the approach from the previous publication in parts, enabling direct comparison of the results obtained from the different IN types. The data is convincing and the conclusions are well-supported, and the manuscript is very well-written.

I see only a few open questions and some inconsistencies in the presentation of the data in the figures (see details below).

more bible quotes. mary loves her bible quotes

God is her savior of all tribulations.

Maybe it is too much to say, Mary.

chosen syndrome continues

odd

didactic

.is.a - on line in browser node development environment

Still managing to have celebrations despite the year being difficult

Dans La Glyptique susienne, il s'agit des empreintes de sceaux-cylindres :

• N^o 1001 — Téhéran, musée Iran Bastan, inv. MT 112
• N^o 1002 — Louvre, inv. Sb 1501
• N^o 1003 — Louvre, inv. Sb 5945
• N^o 1004 — Louvre, inv. Sb 1503
• N^o 1005 — Louvre, inv. Sb 6358
• N^o 1006 — Louvre, inv. Sb 2230
• N^o 1007 — Louvre, inv. Sb 6406
• N^o 1008 — MT 117
• N^o 1009 — Louvre, inv. Sb 1504
• N^o 1010 — Louvre, inv. Sb 1505
• N^o 1011 — Louvre, inv. Sb 6412
• N^o 1012 — Louvre, inv. Sb 4842
• N^o 1013 — Louvre, inv. Sb 6385, 6386, 6387
• N^o 1014 — Cylindre perdu
• N^o 1015 — AS, inv. 4222
• N^o 1016 — Louvre, inv. Sb 6404, Sb 6405
• N^o 1017 — Louvre, inv. Sb 6400

Il s'agit des empreintes de sceaux-cylindres :

• N^o 1001 — Téhéran, musée Iran Bastan, inv. MT 112
• N^o 1002 — Louvre, inv. Sb 1501
• N^o 1003 — Louvre, inv. Sb 5945
• N^o 1004 — Louvre, inv. Sb 1503
• N^o 1005 — Louvre, inv. Sb 6358
• N^o 1006 — Louvre, inv. Sb 2230
• N^o 1007 — Louvre, inv. Sb 6406
• N^o 1008 — MT 117
• N^o 1009 — Louvre, inv. Sb 1504
• N^o 1010 — Louvre, inv. Sb 1505
• N^o 1011 — Louvre, inv. Sb 6412
• N^o 1012 — Louvre, inv. Sb 4842
• N^o 1013 — Louvre, inv. Sb 6385, 6386, 6387
• N^o 1014 — Cylindre perdu
• N^o 1015 — AS, inv. 4222
• N^o 1016 — Louvre, inv. Sb 6404, Sb 6405
• N^o 1017 — Louvre, inv. Sb 6400

it seems like it's been rough...

In a Multiple Instruction stream, Multiple Data stream (MIMD) system, each processor operates with its own cache but shares main memory, storage, and I/O devices. This setup allows for more complex and flexible data sharing and processing among processors, which is important for understanding how MIMD systems manage and coordinate tasks.

DOI: 10.1016/j.isci.2024.110853

Resource: (Abcam Cat# ab154823, RRID:AB_2716802)

Curator: @scibot

SciCrunch record: RRID:AB_2716802

What is this?

Pero si subjetividad y objetividad se derivan de polaridad, y ésta se refiere exclusivamente a el "sentiment strength", nada se puede decir sobre la descriptividad de la información incluida o la neutralidad de la fuente.

El uso de esta herramienta no parece relevante para el tema el artículo. Tratándose de un artículo en el que se mide polaridad, subjetividad y objetividad como aspectos del "sentimiento", habría que destacar las librerías que se usaron para tal fin, en todo caso (que no queda claro si fue TextBlob o NLTK).

website includes lots of both textual and visual representation to better help navigate the links and website

good use of lots of text labels that clearly identifies tabs and click through buttons to navigate the website.

O vapor de uma mistura azeotrópica sempre sai na mesma proporção de sua composição.

Exemplo: álcool etílico (96% álcool, 4% água).

“O Machine! O Machine!” and raised the volume to her lips. Thrice she kissed it, thrice inclined her head, thrice she felt the delirium of acquiescence

Pode ser uma boa alternativa

she has not done anything the entire time except travel. Obviously they do not know was Sabbath is

This is called a good rapport!

En un taller con unos Ing Forestales en Hidalgo mencionaron que la ley agraria es contradictoria, pues aunque existe la posibilidad legal de parcelar o vender, esto no se acepta en todos los tribunales agrarios. Hay muchos juicios, muchos antecedentes, pero no hay caminos claros para realizar este proceso. Dependiendo del estado de la republica en el que se haga y del juez que revise, el resultado varía.

ejercicio: que expliquen cómo eligieron su nombre de correo electrónico y por qué.

¿Hay entonces una manera "correcta" de decir las cosas, de enunciaras? Nosotros no vamo a ver lo correcto o lo incorrecto, sino que vamos a desmontar esos mecanismos para ver formas de pensamiento, ideología, realciones simbólicamente mediadas.

This sentence highlights the author’s intent to elevate the perception of Indigenous peoples, countering the view that they are merely savage. The author aims to show that Indigenous communities possess complex social structures, including political and civil aspects, by noting their organized village life, agricultural practices, and systems of social interaction. This argument serves to acknowledge their humanity and societal sophistication, challenging stereotypes about them as uncivilized.

DOI: 10.1016/j.cell.2024.08.013

Resource: AB_3493878

Curator: @scibot

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What is this?

DOI: 10.1016/j.cmet.2024.08.005

Resource: (Abcam Cat# ab183685, RRID:AB_2686917)

Curator: @scibot

SciCrunch record: RRID:AB_2686917

What is this?

DOI: 10.1016/j.xcrm.2024.101732

Resource: (BioLegend Cat# 102504, RRID:AB_312911)

Curator: @scibot

SciCrunch record: RRID:AB_312911

What is this?

DOI: 10.1016/j.xcrm.2024.101732

Resource: (Proteintech Cat# 55190-1-AP, RRID:AB_11182818)

Curator: @scibot

SciCrunch record: RRID:AB_11182818

What is this?

DOI: 10.1016/j.cell.2024.08.009

Resource: (IMSR Cat# JAX_009253,RRID:IMSR_JAX:009253)

Curator: @vtello

SciCrunch record: RRID:IMSR_JAX:009253

What is this?

DOI: 10.1016/j.cmet.2024.08.006

Resource: RRID:IMSR_JAX:000642

Curator: @vtello

SciCrunch record: RRID:IMSR_JAX:000642

What is this?

6lowpan

desire of clarity and truth and knowledge wich is why it says "eye of golden day o marching light" this is beacuse of what the sun symbols and this is what chorus wants which is to see the reality

Antigone is shocked of the members of the family, can't believe that living with the closest people can be ending up of his death.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

…the degree to which the predictions can vary according to environmental composition remains difficult to quantify, and the work does not address the sensitivity of the modeling predictions beyond a simulated medium containing 33 root exudates. I find this especially important given that relatively few (84 of 243) species were predicted to grow even after cross-feeding, suggesting that a richer medium could lead to different interaction network structures. While the authors do state the importance of environmental composition and have carefully designed an in silico medium, I believe that simulating a broader set of resource pools would add necessary insight into both the predictive power of the models themselves and trophic interactions in the rhizosphere more generally.

The original analyses were indeed focused on a single well-defined environment supporting the growth of only a subset of the species. We have added a paragraph to the discussion section dealing with the potential limitations of this approach. 

On line 289 we write:

"Overall, the successive iterations connected 84 out of 243 native members of the apple rhizosphere GSMM community via trophic exchanges. The inability of the remaining bacteria to grow, despite being part of the native root microbiome, possibly reflects the selectiveness of the root environment, which fully supports the nutritional demands of only part of the soil species, whereas specific compounds that might be essential to other species are less abundant1. It is important to note that the specific exudate profile used here represent a snapshot of the root metabolome as root secretion-profiles are highly dynamic, reflecting both environmental and plant developmental conditions. A possible complementary explanation to the observed selective growth might be the partiality of our simulation platform, which examined only plant-bacteria and bacteria-bacteria interactions while ignoring other critical components of the rhizosphere system such as fungi, archaea, protists and mesofauna, as well as less abundant bacterial species, components all known to metabolically interact2. Finally, the MAG collection, while relatively substantial, represents only part of the microbial community. Accordingly, the iterative growth simulations represent a subset of the overall hierarchical-trophic exchanges in the root environment, necessarily reflecting the partiality of the dataset."

In addition, we have tried to better explain the advantages of a limited/defined medium to such an analysis. On Line 231 we add:

"By avoiding the inclusion of non-exudate organic metabolites, the true-to-source rhizosphere environment was designed to reveal the hierarchical directionality of the trophic exchanges in soil, as rich media often mask various trophic interactions taking place in native communities3"

More generally, beyond the above justification of our specific medium selection, we agree that simulating a broader set of resource pools would contribute to a more comprehensive understanding of the trophic interactions. Therefore, we conducted the analysis in an additional environment, in which cellulose was used as an input. We were able to follow its well-documented degradation via multiple steps, conducted by different community members, to serve as a benchmark to our suggested framework. 

On line 357 we add:

"To validate the ability of MCSM to capture trophic dependencies and succession, we further tested whether it can trace the well-documented example of cellulose degradation - a multi-step process conducted by several bacterial strains that go through the conversion of cellulose and its oligosaccharide derivatives into ethanol, acetate and glucose, which are all eventually oxidized to CO24. Here, the simulation followed the trophic interactions in an environment provided with cellulose oligosaccharides (4 and 6 glucose units) on the 1st iteration (Supp. Table 3). The formed trophic successions detected along iterations captured the reported multi-step process (Supp.

Fig.7)." 

Finally, we have included additional text regarding the challenge of defining our simulation environment in the Discussion section. 

On line 532 we add:

"In the current study, the root environment was represented by a single pool of resources (metabolites). As genuine root environments are highly dynamic and responsive to stimuli, a single environment can represent, at best, a temporary snapshot of the conditions. Conductance of simulations with several sets of resource pools (e.g., representing temporal variations in exudation profile) can add insights regarding their effect on trophic interactions and community dynamics. In parallel, confirming predictions made in various environments will support an iterative process that will strengthen the predictive power of the framework and improve its accuracy as a tool for generating testable hypotheses. Similarly, complementing the genomicsbased approaches used here with additional layers of 'omics information (mainly transcriptomics & metabolomics) can further constrain the solution space, deflate the number of potential metabolic routes and yield more accurate predictions of GSMMs' performances5."

And we add in Line 520:

"For these reasons, among others, the framework presented here is not intended to be used as a stand-alone tool for determining microbial function. The framework presented is designed to be used as a platform to generate educated hypotheses regarding bacterial function in a specific environment in conjunction with actual carbon substrates available in the particular ecosystem under study. The hypotheses generated provide a starting point for experimental testing required to gain actual, targeted and feasible applicable insights6,7. While recognizing its limitations, this framework is in fact highly versatile and can be used for the characterization of a variety of microbial communities and environments. Given a set of MAGs derived from a specific environment and environmental metabolomics data, this computational framework provides a generic simulation platform for a wide and diverse range of future applications." 

Reviewer #2 (Public review):

There are two main drawback approaches like the one described here, both related only partially to the authors' work yet with great impact in the presented framework. First, the usage of automatic GSMM reconstruction requires great caution. It is indicative of how the semicurated AGORA models are still considered reconstructions and expect the user to parameterize those in a model. In this study, CarveMe was used. CarveMe is a well-known tool with several pros [1]. Yet, several challenges need to be considered when using it [2]. For example, the biomass function used might lead to an overestimation of auxotrophies. Also, as its authors admit in their reply paper, CarveMe does gap fill in a way [3]; models are constructed to ensure no gaps and also secure a minimum growth. However, curation of such a high number of GSMMs is probably not an option. Further, even if FVA is way more useful than FBA for the authors' aim, it does not yet ensure that when a species secretes one compound (let's say metabolite A), the same flux vector, i.e. the same metabolic functioning profile, secretes another compound (metabolite B) at the same time, even if the FVA solution suggests that metabolite B could be secreted in general.

We thank Reviewer #2 for highlighting this key limitation of our analysis. Below and in the 'recommendations to authors' section we address these concerns. 

Concerning the first point raised (models' accuracy) we have now clearly acknowledged in the text the limitations of using an automated GSMM reconstruction tool such as CarveMe. More generally, the framework applied here was built in order to meet the challenges of analyzing highthroughput data while acknowledging the inherent potential of introducing inaccuracies. Pros & cons are now discussed. 

On line 507 we write:

"Moreover, the use of an automatic GSMM reconstruction tool (CarveMe8), though increasingly used for depicting phenotypic landscapes, is typically less accurate than manual curation of metabolic models9. This approach typically neglects specialized functions involving secondary metabolism10 and introduces additional biases such as the overestimation of auxotrophies11,12. Nevertheless, manual curation is practically non-realistic for hundreds of MAGs, an expected outcome considering the volume of nowadays sequencing projects. As the primary motivation of this framework is the development of a tool capable of transforming high-throughput, low-cost genomic information into testable predictions, the use of automatic metabolic network reconstruction tools was favored, despite their inherent limitations, in pursuit of addressing the necessity of pipelines systematically analyzing metagenomics data." 

Regarding using FVA solutions, indeed such solutions return all potential metabolic fluxes in GSMMs (ranges of all fluxes satisfying the objective function, which by default is set to biomass increase) in a given environment. However, as indicated by the reviewer, predicted fluxes do not necessarily co-occur (i.e., when a metabolite is secreted another metabolite is not necessarily secreted too), yet, they provide the full set of potential solutions (unlike the single solution provided by FBA). A possible strategy to reduce inflated predictions provided by FVA and further constrain the solution space (reduce the set of metabolic fluxes) can be the incorporation of additional `omics data layers, as for example was done in the work of Zampieri et al5. Such approach could allow for instance limiting active reactions (blocking fluxes) from the network reconstructions if not coming to play in situ, and therefore impose further constraints and narrow the solution space. We now refer in the text to this limitation and to potential routes to overcome it. 

On line 541 we now write:

Similarly, complementing the genomics-based approaches done here with additional layers of 'omics information (mainly transcriptomics & metabolomics) can further constrain the solution space, deflate the number of potential metabolic routes and yield more accurate predictions of GSMMs' performances5.  

Reviewer #3 (Public review):

When presenting a computational framework, best practices include running it on artificial (synthetic) data where the ground truth is known and therefore the precision and accuracy of the method may be assessed. This is not an optional step, the same way that positive/negative controls in lab experiments are not optional. Without this validation step, the manuscript is severely limited. The authors should ask themselves: what have we done to convince the reader that the framework actually works, at least on our minimal synthetic data? 

Thank you for this suggestion. To validate the ability of MCSM to capture trophic succession, we conducted an additional analysis testing whether it can track the well documented example of cellulose degradation - a multi-step process conducted by several bacterial strains. This example has been included in the manuscript to serve as a case study (i.e. positive control) for metabolic interactions occurring within the bacterial community (Supp. Fig. 7). 

On line 357 we add:

"To validate the ability of MCSM to capture trophic dependencies and succession, we further tested whether it can track the well-documented example of cellulose degradation - a multi-step process conducted by several bacterial strains that go through the conversion of cellulose and its oligosaccharide derivatives into ethanol, acetate and glucose, which are all eventually oxidized to CO24. Here, the simulation followed the trophic interactions in an environment provided with cellulose oligosaccharides (4 and 6 glucose units) on the 1st iteration (Supp. Table 3). The formed trophic successions detected along iterations captured the reported multi-step process (Supp. Fig.

7)."  

"Supplementary Figure 7. Application of MCSM over the process of cellulose decomposition as described by Kato et al4. 5-partite network exhibiting the uptake of cellulose oligomers (4 and 6 units of connected D-glucose) by primary decomposers, through secretion of intermediate compounds and their metabolization by secondary decomposers to CO2. Distribution of phyla of primary and secondary decomposers is denoted by pie charts. Though MAGs were not constructed for the original species as in Kato et al., among the primary consumers, species corresponding to the Acidobacteria (Acidobacteriales)13, Actinobacteria14, Bacteriodetes15, Proteobacteria (Xanthomonadales)16 and Verrucobacteria17 groups are found to be capable of degrading cellulose compounds via enzymatic mechanisms."

More generally, beyond the above addition, the relevance of the framework to the analysis of the data is discussed throughout the analysis (in the original version of the manuscript). We have scrutinized each of our observations in light of current available information and provided a corroborating evidence as well as a few discrepancies for multiple steps in the analysis.  Examples include the following discussions:

On line 312, we discuss the biological relevance of taxonomic classes classified as primary versus secondary degraders

"As in the full GSMM data set (Community bar, Fig. 3C), most of the species which grew in the 1st iteration belonged to the phyla Acidobacteriota, Proteobacteria, and Bacteroidota. This result concurred with findings from the work of Zhalnina et al, which reported that bacteria assigned to these phyla are the primary beneficiaries of root exudates18. Species from three out of the 17 phyla that did not grow in the first iteration - Elusimicrobiota, Chlamydiota, and Fibrobacterota, did grow on the 2nd iteration (Fig. 3C). Members of these phyla are known for their specialized metabolic dependencies. Such is the case for example with members of the Elusimicrobiota phylum, which include mostly uncultured species whose nutritional preferences are likely to be selective19.

At the order level, bacteria classified as Sphingomonadales (class Alphaproteobacteria), a group known to include typical inhabitants of the root environment20, grew in the initial Root environment. In comparison, other root-inhabiting groups including the orders Rhizobiales and Burkholderiales_20, did not grow in the first iteration. _Rhizobiales and Burkholderiales did, however, grow in the second and third iterations, respectively, indicating that in the simulations, the growth of these groups was dependent on exchange metabolites secreted by other community members (Supp. Fig. 4)."

On line 331, we provide support to the classification of specific metabolites as exchange molecules

"Overall, 158 organic compounds were secreted throughout the MCSM simulation (from which 12 compounds overlapped with the original exudate medium). These compounds varied in their distribution and were mapped into 12 biochemical categories (Fig. 3D). Whereas plant secretions are a source of various organic compounds, microbial secretions provide a source of multiple vitamins and co-factors not secreted by the plant. Microbial-secreted compounds included siderophores (staphyloferrin, salmochelin, pyoverdine, and enterochelin), vitamins (pyridoxine, pantothenate, and thiamin), and coenzymes (coenzyme A, flavin adenine dinucleotide, and flavin mononucleotide) – all known to be exchange compounds in microbial communities21,22. In addition, microbial secretions included 11 amino acids (arginine, lysine, threonine, alanine, serine, phenylalanine, tyrosine, leucine, glutamate, isoleucine, and methionine), also known as a common exchange currency in microbial communities23. Some microbial-secreted compounds, such as phenols and alkaloids, were reported to be produced by plants as secondary metabolites24,25. Additional information regarding mean uptake and secretion degrees of compounds classified to biochemical groups is found in Supp. Fig. 5."

On line 432, we provide corroborative support to the classification of exudates as associated with beneficial/non beneficial root communities

"Notably, the S-classified root exudates included compounds reported to support dysbiosis and ARD progression. For example, the S-classified compounds gallic acid and caffeic acid (3,4-dihidroxy-trans-cinnamate) are phenylpropanoids – phenylalanine intermediate phenolic compounds secreted from plant roots following exposure to replant pathogens26. Though secretion of these compounds is considered a defense response, it is hypothesized that high levels of phenolic compounds can have autotoxic effects, potentially exacerbating ARD. Additionally, it was shown that genes associated with the production of caffeic acid were upregulated in ARD-infected apple roots, relative to those grown in γ-irradiated ARD soil27,28, and that root and soil extracts from replant-diseased trees inhibited apple seedling growth and resulted in increased seedling root production of caffeic acid29."

On line 446, we provide a supporting evidence to the classification of secreted compounds as associated with beneficial/non beneficial root communities

"Several secreted compounds classified as healthy exchanges (H) were reported to be potentially associated with beneficial functions. For instance, the compounds L-Sorbose (EX_srb__L_e) and Phenylacetaladehyde (EX_pacald_e), both over-represented in H paths (Fig. 5C), have been shown to inhibit the growth of fungal pathogens associated with replant disease30,31.

Phenylacetaladehyde has also been reported to have nematicidal qualities32."

On line 453 we discuss the correspondence of specific exudate uptakes and compound secretions via specific subnetwork motifs (PM) and their literature/experimental evidence 

"Combining both exudate uptake data and metabolite secretion data, the full H-classified PM path 4-Hydroxybenzoate; GSMM_091; catechol (Fig. 4C; the consumed exudate, the GSMM, and the secreted compound, respectively) provides an exemplary model for how the proposed framework can be used to guide the design of strategies which support specific, advantageous exchanges within the rhizobiome. The root exudate 4-Hydroxybenzoate is metabolized by GSMM_091 (class Verrucomicrobiae, order Pedosphaerales) to catechol. Catechol is a precursor of a number of catecholamines, a group of compounds which was recently shown to increase apple tolerance to ARD symptoms when added to orchard6,33. This analysis (PM; Fig 4C), leads to formulating the testable prediction that 4-Hydroxybenzoate can serve as a selective enhancer of catecholamine synthesizing bacteria associated with reduced ARD symptoms, and therefore serve as a potential source for indigenously produced beneficial compounds."

Moreover, we perceive our analysis as a strategy for integrating high throughput genomic data into testable predictions allowing narrowing the solution space while acknowledging potential inaccuracies that are inherent to the analysis. We have revised the text in order to clearly acknowledge this limitation.

On line 497 we write: 

"The framework we present is currently conceptual."

On line 520 we write: 

"For these reasons, among others, the framework presented here is not intended to be used as a stand-alone tool for determining microbial function. The framework presented is designed to be used as a platform to generate educated hypotheses regarding bacterial function in a specific environment in conjunction with actual carbon substrates available in the particular ecosystem under study. The hypotheses generated provide a start point for experimental testing required to gain actual, targeted and feasibly applicable insights6,7."

On line 532 we add: 

"In the current study, the root environment was represented by a single pool of resources (metabolites). As genuine root environments are highly dynamic and responsive to stimuli, a single environment can represent, at best, a temporary snapshot of the conditions. Conductance of simulations with several sets of resource pools (e.g., representing temporal variations in exudation profile) can add insights regarding their effect on trophic interactions and community dynamics. In parallel, confirming predictions made in various environments will support an iterative process that will strengthen the predictive power of the framework and improve its accuracy as a tool for generating testable hypotheses. Similarly, complementing the genomicsbased approaches used here with additional layers of 'omics information (mainly transcriptomics & metabolomics) can further constrain the solution space, deflate the number of potential metabolic routes and yield more accurate predictions of GSMMs' performances5."

Recommendations for the authors:

Reviewer #1( Recommendations for the authors):

(1) Line 219: "Feasibility" - this term/concept may be difficult to understand for readers unfamiliar with GSMMs. I would recommend either clarifying or rephrasing, perhaps as "simulations confirmed the existence of a feasible solution space for all the 243 models, as well as their capacity to predict growth in the respective environment."

Thanks, done. We have modified this section as suggested (line 221). 

(2) Line 244: How does MCSM fit within/build upon existing frameworks that simulate patterns of niche construction and cross-feeding with constraint-based modeling?

This is now addressed. On line 250 we write:  

"Unlike tools designed for modelling microbial interactions34,35, MCSM bypasses the need for defining a community objective function as the growth of each species is simulated individually. Trophic interactions are then inferred by the extent to which compounds secreted by bacteria could support the growth of other community members."

(3) Figure 4A: While illustrating the general complexity of the predicted trophic interactions, the density of the network makes it very difficult to interpret specific exchanges. Moreover, the naming conventions of the metabolites make it difficult to understand what they represent. I would recommend either restructuring the graph such that the label of each node is legible, or removing the labels altogether.

Thanks, done. Labels were removed and a zoom-in-window to the exchanges highlighted in Figure 4C were added. Caption was revised to indicate that node colors correspond to differential abundance classification of GSMMs in the different plots (H, S, NA are Healthy, Sick, Not-Associated, respectively).

Reviewer #2 (Recommendations for the authors):

CarveMe solves a Mixed Integer Linear Program (MILP) that enforces network connectivity, thus requiring gapless pathways. It's puzzling how to deal with such a great number of GSMMs that is for sure, especially when coming from such an environment as soil and the vast majority of their corresponding MAGs represent most likely novel taxa. One alternative approach for using CarveMe might be to use the rich medium as a medium to gap-fill during the reconstruction. In this case, the gene annotation scores that CarveMe calculates in its initial step, are used to prioritise the reactions selected for gap-filling. This would lead to a new series of challenges but might be a useful comparison with the current GSMMs of the study.

Though indeed CraveMe includes a gap-filling option, here we have purposely avoided the gapfilling option as we aimed to adhere to genomic content of the corresponding genomes and to avoid masking their metabolic dependencies emerging due to their incompleteness. This is noted in the Methods section, which we revised to emphasize the adherence to the genomic content of the models: 

On line 615 we now write:

"All GSMMs were drafted without gap filling in order to adhere to genomic content and to avoid masking metabolic co-dependencies51"

More generally, we now refer to the limitation of automatic reconstruction in the context of the current analysis. On line 507 we write:

"Moreover, the use of an automatic GSMM reconstruction tool (CarveMe8), though increasingly used for depicting phenotypic landscapes, is typically less accurate than manual curation of metabolic models9. This approach typically neglects specialized functions involving secondary metabolism10 and introduces additional biases such as the overestimation of auxotrophies11,12. Nevertheless, manual curation is practically non-realistic for hundreds of MAGs, an expected outcome considering the volume of nowadays sequencing projects. As the primary motivation of this framework is the development of a tool capable of transforming high-throughput, low-cost genomic information into testable predictions, the use of automatic, semi-curated, metabolic network reconstruction tools was favored, despite their inherent limitations, in pursuit of developing pipelines for the systematic analysis of metagenomics data."

Thermodynamically infeasible loops have been a challenge in constraint-based analysis [1].

However, for the case of FBA and FVA time efficient implementations are already available. Therefore, I would suggest using the loopless flag of the cobrapy package when performing FVA. 

Also, it would be nice to show/discuss how many exchange reactions each GSMM includes and what is the number of those with at least a non-zero minimum or maximum in the FVA using each of the three media.

Done. In Supplementary Figure 4, we added a graphic summary of active FVA ranges for each GSMM in the three different environments (exchange reactions, non-zero flux). Additionally, we analyzed a subset of models and compared their regular FVA results vs loopless FVA results.

On line 217 we write:

"The number of active exchange fluxes in each medium corresponds with the respective growth performances displaying noticably higher number of potentially active fluxes in the rich environment (also when applying loopless FVA) (Supp. Fig. 4). Overall, Simulations confirmed the existence of a feasible solution space for  all the 243 models as well as their capacity to predict growth in the respective environemnt (Supp. Data 5)."

"Supplementary Figure 4. FVA performances of GSMMs in different environments (Supp. Fig.

3; Supp. Data 5). A. Distribution of potentially active exchange reactions (non-zero minimum FVA flux) in the different environments. Solid line inside each violin indicates the interquartile range (IQR). White point in IQR indicates the median value. Whiskers extending from the IQR indicate the range within 1.5 times the IQR from the quartiles. Violin width at a given value represents the density of data points at that value. B. Loopless FVA scores compared to regular FVA for models in the 3 different environments. Bars indicate the count of active fluxes (nonzero minimum FVA flux). Only a subset of models was used for this analysis."

This brings us to the main challenge of your framework in my opinion: FVA returns the minimum and the maximum a flux may get. However, it does not ensure that when a metabolite is being secreted, another does the same too. That could lead to an overrepresentation of secreted metabolites after each iteration. To my understanding, unbiased methods focusing on metabolite exchanges would be a much better alternative for such questions. Unbiased constraint-based methods are known for requiring essential computational requirements, yet when focusing on specific parts of the models, recent implementations support them. A great showcase of such techniques is presented in [2].

Indeed, FVA solutions return all potential metabolic fluxes in GSMMs (ranges of all fluxes satisfying the objective function, which by default is set to biomass increase) but they do not ensure that all fluxes actually co-occur (i.e., when a metabolite is secreted necessarily another metabolite is secreted too). However, though FVA solutions do not necessarily ensure cooccurrence regarding secretion and uptake, they provide a broader metabolic picture (the full set of potential solutions), unlike the arbitrary single solution provided by FBA, which is limited in providing information about potential secretions and uptakes in a specific environment. Here, we tried to elucidate the connection between a specific environment (root exudates) and the growth and metabolic capabilities of native bacteria. To the best of our understanding,  unbiased approaches (such as the one displayed in Wedmark et al.36) are not environment dependent but rather calculate all possible metabolic elements and routes within a metabolic network. Therefore, using FVA is well adapted to explore environment-dependent growth. The sensitivity of FVA predicted active fluxes to the environments is now also implied by Sup. Fig. 3B demonstrating the number of potential active fluxes is proportional to growth performances.  In addition, inquiring all possible metabolic routes across a large dataset of hundreds of MAGS, is central to the current analysis, thus the easy implementation of FVA further justifies its use in the current study.

An alternative strategy to reduce inflated FVA predictions and further constrain the solution space of predicted active fluxes can be the incorporation of additional layers of `omics data, as for example was done in the work of Zampieri et al5. Such approach could allow for instance removing reactions from the network reconstructions if not coming to play in situ, and therefore impose further constraints and narrow down the solution space. Currently, the complexity of the soil community might impede or at least constrain a high coverage recovery of transcriptomic data, though future works utilizing additional layers of `omics data are expected to significantly reduce the number of potential solutions and thus improve the accuracy of GEMs predictions. 

This is now discussed in the text. In line 541 we write:

"Similarly, complementing the genomic-based approaches done here, with additional layers of 'omics information (mainly transcriptomics & metabolomics) can further constrain the solution space, deflate the number of potential metabolic routes and yield more accurate predictions of GSMMs' performances5."  

In case it was the first version of CheckM used, the authors could consider repeating this check with CheckM2. As they state in line 293, Archaea may play an essential role in the community. Yet, among the high-quality MAGs only one corresponded to Archaea. However, that is quite possible to be the case because CheckM underestimates the completeness of archaeal genomes. If CheckM2 suggests that archaeal MAGs could be used, these would probably benefit a lot for the aim of the study.

The analysis was conducted with the first version of CheckM to assess MAGs quality. In future analyses we will use CheckM2. However, also before MAG recovery, we already know from the work of Beirhu et al., that Archaea species have a very low representation in the metagenomics data used here (Berihu et al., Additional data 2. Supp. fig. 4; "others" group)6, with less than 0.5% of the contigs mapped to archaeal genomes. The overall taxonomic distribution of the high-quality MAGs was compared to the distribution inferred from the non-binned data (contigs) and amplicon sequencing and the three different data sets are very similar (Fig. 2). 

On line 130 we write:

"Overall, the taxonomic distribution of the MAG collection corresponded with the profile reported for the same samples using alternative taxonomic classification approaches such as 16S rRNA amplicon sequencing and gene-based taxonomic annotations of the non-binned shotgun contigs

(Fig. 2B)."

The visualisation of the network in Figure 4A is hard to follow. An alternative could be a 5partite plot having taxa in columns one, three, and five and compounds in the other two. An alternative visualisation is necessary.

The full list of the 5 and 3 partite graphs is provided in supplementary data 10 (also noted in the figure legend now). Figure 4 was revised to improve its visualization. Labels were removed and a zoom in to 5 and 3 partite plots were added (PMM and PM subnetworks, respectively). 

Line 509: If I get the point of the authors right, they refer to the "from shotgun data to GEMs" approach. I would suggest skipping this statement. Here is a recent study implementing this: https://doi.org/10.1016/j.crmeth.2022.100383.

Thank you for your comment and reference. The intention behind the phrase in line 509 (in previous version) was to refer to going from metagenomics data to GEMs in soil-rhizosphere microbiome while linking environmental inputs (crop-plants exudates metabolomics data) and the agricultural-related metabolic function of bacteria. This phrase has been modified to clearly make a more modest claim while acknowledging other related studies.

On line 548 we write

"Where recent studies begin to apply GSMM reconstruction and analysis starting from MAGs5,37 , this work applies the MAGs to GSMMs approach to conduct a large-scale CBM analysis over highquality MAGs derived from a native rhizosphere and explore the complex network of interactions in light of the functioning of the respective agro-ecosystem. "

Line 820: Reference format is broken.

Corrected.

In the caption of Figure 4, please add the meaning of H, S, and NA so it is selfexplanatory.

Done. In Figure 4 legend we added:

"Node colors correspond to differential abundance classification of GSMMs in the different plots; H, S, NA are Healthy, Sick, Not-Associated, respectively."

Reviewer #3 (Recommendations for the authors):

(1) Figure 4A is unreadable. It is not clear what insight the reader could gain by examining this figure.

Thanks. Figure was revised. Labels were removed and a zoom-in-window to the exchanges highlighted in Figure 4C were added. Caption was revised to indicate that node colors correspond to differential abundance classification of GSMMs in the different plots (H, S, NA are Healthy, Sick, Not-Associated, respectively).

(2) In Figure 5, it is not apparent what the units of "prevalence" are, that is, what is the scale. What does 140 mean? How does that compare to 350?

Thanks. Prevalence in the context of Figure. 5B,C refers to the count of the compounds in each category (significantly affiliated with either healthy or symptomized soils) in sub-network motifs corresponding to this DA classification. We revised the figures (Y axes) and legend to be more specific (B: # of exudates; C: # of secreted compounds).

"B. Bar plot indicating the number of exudates significantly associated with H or S-classified PM sub-networks (Hypergeometric test; FDR <= 0.05; green: healthy-H, red: sick-S). C. Bar plots indicate the number of secreted compounds in PM sub-networks, which are significantly associated with H-classified (upper, colored green), or S-classified (lower, colored red) (Hypergeometric test; FDR <= 0.05)."

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Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

It is suggested that for each limb the RG (rhythm generator) can operate in three different regimes: a non-oscillating state-machine regime, and in a flexor driven and a classical half-center oscillatory regime. This means that the field can move away from the old concept that there is only room for the classic half-center organization

Strengths:

A major benefit of the present paper is that a bridge was made between various CPG concepts ( "a potential contradiction between the classical half-center and flexor-driven concepts of spinal RG operation"). Another important step forward is the proposal about the neural control of slow gait ("at slow speeds ({less than or equal to} 0.35 m/s), the spinal network operates in a state regime and requires external inputs for phase transitions, which can come from limb sensory feedback and/or volitional inputs (e.g. from the motor cortex").

Weaknesses:

Some references are missing

We thank the Reviewer for the thoughtful and constructive comments. We have added additional text to meet the specific Reviewer’s recommendations and several references suggested by the Reviewer.  

Reviewer #2 (Public Review):

Summary:

The biologically realistic model of the locomotor circuits developed by this group continues to define the state of the art for understanding spinal genesis of locomotion. Here the authors have achieved a new level of analysis of this model to generate surprising and potentially transformative new insights. They show that these circuits can operate in three very distinct states and that, in the intact cord, these states come into successive operation as the speed of locomotion increases. Equally important, they show that in spinal injury the model is "stuck" in the low speed "state machine" behavior.

Strengths:

There are many strengths for the simulation results presented here. The model itself has been closely tuned to match a huge range of experimental data and this has a high degree of plausibility. The novel insight presented here, with the three different states, constitutes a truly major advance in the understanding of neural genesis of locomotion in spinal circuits. The authors systematically consider how the states of the model relate to presently available data from animal studies. Equally important, they provide a number of intriguing and testable predictions. It is likely that these insights are the most important achieved in the past 10 years. It is highly likely proposed multi-state behavior will have a transformative effect on this field.

Weaknesses:

I have no major weaknesses. A moderate concern is that the authors should consider some basic sensitivity analyses to determine if the 3 state behavior is especially sensitive to any of the major circuit parameters - e.g. connection strengths in the oscillators or?

We thank the Reviewer for the thoughtful and constructive comments. The sensitivity analysis has been included as Supplemental file.

Reviewer #3 (Public Review):

Summary:

This work probes the control of walking in cats at different speeds and different states (split-belt and regular treadmill walking). Since the time of Sherrington there has been ongoing debate on this issue. The authors provide modeling data showing that they could reproduce data from cats walking on a specialized treadmill allowing for regular and split-belt walking. The data suggest that a non-oscillating state-machine regime best explains slow walking - where phase transitions are handled by external inputs into the spinal network. They then show at higher speeds a flexor-driven and then a classical halfcenter regime dominates. In spinal animals, it appears that a non-oscillating state-machine regime best explains the experimental data. The model is adapted from their previous work, and raises interesting questions regarding the operation of spinal networks, that, at low speeds, challenge assumptions regarding central pattern generator function. This is an interesting study. I have a few issues with the general validity of the treadmill data at low speeds, which I suspect can be clarified by the authors.

Strengths:

The study has several strengths. Firstly the detailed model has been well established by the authors and provides details that relate to experimental data such as commissural interneurons (V0c and V0d), along with V3 and V2a interneuron data. Sensory input along with descending drive is also modelled and moreover the model reproduces many experimental data findings. Moreover, the idea that sensory feedback is more crucial at lower speeds, also is confirmed by presynaptic inhibition increasing with descending drive. The inclusion of experimental data from split-belt treadmills, and the ability of the model to reproduce findings here is a definite plus.

Weaknesses:

Conceptually, this is a very useful study which provides interesting modeling data regarding the idea that the network can operate in different regimes, especially at lower speeds. The modelling data speaks for itself, but on the other hand, sensory feedback also provides generalized excitation of neurons which in turn project to the CPG. That is they are not considered part of the CPG proper. In these scenarios, it is possible that an appropriate excitatory drive could be provided to the network itself to move it beyond the state-machine state - into an oscillatory state. Did the authors consider that possibility? This is important since work using L-DOPA, for example, in cats or pharmacological activation of isolated spinal cord circuits, shows the CPG capable of producing locomotion without sensory or descending input.

We thank the Reviewer for the thoughtful and constructive comments. We have added additional texts, references, and discussed the issues raised by the Reviewer. Particularly, in section “Model limitations and future directions” we now admit that afferent feedback can provide some constant level excitation to the RG circuits after spinal transection which can partly compensate for the lack of supraspinal drive and hence affect (shift) the timing of transitions between the considered regimes. We mentioned that this is one of the limitations of the present model. The potential effects of neuroactive drugs, like DOPA, on CPG circuits after spinal transection were left out because they are outside the scope of the present modeling studies.    

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

specific feedback to the authors:

Nevertheless, there are some minor points, worth considering.

Link to HUMAN DATA

Here the authors may be interested to know that human data supports their proposal. This is relevant since there is ample evidence for the operation of spinal CPG's in humans (Duysens and van de Crommert,1998). The present model predicts that the basic output of the CPG remains even at very slow speeds, thus leading to similarity in EMG output. This prediction fits the experimental data (den Otter AR, Geurts AC, Mulder T, Duysens J. Speed related changes in muscle activity from normal to very slow walking speeds. Gait Posture. 2004 Jun;19(3):270-8). To investigate whether the basic CPG output remains basically the same even at very slow speeds (as also predicted by the current model), humans walked slowly on a treadmill (speeds as slow as 0.28 m s−1). Results showed that the phasing of muscle activity remained relatively stable over walking speeds despite substantial changes in its amplitude. Some minor additions were seen, consistent with the increased demands of postural stability. Similar results were obtained in another study: Hof AL, Elzinga H, Grimmius W, Halbertsma JP. Speed dependence of averaged EMG profiles in walking. Gait Posture. 2002 Aug;16(1):78-86. doi:

10.1016/s0966-6362(01)00206-5. PMID: 12127190.

These authors wrote: "The finding that the EMG profiles of many muscles at a wide range of speeds can be represented by addition of few basic patterns is consistent with the notion of a central pattern generator (CPG) for human walking". The basic idea is that the same CPG can provide the motor program at slow and fast speeds but that the drive to the CPG differs. This difference is accentuated under some conditions in pathology, such as in Parkinson's Kinesia Paradoxa. It was argued that the paradox is not really a paradox but is explained as the CPGs are driven by different systems at slow and at fast speeds (Duysens J, Nonnekes J. Parkinson's Kinesia Paradoxa Is Not a Paradox. Mov Disord. 2021 May;36(5):1115-1118. doi: 10.1002/mds.28550. Epub 2021 Mar 3. PMID: 33656203.)

These ideas are well in line with the current proposal ("Based on our predictions, slow (conditionally exploratory) locomotion is not "automatic", but requires volitional (e.g. cortical) signals to trigger stepby-step phase transitions because the spinal network operates in a state-machine regime. In contrast, locomotion at moderate to high speeds (conditionally escape locomotion) occurs automatically under the control of spinal rhythm-generating circuits receiving supraspinal drives that define locomotor speed, unless voluntary modifications or precise stepping are required to navigate complex terrain").

As mentioned in the present paper, other examples exist from pathology ("...Another important implication of our results relates to the recovery of walking in movement disorders, where the recovered pattern is generally very slow. For example, in people with spinal cord injury, the recovered walking pattern is generally less than 0.1 m/s and completely lacks automaticity 77-79. Based on our predictions, because the spinal locomotor network operates in a state-machine regime at these slow speeds, subjects need volition, additional external drive (e.g., epidural spinal cord stimulation) or to make use of limb sensory feedback by changing their posture to perform phase transitions"). As mentioned above, another example is provided by Parkinson's disease. The authors may also be interested in work on flexible generators in SCI: Danner SM, Hofstoetter US, Freundl B, Binder H, Mayr W, Rattay F, Minassian K. Human spinal locomotor control is based on flexibly organized burst generators. Brain. 2015 Mar;138(Pt 3):577-88. doi: 10.1093/brain/awu372. Epub 2015 Jan 12. PMID: 25582580; PMCID: PMC4408427.

We thank the reviewer for these additional and interesting insights. We added a new paragraph in the Discussion to bolster the link with human data that includes references suggested by the Reviewer.

CHAIN OF REFLEXES

It reads: "... in opposition to the previously prevailing viewpoint of Charles Sherrington 21,22 that locomotion is generated through a chain of reflexes, i.e., critically depends on limb sensory feedback (reviewed in 23)." This is correct but incomplete. The reference cited (23: Stuart, D.G. and Hultborn, H, "Thomas Graham Brown (1882--1965), Anders Lundberg (1920-), and the neural control of stepping," Brain Res. Rev. 59(1), 74-95 (2008)) actually reads: "Despite the above findings, the doctrinaire position in the early 1900s was that the rhythm and pattern of hind limb stepping movements was attributable to sequential hind limb reflexes. According to Graham Brown (1911c) this viewpoint was largely due to the arguments of Sherrington and a Belgian physiologist, Maurice Philippson (1877-1938). Philippson studied stepping movements in chronically maintained spinal dogs, using techniques he had acquired in the Strasbourg laboratory of the distinguished German physiologist, Friedrich Goltz (1834-1902). He also analyzed kinematically moving pictures of dog locomotion, which had been sent to him by the renowned French physiologist, Etienne-Jules Marey (1830-1904). Philippson (1905) certainly presented arguments explaining his perception of how sequential spinal reflexes contributed to the four phases of the step cycle (see Fig. 1 in Clarac, 2008). In retrospect, it is likely that Graham Brown was correct in attributing to Philippson and Sherrington the then-prevailing viewpoint that reflexes controlled spinal stepping. It is puzzling, nonetheless, that far less was said then and even now about Philippson's belief that the spinal control was due to a combination of central and reflex mechanisms (Clarac, 2008),4,5 4 We are indebted to François Clarac for drawing to our attention Philippson's statement on p. 37 of his 1905 article that "Nos expériences prouvent d'une part que la moelle lombaire séparée du reste de l'axe cérébro-spinal est capable de produire les mouvements coordonnés dans les deux types de locomotion, trot et gallop. [Our experiments prove that one side of the spinal cord separated from the cerebro-spinal axis is able to produce coordinated movements in two types of locomotion, trot and gallop]." Then, on p. 39 Philippson (1905) states that "Nous voyons donc, en résumé que la coordination locomotrice est une fonction exclusivement médullaire, soutenue d'une part par des enchainements de réflexes directs et croisés, dont l'excitant est tantot le contact avec le sol, tantot le mouvement même du membre. [In summary, we see that locomotor coordination is an exclusive function of the spinal cord supported by a sequencing of direct and crossed reflexes, which are activated sometimes by contact with the ground and sometimes even by leg movement]. A coté de cette coordination basée sur des excitations périphériques, il y a une coordination centrale provenant des voies d'association intra-médullaires. [In conjunction with this peripherally excited coordination, there is a central coordination arising from intraspinal pathways]." (The English translations have also been kindly supplied by François Clarac.) Clearly, Philippson believed in both a central spinal and a reflex control of stepping! 5 In part 1 of his 1913/1916 review Graham Brown discussed Philippson's 1905 article in much detail (pp. 345-350 in Graham Brown, 1913b). He concludes with the statement that "... Philippson die wesentlichen Factoren des Fortbewegungsaktes in das exterozeptive Nervensystem verlegt. Er nimmt an, dass die zyklischen Bewegungen automatisch durch äussere Reize erhalten werden, welche in sich selbst thythmisch als Folge der Reflexakte welche sie selbst erzeugen, wiederholt werden. [Philippson assigns the important factors of the act of locomotion to the exteroceptive nervous system. He assumes that the cyclic movements are automatically maintained by external stimuli which, by themselves, are rhythmically repeated as a consequence of the reflexive actions that they generate themselves]." (English translation kindly supplied by Wulfila Gronenberg). This interpretation clearly ignores Philippson's emphasis on a central spinal component in the control of stepping....). "

Hence it is a simplification to give all credits to Sherrington and ignoring the role of Philippson concerning the chain of reflexes idea.

We again thank the Reviewer for these additional and interesting insights. We added the Philippson (1905) and Clarac (2008) references. The important contribution of Philippson is now indicated.

GTO Ib feedback

It reads: "This effect and the role of Ib feedback from extensor afferents has been demonstrated and described in many studies in cats during real and fictive locomotion 2,57-59."

These citations are appropriate but it is surprising to see that the Hultborn contribution is limited to the Gossard reference while the even more important earlier reference to Conway et al is missing (Conway BA, Hultborn H, Kiehn O. Proprioceptive input resets central locomotor rhythm in the spinal cat. Exp Brain Res. 1987;68(3):643-56. doi: 10.1007/BF00249807. PMID: 3691733).

Yes, the Conway et al. reference has been added.

Other species

The authors may also look at other species. The flexible arrangement of the CPGs, as described in this article, is fully in line with work on other species, showing cpg networks capable to support gait, but also scratching, swimming ..etc (Berkowitz A, Hao ZZ. Partly shared spinal cord networks for locomotion and scratching. Integr Comp Biol. 2011 Dec;51(6):890-902. doi: 10.1093/icb/icr041. Epub 2011 Jun 22. PMID: 21700568. Berkowitz A, Roberts A, Soffe SR. Roles for multifunctional and specialized spinal interneurons during motor pattern generation in tadpoles, zebrafish larvae, and turtles. Front Behav Neurosci. 2010 Jun 28;4:36. doi: 10.3389/fnbeh.2010.00036. PMID: 20631847; PMCID: PMC2903196.)

Similar ideas about flexible coupling can also be found in: Juvin L, Simmers J, Morin D. Locomotor rhythmogenesis in the isolated rat spinal cord: a phase-coupled set of symmetrical flexion extension oscillators. J Physiol. 2007 Aug 15;583(Pt 1):115-28. doi: 10.1113/jphysiol.2007.133413. Epub 2007 Jun 14. PMID: 17569737; PMCID: PMC2277226. Or zebrafish: Harris-Warrick RM. Neuromodulation and flexibility in Central Pattern Generator networks. Curr Opin Neurobiol. 2011 Oct;21(5):685-92. doi: 10.1016/j.conb.2011.05.011. Epub 2011 Jun 7. PMID: 21646013; PMCID: PMC3171584.

We added a sentence in the Discussion along with supporting references.

Standing

In the view of the present reviewer, the model could even be extended to standing in humans. It reads: "at slow speeds ({less than or equal to} 0.35 m/s), the spinal network operates in a state regime and requires external inputs"; similarly (personal experience) when going from sit to stand: as soon as weight is over support, extension is initiated and the body raises, as one would expect when the extensor center is activated by reinforcing load feedback, replacing GTO inhibition (Faist M, Hoefer C, Hodapp M, Dietz V, Berger W, Duysens J. In humans Ib facilitation depends on locomotion while suppression of Ib inhibition requires loading. Brain Res. 2006 Mar 3;1076(1):87-92. doi:

Yes, we agree that the model could be extended to standing and the transition from standing to walking is particularly interesting. However, for this paper, we will keep the focus on locomotion over a range of speeds.

Reviewer #2 (Recommendations For The Authors):

The presentation is exceedingly well done and very clear.

A moderate concern is that the authors do not make use of the capacity of computer simulations for sensitivity analyses. Perhaps these have been previously published? In any case, the question here is whether the 3 state behavior is especially sensitive to excitability of one of the main classes of neurons or a crucial set of connections.

The sensitivity analysis has been made and included as Supplemental file.

Minor point. I have but two minor points. A bit more explanation should be provided for the use of the terms "state machine" to describe the lowest speed state. Perhaps this is a term from control theory? In any case, it is not clear why this is term is appropriate for a state in which the oscillator circuits are "stuck" in a constant output form and need to be "pushed" by sensory input.

Yes, we now provide a definition in the Introduction.

Minor point: it is of course likely that neuromodulation of multiple types of spinal neurons occurs via inputs that activate G protein coupled receptors. These types of inputs are absent from the model, which is fine, but some sort of brief discussion should be included. One possibility is to note that the circuit achieves transitions between different states without the need for neuromodulatory inputs. This appears to me to be a very interesting and surprising insight.

In section “Model limitations and future directions” in the Discussion, we now mention that the term “supraspinal drive” in our model is used to represent supraspinal inputs providing both electrical and neuromodulator effects on spinal neurons increasing their excitability, which disappear after spinal transection.” We think that it is so far too early to simulate the exact effects of the descending neuromodulation, since there is almost no data on the effect of different modulators on specific types of spinal interneurons.

Reviewer #3 (Recommendations For The Authors):

Minor Comments  

Page numbers would be useful.

Abstract

Following spinal transection, the network can only operate in a state-machine regime. This is a bit strong since it applies to computational data. Clarify this statement.

We agree. Sentence has been changed to: “Following spinal transection, the model predicts that the spinal network can only operate in the state-machine regime.”

Introduction

Intro - "This is somewhat surprising...". It gives the impression that spinal cats are autonomously stable on the belt. They are stabilized by the experimenter.

The text has been changed to: “This is somewhat surprising because intact and spinal cats rely on different control mechanisms. Intact cats walking freely on a treadmill engage vision for orientation in space and their supraspinal structures process visual information and send inputs to the spinal cord to control locomotion on a treadmill that maintains a fixed position of the animal relative to the external space. Spinal cats, whose position on the treadmill relative to the external space is fixed by an experimenter, can only use sensory feedback from the hindlimbs to adjust locomotion to the treadmill speed.”

"Cannot consistently perform treadmill locomotion" - likely a context-dependent result. Certainly, cats can do this easily off a treadmill - stalking, for example. Perhaps somewhere, mention that treadmill locomotion is not entirely similar to overground locomotion.

We completely agree. Stalking is an excellent example showing that during overground locomotion slow movements (and related phase transitions) can be controlled by additional voluntary commands from supraspinal structures, which differs from simple treadmill locomotion, performing out of specific goalor task-dependent contexts. Based on this, we suggest a difference between a relatively slow (exploratory-type, including stalking) and relatively fast (escape-type) overground locomotion. We added the following sentence to the introduction:” This is evidently context dependent and specific for the treadmill locomotion as cats, humans  and other animals can voluntarily decide to perform consistent overground locomotion at slow speeds.”

The authors introduce the concept of the state machine regime. In my opinion, this could use some more explanation and citations to the literature. Was it a term coined by the authors, or is there literature reinforcing this point?

This is a computer science and automata theory term that has already been used in descriptions of locomotion (see our references in the 2nd paragraph of Discussion). We added a definition and corresponding references in the Introduction.

In terms of sensory feedback, particularly group II input, it would be interesting to calculate if the conduction delay to the spinal cord at higher speeds would have a certain cutoff point at which it would no longer be timed effectively for phase transitions. This could reinforce your point.

This is an interesting proposition but it is unlikely to be a factor over the range of speeds that we investigated (0.1 to 1.0 m/s). Assuming that group II afferents transmit their signals to spinal circuits at a latency of 10-20 ms, this is more than enough time to affect phase transitions, even at the highest speed considered. This might be a factor at very high speeds (e.g. galloping) or in small animals with high stepping frequencies.

Results.

The assertion that intact cats are inconsistent in terms of walking at slow speeds needs to be bolstered. For example, if a raised platform were built for a tray of food, would the intact cat consistently walk at slower speeds and eat? I suspect so. By the same token, would they walk slowly during bipedal walking? It is pretty easy to check this. Also, reports from the literature show differential effects of runway versus treadmill gait analysis, specifically when afferent input is removed.

The Reviewer is correct that raising a platform for a food tray or even having intact cats walk with their hindlimbs only (with forelimbs on a stationary platform) may allow for consistent stepping at slow speeds (0.1 – 0.3 m/s). However, this effectively removes voluntary control of locomotion and makes the pattern more automatic (spinal + limb sensory feedback). These examples provide additional specific contexts, and we have already mentioned (see above) that slow locomotion of intact cat is context dependent. 

"We believe that intact animals walking on a treadmill..." Citations for this? Certainly, this is not a new point.

No, this is not new. We changed the sentence and added a reference to the statement: “Intact animals walking on a treadmill use visual cues and supraspinal signals to adjust their speed and maintain a fixed position relative to the external space with reference to Salinas et al. (Salinas, M.M., Wilken, J M, and Dingwell, J B, "How humans use visual optic flow to regulate stepping during walking," Gait. Posture. 57, 15-20, 2017).

The presentation of the results is somewhat disjointed. The intact data is presented for tied and splitbelt results, but this is not addressed explicitly until figure 4. Would it not be better to create a figure incorporating both intact and modelling data and present the intact data where appropriate?

We tried to do this initially, but this way required changing the style of the whole paper and we decided against this idea. Therefore, we prefer to keep the presentation of results as it is now. 

Regarding the role of sensory feedback being especially important at low speeds, it is interesting that egr3+ mice (lacking spindle input) show an inability to walk at high speeds >40 cm/s but can walk at lower speeds (up to 7 cm/s) (Takeoka et al 2014). Similar findings were found with a lesion affecting Group I afferents in general (Takeoka and Arber 2019). Also, Grillner and colleagues show that cats can produce fictive locomotion in the absence of sensory input.

In the Takeoka experiments it is difficult to assess the effect of removing somatosensory feedback because animals can simply decide to not step at higher speeds to avoid injury. Their mice deprived of somatosensory feedback can walk at slow speeds, likely thanks to voluntary commands, and cannot do so at higher speeds because (1) maybe somatosensory feedback is indeed necessary and/or (2) because they feel threatened because of impaired posture and poor control in general. In other words, they choose to not walk at faster speeds to avoid injury.

Fictive locomotion by definition is without phasic somatosensory feedback as the animals are curarized or studies are performed in isolated spinal cord preparations. Depending on the preparation, pharmacology or brainstem stimulation is required to evoke fictive locomotion. If animals are deafferented, pharmacology or brainstem stimulation are required to induce fictive locomotion to offset the loss of spinal neuronal excitability provided by primary afferents. At the same time, our preliminary analysis of old fictive locomotion data in the University of Manitoba Spinal Cord center (Drs. Markin and Rybak had an official access to these data base during our collaboration with Dr. David McCrea) has shown that the frequency of stable fictive locomotion in cats usually exceeded 0.6 - 0.7 Hz, which approximately corresponds to the speed above 0.3 - 0.4 m/s. These data and estimation are just approximate; they have not been statistically analyzed and published and hence have not been included in our paper.

Discussion. The statement that sensory feedback is required for animals to locomote may need to be qualified. Animals need some sensory feedback to locomote is perhaps better. For example, lesion studies by Rossignol in the early 2000s showed that cutaneous feedback from the paw was seemingly quite critical (in spinal cats). Also, see previous comments above.

We changed this to: “… requires some sensory feedback to locomote, …”

Figures

Figure 1C. This figure is somewhat confusing. If intact cats do not walk (arrow), how are the data for swing and stance computed? Also raw traces would be useful to indicate that there is variability. Also, while duration is useful, would you not want to illustrate the co-efficient of variation as well as another way to show that the stepping pattern was inconsistent?

This is probably a misunderstanding. The left panel of Fig. 1C superimposes data of intact cats from panel A (with speed range from 0.4 m/s to 1.0 m/s) and data from spinal cats from panel B (with speed range from 0.1 m/s and 1.0 m/s). Therefore, the left part of this left panel 1C (with speed range from 0.1 m/s to 0.4 m/s (pointed out by the arrow) corresponds only to spinal cats (not to intact cats). The standard deviations of all measurements are shown. All these figures were reproduced from the previous publications. We did not apply new statistical analysis to these previously published data/figures.

Figure 4. 'All supraspinal drives (and their suppression of sensory feedback) are eliminated from the schematic shown in A. ' However, it is labelled 'brainstem drives,' which is confusing. Moreover, many of the abbreviations are confusing. Do you need l-SF-E1 in the figure, or could you call it 'Feedback 1' and then refer to l-SF-E1 in the legend? The same goes for βr, etc. Can they move to the legend?

In the intact model (Fig. 4A), we have supraspinal drives (𝛼𝐿 and 𝛼𝑅, and  𝛾𝐿 and 𝛾𝑅 ), some of which provide presynaptic inhibition of sensory feedback (SF-E1 and SF-E2) as shown in Fig. 4A. In spinaltransected model (Fig. 4B), the above brainstem drives and their effects (presynaptic inhibition) on both feedback types are eliminated (therefore, there is no label “Brainstem drives in Fig. 4B). Also, we do not see a strong reason to change the feedback names, since they are explained in the text.

I appreciate the detail of these figures, but they are difficult to conceptualize. They are useful in the context of 3C. Perhaps move this figure to supplementary and then show the proposed schematics for the system operating at slow, medium, and fast speeds in a replacement figure?

We apologize for the resistance, but we would like to keep the current presentation.

There is a lack of raw data (models or experimental) data reinforcing the figures. I would add these to all figures, which would nicely complement the graphs.

These raw data can be found in the cited manuscripts. It would be the same figures.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

Manuscript number: RC-2024-02546

Corresponding author: Woo Jae, Kim

1. General Statements

The goal of this study is to provide the insights of one specific neuron ‘SIFa’ controls interval timing behavior by its receptor ‘SIFaR’ through neuropeptide relay. Interval timing, or the sense of time in the seconds to hours range, is important in foraging, decision making, and learning in humans via activation of cortico-striatal circuits. Interval timing requires completely distinct brain processes from millisecond or circadian timing. In summary, interval timing allows us to subjectively sense the passage of physical time, allowing us to integrate action sequences, thoughts, and behavior, detect developing trends, and predict future consequences.

Many researchers have tried to figure out how animals, including humans, can estimate time intervals with such precision. However, most investigations have been conducted in the realm of psychology rather than biology thus far. Because the study of interval timing was limited in its ability to intervene in the human brain, many psychologists concentrated on developing convincing theoretical models to explain the known occurrence of interval timing.

To overcome the limits of studying interval timing in terms of genetic control, we have reported that the time investment strategy for mating in Drosophila males can be a suitable behavioral platform to genetically dissect the principle of brain circuit mechanism for interval timing. For example, we previously reported that males prolong their mating when they have previously been exposed to rivals (Kim, Jan & Jan, "Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals" Nature Neuroscience, 2012) (Kim et al, 2012), and this behavior is regulated by visual stimuli, clock genes, and neuropeptide signaling in a subset of neurons (Kim, Jan & Jan, “A PDF/NPF Neuropeptide Signaling Circuitry of Male Drosophila melanogaster Controls Rival-Induced Prolonged Mating” Neuron, 2013) (Kim et al, 2013). And we also reported that the sensory inputs are required for sexual experienced males to shorten their mating time (Lee, Sun, et al, “Taste and pheromonal inputs govern the regulation of time investment for mating by sexual experience in male Drosophila melanogaster” PLOS genetics, 2023) (Lee et al, 2023).

Throughout their lives, all animals must make decisions in order to optimize their utility function. Male reproductive success is determined by how many sperms successfully fertilize an egg with a restricted number of investment resources. To optimize male reproductive fitness, a time investment strategy has been devised. As a consequence, we believe that the flexible responses of mating duration to different environmental contexts in Drosophila males might be an excellent model to investigate neural circuits for interval timing.

The most well-known features of mammalian modulating energy homeostasis between the gut and the brain is one of the most intensively studied neuro-modulatory circuits via the neuronal relay of neuropeptides. In this article, we report that SIFa controls two alternate interval timing behaviors through neuropeptide relay signaling by SIFaR and other important neuropeptides and transmits the internal states of the male brain into decision making. According to our findings, male Drosophila utilize SIFa-SIFaR signaling modulating LMD and SMD behaviors. During our investigation in this regulation, we found a subset of cells that express SIFaR in SOG and AG region are important for the modulation of interval timing behaviors. Furthermore, we discovered a neuropeptide named Corazonin (Crz) which expressed in SIFaR is important for both LMD and SMD behaviors.

Our discovery of neuropeptide relay of SIFa-SIFaR-Crz-CrzR in male Drosophila in modulating interval timing behaviors will be a huge step forward in our knowledge of interval timing behavior.

2. Point-by-point description of the revisions

Reviewer #1

Comment 1. The authors are to be commended for the sheer quantity of data they have generated, but I was often overwhelmed by the figures, which try to pack too much into the space provided. As a result, it is often unclear what components belong to each panel. Providing more space between each panel would really help.

__ Answer:__ We are grateful for the insightful feedback regarding the structure of our data presentation. In response to your valuable suggestion, we have made adjustments in this revised version by downsizing the diagram and ensuring the spacing between the panels.

Comment 2. The use of three independent RNAi lines to knock down SIFaR expression is experimentally solid, as the common phenotype observed with all 3 lines supports the conclusion that the SIFaR is important for mating duration choice. However, the authors have not tested whether these lines effectively reduce SIFaR expression, nor whether the GAL80 constructs used to delimit knockdown are able to effectively do so. This makes it hard to make definitive conclusions with these manipulations, especially in the face of negative results. A lack of complete knockdown is suggested by the fact that the F24F06 driver rescues lethality when used to express SIFaR in the B322 mutant background, but does not itself produce lethality when used to express SIFaR RNAi. The authors should either conduct experiments to determine knockdown efficiency or explicitly acknowledge this limitation in drawing conclusions from their experiments. A similar concern relates to the CrzR knockdown experiments (eg Figure 7).

   __Answer:__ We appreciate the reviewer's attention to the details of our experimental design. Indeed, the validation of SIFaR-RNAi efficiency is crucial for interpreting our results accurately. In our initial experiments, we focused on the consistent phenotypic outcomes across the three independent RNAi lines, which collectively suggest the importance of SIFaR in LMD and SMD behaviors. However, we recognize the importance of confirming the effectiveness of our RNAi constructs in reducing SIFaR expression. Initially, we incorporated experiments utilizing *elav-GAL80* to demonstrate that the SIFaR knockdown mediated by the *elavc155* driver is sufficient to eliminate LMD and SMD behaviors. The corresponding results are presented in Figure 1C-D, with a detailed description provided in the manuscript as detailed below.

"The inclusion of elav-GAL80, which suppresses GAL4 activity in a pan-neuronal context, was found to restore both LMD and SMD behaviors when SIFaR was knocked down by a pan-neuronal elavc155 driver (Fig. 1C-D). This observation suggests that the reduction in SIFaR expression mediated by the elavc155 driver is sufficient to significantly impair LMD and SMD behaviors."

   In response to the comments, we have conducted a thorough reevaluation in our revised manuscript. Specifically, we have confirmed the efficiency of the SIFaR-RNAi line HMS00299, which exhibited the most pronounced phenotype when co-expressed with the tub-GAL4 and nSyb-GAL4 drivers, using quantitative real-time PCR (qRT-PCR). It has come to our attention that we omitted mentioning the embryonic lethality induced by the HMS00299 line when combined with either tub-GAL4 or nSyb-GAL4 drivers, which is consistent with the homozygous lethality observed in the *SIFaRB322* mutant. To address this, we have performed qRT-PCR experiments by crossing the HMS00299 line with tub-GAL4; tub-GAL80ts, allowing for the temporary knockdown of SIFaR specifically during the adult stage. We utilized w-/SIFaR-RNAis as a control in these experiments. The outcomes are illustrated in Figure 1E, and we have made the necessary modifications and additions to the manuscript to accurately reflect the efficiency of the SIFaR-RNAi line as detailed below.

"To ensure that RNAi did not have an off-target effect, we tested three independent RNAi strains and found that all three RNAi successfully disrupted LMD/SMD when expressed in neuronal populations. (Fig. S1E-J). We chose to use the HMS00299 line as SIFaR-RNAi for all our experiments because it efficiently disrupts LMD/SMD without UAS-dicer expression. Employment of broad drivers, including the tub-GAL4 and the strong neuronal driver nSyb-GAL4, with HMS00299 line consistently results in 100% embryonic lethality (data not shown). This phenotype mirrors the homozygous lethality observed in the SIFaRB322 mutant. The efficiency of HMS00299 SIFaR-RNAi lines was also validated through quantitative PCR analysis (Fig. 1E). Consequently, we infer that the knockdown of SIFaR using the HMS00299 line nearly completely diminishes the levels of the SIFaR protein."

We also examined the knockdown efficiency of CrzR in the experiments related to Figure 8 (revised version), following a similar approach (Fig. S7K).

Comment 3. Most of the behavioral experiments lack traditional controls, for example flies that contain either the GAL4 or UAS elements alone. The authors should explain their decision to omit these control experiments and provide an argument for why they are not necessary to correctly interpret the data. In this vein, the authors have stated in the methods that stocks were outcrossed at least 3x to Canton-S background, but 3 outcrosses is insufficient to fully control for genetic background.

• *Answer: We sincerely thank the reviewer for insightful comments regarding the absence of traditional genetic controls in our study of LMD and SMD behaviors. We acknowledge the importance of such controls and wish to clarify our rationale for not including them in the current investigation. The primary reason for not incorporating all genetic control lines is that we have previously assessed the LMD and SMD behaviors of GAL4/+ and UAS/+ strains in our earlier studies. Our past experiences have consistently shown that 100% of the genetic control flies for both GAL4 and UAS exhibit normal LMD and SMD behaviors. Given these findings, we deemed the inclusion of additional genetic controls to be non-essential for the present study, particularly in the context of extensive screening efforts. Consequently, in accordance with the reviewer's recommendation, we conducted genetic validation experiments on novel genetic crosses, including SIFaR-RNAi/+, CrzR-RNAi/+, and GAL4NP5270/+, and incorporated the results in the supplementary figures (Supplementary information 1). We have made the necessary modifications and additions to the manuscript as below.

"Given those genetic controls, as evidenced by consistent exhibition of normal LMD and SMD behaviors (Supplemental information 1), the observed reduction in SIFaR expression, driven by elavc155, is deemed sufficient to induce significant disruptions in LMD and SMD behaviors."

However, we understand the value of providing a clear rationale for our methodology choices. To this end, we have added a detailed explanation in the "MATERIALS AND METHODS" section and the figure legends of Figure 1. This clarification aims to assist readers in understanding our decision to omit traditional controls, as outlined below.

"__Mating Duration Assays for Successful Copulation__The mating duration assay in this study has been reported (Kim et al. 2012; Kim et al. 2013; Lee et al. 2023). To enhance the efficiency of the mating duration assay, we utilized the Df(1)Exel6234 (DF here after) genetic modified fly line in this study, which harbors a deletion of a specific genomic region that includes the sex peptide receptor (SPR) (Parks et al. 2004; Yapici et al. 2008). Previous studies have demonstrated that virgin females of this line exhibit increased receptivity to males (Yapici et al. 2008). We conducted a comparative analysis between the virgin females of this line and the CS virgin females and found that both groups induced SMD. Consequently, we have elected to employ virgin females from this modified line in all subsequent studies. For group reared (naïve) males, 40 males from the same strain were placed into a vial with food for 5 days. For single reared males, males of the same strain were collected individually and placed into vials with food for 5 days. For experienced males, 40 males from the same strain were placed into a vial with food for 4 days then 80 DF virgin females were introduced into vials for last 1 day before assay. 40 DF virgin females were collected from bottles and placed into a vial for 5 days. These females provide both sexually experienced partners and mating partners for mating duration assays. At the fifth day after eclosion, males of the appropriate strain and DF virgin females were mildly anaesthetized by CO2. After placing a single female into the mating chamber, we inserted a transparent film then placed a single male to the other side of the film in each chamber. After allowing for 1 h of recovery in the mating chamber in 25℃ incubators, we removed the transparent film and recorded the mating activities. Only those males that succeeded to mate within 1 h were included for analyses. Initiation and completion of copulation were recorded with an accuracy of 10 sec, and total mating duration was calculated for each couple. Genetic controls with GAL4/+ or UAS/+ lines were omitted from supplementary figures, as prior data confirm their consistent exhibition of normal LMD and SMD behaviors (Kim et al. 2012; Kim et al. 2013; Lee et al. 2023; Huang et al. 2024; Zhang et al. 2024). Hence, genetic controls for LMD and SMD behaviors were incorporated exclusively when assessing novel fly strains that had not previously been examined. In essence, internal controls were predominantly employed in the experiments, as LMD and SMD behaviors exhibit enhanced statistical significance when internally controlled. Within the LMD assay, both group and single conditions function reciprocally as internal controls. A significant distinction between the naïve and single conditions implies that the experimental manipulation does not affect LMD. Conversely, the lack of a significant discrepancy suggests that the manipulation does influence LMD. In the context of SMD experiments, the naïve condition (equivalent to the group condition in the LMD assay) and sexually experienced males act as mutual internal controls for one another. A statistically significant divergence between naïve and experienced males indicates that the experimental procedure does not alter SMD. Conversely, the absence of a statistically significant difference suggests that the manipulation does impact SMD. Hence, we incorporated supplementary genetic control experiments solely if they deemed indispensable for testing. All assays were performed from noon to 4 PM. We conducted blinded studies for every test."

   We appreciate the reviewer's inquiry regarding the genetic background of our experimental lines. In response to the comments, we would like to clarify the following. All of our GAL4, UAS, or RNAi lines, which were utilized as the virgin female stock for outcrosses, have been backcrossed to the Canton-S (CS) genetic background for over ten generations. The majority of these lines, particularly those employed in LMD assays, have been maintained in a CS backcrossed status for several years, ensuring a consistent genetic background across multiple generations. Our experience has indicated that the genetic background, particularly that of the X chromosome inherited from the female parent, plays a pivotal role in the expression of certain behavioral traits. Therefore, we have consistently employed these fully outcrossed females as virgins for conducting experiments related to LMD and SMD behaviors. It is noteworthy that, in contrast to the significance of genetic background for LMD behaviors, we have previously established in our work (Lee *et al*, 2023) that the genetic background does not significantly influence SMD behaviors. This distinction is important for the interpretation of our findings. To provide a comprehensive understanding of our experimental design, we have detailed the genetic background considerations in the __"Materials and Methods"__ section, specifically in the subsection __"Fly Stocks and Husbandry"__ as outlined below.

"To reduce the variation from genetic background, all flies were backcrossed for at least 3 generations to CS strain. For the generation of outcrosses, all GAL4, UAS, and RNAi lines employed as the virgin female stock were backcrossed to the CS genetic background for a minimum of ten generations. Notably, the majority of these lines, which were utilized for LMD assays, have been maintained in a CS backcrossed state for long-term generations subsequent to the initial outcrossing process, exceeding ten backcrosses. Based on our experimental observations, the genetic background of primary significance is that of the X chromosome inherited from the female parent. Consequently, we consistently utilized these fully outcrossed females as virgins for the execution of experiments pertaining to LMD and SMD behaviors. Contrary to the influence on LMD behaviors, we have previously demonstrated that the genetic background exerts negligible influence on SMD behaviors, as reported in our prior publication (Lee et al, 2023). All mutants and transgenic lines used here have been described previously."

Comment 4. Throughout the manuscript, the authors appear to use a single control condition (sexually naïve flies raised in groups) to compare to both males raised singly and males with previous sexual experience. These control conditions are duplicated in two separate graphs, one for long mating duration and one for short mating duration, but they are given different names (group vs naïve) depending on the graph. If these are actually the same flies, then this should be made clear, and they should be given a consistent name across the different "experiments".

* * Answer: We are grateful to the reviewer for highlighting the potential for confusion among readers regarding the visualization methods used in our figures. In response to this valuable feedback, we have now included a more detailed explanation of the graph visualization techniques in the legends of Figure 1, as detailed below. This additional information should enhance the clarity and understanding of the figure for all readers.

"In the mating duration (MD) assays, light grey data points denote males that were group-reared (or sexually naïve), whereas blue (or pink) data points signify males that were singly reared (or sexually experienced). The dot plots represent the MD of each male fly. The mean value and standard error are labeled within the dot plot (black lines). Asterisks represent significant differences, as revealed by the unpaired Student’s t test, and ns represents non-significant differences (*p* *

Comment 5.* The authors have consistently conflated overlap of neuronal processes with synaptic connections. Claims of synaptic connectivity deriving solely from overlap of processes should be tempered and qualified.

• For example, they say (Lines 201-202) "These findings suggest that SIFa neurons and GAL424F06-positive neurons form more synapses in the VNC than in the brain." This is misleading. Overlap of 24F06-LexA>CD8GFP and SIFa-GAL4>CD8RFP tells us nothing about synapse number, or even whether actual synapses are being formed.*

• *Answer: We sincerely thank the reviewer for their insightful and constructive feedback regarding the interpretation of our data. We acknowledge the important point raised about the limitations of inferring synapse numbers from the overlap of membrane GFP and RFP signals. We fully concur that more specific techniques, such as the GRASP method, are necessary to accurately quantify synapse numbers, as we have demonstrated in subsequent sections of our manuscript. In the section where we describe the SIFa-SIFaR neuronal architecture labeled with membrane GFP and RFP, we recognize the need for caution in not overstating the implications of these findings as indicative of synapse formation. In light of the reviewer's comments, we have revised our discussion to more accurately reflect the nature of the SIFa-SIFaR neuronal arborizing patterns, as detailed below. This revision aims to provide a more nuanced interpretation of our observations and to align with the current scientific understanding of synaptic quantification.

"As previously reported, SIFa neurons arborize extensively throughout the CNS, but the neuronal processes of GAL424F06-positive neurons are enriched in the optic lobe (OL), sub-esophageal ganglion (SOG), and abdominal ganglion (AG) (GFP signal in Fig. 2F). Neuronal processes that are positive for SIFa and SIFaR strongly overlap in the prow (PRW), prothoracic and metathoracic neuromere (ProNm and MesoNm), and AG regions (yellow signals in Fig. S3A). We quantified these overlapping neuronal processes between SIFa- and SIFaR-positive neurons and found that approximately 18% of SIFa neurons and 52% of GAL424F06-positive neurons overlap in brain (Fig. S3B, C), whereas approximately 48% of SIFa and 54% of GAL424F06-positive neurons overlap in VNC (Fig. S3D, E). These findings suggest that SIFa neurons and GAL424F06-positive neurons form more neuronal processes in the VNC than in the brain."

• * Lines 210-211: "The overlap of DenMark and syt.EGFP signals was highly enriched in both SOG and ProNm regions, indicating that these regions are where GAL424F06 neurons form interconnected networks". This is misleading. Overlap of DenMark and syt.EGFP does not indicate synapses (especially since these molecules can be expressed outside the expected neuronal compartment if driven at high enough levels).*

• *Answer: We are grateful for the reviewer's critical insights regarding our interpretation of the DenMark and syt.eGFP experiments. We acknowledge the reviewer's point that the overlap of DenMark and syt.eGFP signals does not conclusively indicate synapses and that some of these signals can be expressed outside the expected neuronal compartments, particularly at high levels.

  It is important to note that DenMark and syt.eGFP are markers of synaptic polarity. In the original publication of DenMark, the authors demonstrated that while these two markers are closely apposed, they do not necessarily overlap, as seen in the labeled yellow areas. They concluded that these areas could represent closely apposed regions where "LNv neurons establish presynaptic contacts within the aMe, suggesting that these contacts are on the postsynaptic sites of the LNv neurons themselves. (Nicolaï et al, 2010)" The authors also observed that DenMark-enriched structures appear juxtaposed to, rather than coexpressed with, syt.eGFP, indicating a potential for synapse formation between R neurons within the eb. In contrast, projections to the suboesophageal ganglion, which show strong Syt–GFP expression, are devoid of DenMark, suggesting a different interpretation of the signals (Nicolaï et al, 2010).

  Building on these findings, we have reanalyzed our data with caution (as shown in Figure S3). In the SOG region, where we observed strong yellow signals, these were not limited to cell bodies but also extended to the middle region filled with neural processes. Upon close examination of the DenMark and syt.eGFP signals, we confirmed that these yellow signals are closely juxtaposed, suggesting the possibility of synapse formation between SIFaR24F06 neurons within the SOG. We emphasize that this interpretation is based on the original findings from the DenMark study. To provide clarity for general readers, we have added further explanations regarding the interpretation of these signals, as detailed below. We believe that our revised analysis and the additional explanations will help to clarify the potential implications of our findings, while also acknowledging the limitations and the need for further investigation.

"DenMark-enriched structures, localized within the SOG, are observed in close apposition to syt.eGFP signals, as indicated by the white-dashed circles (Fig. S3Fa). This spatial relationship suggests that SIFaR-expressing neurons, identified by GAL424F06 labeling, may form synapses with one another within the SOG. The colocalization of yellow signals resulting from the interaction between DenMark and syt.eGFP has been previously interpreted and validated by other researchers, supporting our observation (Nicolaï 2010,Kennedy 2018). In contrast to the yellow signals observed in the SOG, which are indicative of neural processes, the yellow signals detected in the ProNm appear to be associated with cell bodies rather than neural processes, as DenMark signals are often observed to leak out (as shown in Fig. S3Fb) (Nicolaï 2010,Kennedy 2018). Despite the presence of juxtaposed DenMark and syt.GFP signals in the ProNm, the interpretation of the yellow signals as potential synapses between SIFaR neurons remains an open question (indicated by the question mark in Fig. S3K).

• • Lines 320-322: "Neurons expressing Crz exhibit robust synaptic connections with SIFaR24F06 neurons located in the PRW region of the SOG in the brain (panels of Brain and SOG in Fig. 5A)". This is again misleading. They are not actually measuring synapses here, but instead looking at area of overlap between neuronal processes of Crz and SIFaR cells.*

  Answer: We sincerely appreciate the reviewer's critical feedback regarding our initial data interpretation. We acknowledge the important distinction that overlapping membrane markers do not provide a direct measure of synapse formation. In line with the reviewer's suggestion, we have revised the relevant sentence to more accurately reflect this understanding, as detailed below.

"Neurons expressing Crz were observed in close proximity to SIFaR24F06-expressing neurons within the PRW-SOG of the brain (panels of Brain and SOG in Fig. 6A)."

• * In Figs 3B and S4A, they are claiming that all neuronal processes within a given delineated brain area are synapses. The virtual fly brain and hemibrain resource have a way to actually identify synapses. This should be used in addition to the neuron skeleton. Otherwise, it is misleading to label these as synapses.*

  Answer: We are grateful for the reviewer's insightful comments that highlighted the potential for misleading information in our previous submission. Upon careful reexamination of the virtual fly brain model, we have made the necessary corrections and updated the figures in our revised manuscript (Figure 3B and S4B). This reanalysis has allowed us to further substantiate our findings, confirming that SIFa neurons indeed establish dense synaptic connections with multiple regions of the central brain.

• * Furthermore, measuring the area of GRASP signal is not the same as quantifying synapses. We don't know if synapse number changes (eg in lines 240-242).*

• Answer: We sincerely appreciate the reviewer's valuable suggestion regarding our quantification methods for assessing synaptic changes using GRASP signals. We acknowledge the reviewer's accurate observation that GRASP signals alone cannot provide an exact quantification of synapse number changes. In response to this feedback, we have employed the 'Particle analysis' function of ImageJ to infer the number of synapses from GRASP signals, clearly labeling them as 'number of particles' (as exemplified in Figures S4G and S4J). Additionally, we have compared the average size of each particle to enable a more precise comparison of synapse number changes (as shown in Figures S4H and S4K). While it is true that GRASP signals should not be directly equated with synapse counts, the quantification of GRASP signal intensity can still provide insights into the underlying synaptic connectivity, as described in the original GRASP paper (Feinberg et al, 2008a). Following this approach, previous studies have used signal intensity quantifications to draw conclusions about changes in synaptic specificity in various mutants. Since our methods for measuring GRASP intensity are consistent with the original techniques, we have updated our Y-axis labeling to reflect 'normalized GFP intensity (Norm. GFP Int.)', as exemplified in Figure 4. This change aims to provide a clearer and more accurate representation of our data.

Comment 6.* In general, the first part of the manuscript (implicating SIFaR in mating duration) is much stronger than the second part, which attempts to demonstrate that SIFa acts through Crz-expressing neurons to induce its effects. The proof that SIFa acts through Crz-expressing neurons to modify mating duration is tenuous. The most direct evidence of this, achieved via knockdown on Crz in SIFaR-expressing cells, is relegated to supplemental figures. The calcium response of the Crz neurons to SIFa neuron activation (Fig. 6) is more of a lack of a decrease that is observed in controls. Also, this is only done in the VNC. Why not look in the brain, because the authors previously stated a hypothesis that the "transmission of signals through SIFaR in Crz-expressing neurons is limited to the brain" (lines 381-382)?

Furthermore, the authors suggest that Crz acts on cells in the heart to regulate mating duration. It would be useful to add a discussion/speculation as to possible mechanisms for heart cells to regulate mating decisions. Is there evidence of CrzR in the heart? The SCope data presented in Fig. 7I-L and S7G-H is hard to read.*

• Answer: We sincerely appreciate the reviewer's constructive feedback on the section of our manuscript that discusses the role of the SIFa-SIFaR connection in regulating mating duration. We understand that the initial presentation may not have been sufficiently convincing. As we detailed in our previous biorXiv preprint (Wong et al, 2019), we conducted a comprehensive screen of numerous neuropeptides and their receptors that mediate SIFa signals through SIFaR and added those data in Supplementary Table S1 and S2. Among these, Crz was identified as a key neuropeptide in this pathway and is also well-documented for its role in mating duration (Tayler et al, 2012). Our data clearly demonstrate that Crz neurons are responsive to the activity of SIFa neurons, supporting the validity of this connection. Additionally, in another manuscript focusing on the input signals for SIFa (Kim et al*, 2024), we established that CrzR does not function in SIFa neurons, confirming the bidirectional nature of SIFa-to-Crz signaling.

  Inadvertently, we had relegated the Crz knockdown results to supplementary figures, under the assumption that our screening results regarding the relationship between SIFaR and neuropeptides were already well-covered (Wong et al, 2019). In light of the reviewer's comments, we have now relocated the Crz knockdown results, particularly those involving SIFaR-expressing cells, to the main figures (Figure 6F-G). We have also included a more detailed description of our previous screening results within the manuscript, as outlined below, to provide a more comprehensive understanding of our findings.

"Furthermore, the Crz peptide and Crz-expressing neurons have been characterized as pivotal relay signals in the SIFa-to-SIFaR pathway, which is essential for modulating interval timing behaviors (Wong 2019)."

   We greatly appreciate the reviewer's critical and constructive feedback regarding the detection of SIFa-to-Crz long-distance signaling, particularly their observation that this signaling is detectable from the brain to the VNC but not between brain regions. In response to the reviewer's suggestions, we have made the following adjustments to our manuscript:

1. We have relocated our SIFa-Crz GCaMP data pertaining to the VNC region to the Figures 6L-O) to maintain focus on the primary findings within the main text.
2. Our deeper analysis has led to the identification of two cells in the Super Intermediate Protocerebrum (SIP) regions that coexpress both Crz and SIFaR24F06, as well as OL cells (Figure 6D-E).
3. We have included GCaMP data from the brain region in the main figure to provide a comprehensive view of the signaling dynamics (Fig. 6P-R and Fig. S6N-P).
4. Upon examining the SIFa-to-Crz signaling through GCaMP calcium imaging, we observed that the calcium levels in Crz+/SIFaR+ SIP neurons consistently decreased upon SIFa activation (Figure 6P-R). In contrast, the calcium signals in Crz+/SIFaR+ OL neurons increased upon SIFa activation, similar to the pattern observed in Crz+ AG neurons in the VNC (Figure 6M-O and Figure S6N-P).

5. We have summarized these findings in Figure 6S and provided a detailed description of the results in the manuscript, as outlined below. "To elucidate the direct response of Crz neurons to the activity of SIFa neurons, we conducted live calcium (Ca2+) imaging in the Super Intermediate Protocererbrum (SIP), OL and AG region of the VNC, where Crz neurons are situated (Fig. 6D, Fig. S6M). Upon optogenetic stimulation of SIFa neurons, we observed a significant increase in the activity of Crz in OL and AG region (Fig. 6L-O, Fig. S6N-P), evidenced by a sustained elevation in intracellular Ca2+ levels that persisted in a high level before gradually declining to baseline levels, where the cells in top region of the SIP exhibit consistently drop down after stimulated the SIFa neurons (Fig. 6P-R). These calcium level changes were in contrast to the control group (without all-trans retinal, ATR) (Fig. 6L-R, Fig. S6N-P). These findings confirm that Crz neurons in OL and AG are activated in response to SIFa neuronal activity, but the activity of Crz neurons in SIP are inhibited by the activition of SIFa neuron, reinforcing their role as postsynaptic effectors in the neural circuitry governed by SIFa neurons. Moreover, these results provide empirical support for the hypothesis that SIFa-SIFaR/Crz-CrzR long-range neuropeptide relay underlies the neuronal activity-based measurement of interval timing."

   We are grateful for the reviewer's opportunity to elaborate on the intriguing findings concerning the expression of CrzR in the heart and its potential link to mating duration. In the context of traditional interval timing models (Meck et al, 2012; Matell, 2014; Buhusi & Meck, 2005), the role of a pacemaker in generating a temporal flow for measuring time is considered essential. The heart, being a well-known pacemaker organ in animals, provides a compelling framework for our discussion. In response to the reviewer's insightful comments, we have expanded upon our hypotheses in the DISCUSSION section, exploring the possible connections between cardiac function and the regulation of mating duration. Our reflections on this topic are detailed as follows:

"It has been reported that the interaction between the brain and the heart can influence time perception in humans (Khoshnoud et al, 2024). Heart rate is governed by intrinsic mechanisms, such as the muscle pacemaker, as well as extrinsic factors including neural and hormonal inputs (Andersen et al, 2015). Moreover, the pacemaker function is essential for the generation of interval timing capabilities (Meck et al, 2012; Matell, 2014; Buhusi & Meck, 2005), with the heart being recognized as the primary pacemaker organ within the animal body. Consequently, the CrzR in the fly heart may respond to the Crz signal sent by SIFaR+/Crz+ cells and modulate the heart rate, thereby impacting the perception of time in male flies."

   We appreciate the reviewer's interest in the expression of CrzR in the heart and its potential implications for our study. In response to the reviewer's comments, we have conducted a thorough examination of the fly SCope RNAseq dataset. Our analysis revealed that CrzR is indeed broadly expressed in heart tissue, particularly in areas where the Hand gene is also expressed. This significant finding has been incorporated into our manuscript and is depicted in Figure 8L. As illustrated in Figures 8I-L, which present the SCope tSNE plot for various cell types including neurons, glial cells, muscle systems, and heart, the heart tissue exhibits the most robust expression of CrzR. This observation suggests that the Hand-GAL4 mediated CrzR knockdown experiments may provide insights into the role of CrzR expression in the heart and its influence on the interval timing behavior of male fruit flies. We have expanded upon this interpretation in the relevant sections of our manuscript to ensure a clear and comprehensive understanding of our results.

• *

Comment 7. In several cases, the effects of being raised single are opposite the effects of sexual experience. For example, in Fig. 4T, calcium activity is increased in the AG following sexual experience, but decreased in flies raised singly. Likewise, Crz-neurons in the OL have increased CaLexA signal in singly-raised flies but reduced signals in flies with previous sexual experience. In some cases, manipulations selectively affect LMD or SMD. It would be useful to discuss these differences and consider the mechanistic implications of these differential changes, when they all result in decreased mating duration. This could help to clarify the big picture of the manuscript.

   __Answer:__ We sincerely appreciate the reviewer's insightful suggestions regarding the potential mechanistic underpinnings of how differential calcium activities may modulate LMD and SMD behaviors. In response to this valuable input, we have expanded our discussion to include a hypothesis on how neuropeptide relays could potentially induce context-dependent modulation of synaptic changes and calcium activities within distinct neuronal subsets. This addition aims to provide a more comprehensive understanding of the complex interactions at play, as detailed in the revised manuscript.

"Employing two distinct yet comparable models of interval timing behavior, LMD and SMD, we demonstrated that differential SIFa to SIFaR signaling is capable of modulating context-dependent behavioral responses. Synaptic strengths between SIFa and SIFaR neurons was notably enhanced in group-reared naive males. However, these synaptic strengths specifically diminished in the OL, CB, and AG when males were singly reared, with a particular decrease in the AG region when males were sexually experienced (Fig. 4A-J). Intriguingly, overall calcium signaling within SIFaR24F06 neurons was significantly reduced in group-reared naive males, yet these signals surged dramatically in the OL with social isolation and in the AG with sexual experience (Fig. 4K-T). These calcium signals, as reported by the transcriptional calcium reporter CaLexA, were corroborated by GCaMP live imaging in both the AG and OL regions (Fig. 6L-O and Fig. S6N-P), indicating a close association between elevated calcium levels and LMD and SMD behaviors. The modulation of context-dependent synaptic plasticity and calcium dynamics by the SIFa neuropeptide through a single SIFaR receptor raises the question of how a single receptor can elicit such diverse responses. Recent neuroscientific studies in Drosophila have shown that individual neurons can produce multiple neurotransmitters and that neuropeptides are often colocalized with small molecule neurotransmitters (Nässel 2018,Deng 2019,Croset 2018,Kondo 2020). Consistent with this, we have previously reported that SIFa neurons utilize a variety of neurotransmitters, including glutamate, dopamine, and tyramine (Kim 2024). Therefore, we propose that the SIFa-SIFaR-Crz-CrzR neuropeptidergic relay circuitry may interact with different neurotransmitters in distinct neuronal subpopulations to regulate context-dependent behaviors. Supporting this hypothesis, glutamate, known to function as an inhibitory neurotransmitter in the olfactory pathway of Drosophila (Liu 2013), may be one such candidate. We speculate that neuropeptide cotransmission could underlie the mechanisms facilitating these complex, context-dependent behavioral patterns. Further research is warranted to elucidate how such cotransmission contributes to the intricate behavioral repertoire of the fly."

Minor Comments: Comment 8. For CaLexA experiments (eg Fig 7A-D), signal intensity should be quantified in addition to area covered. Increased intensity would indicate greater calcium activity within a particular set of neurons.

• *Answer: We appreciate the reviewer's insightful comments and acknowledge the importance of using intensity measurements in our analysis of CaLexA signals. We concur that the intensity of these signals is indeed correlated with the area measurements, which is a critical factor to consider. In response to the reviewer's valuable suggestion, we have revised our approach and now present our data based on intensity measurements. These have been incorporated as a primary dataset in all our CaLexA results to provide a more accurate representation of our findings. Additionally, we have updated the labeling of our Y-axis to "Norm. GFP Int.", which stands for "normalized GFP intensity". This change ensures clarity and consistency in the presentation of our data, aligning with the reviewer's recommendations and enhancing the overall quality of our manuscript.

Comment 9. In Figure 5K: quantification of cell overlap is missing. In the text they state that there are ~100 neurons that co-express SIFaR24F06 and Crz. How was this determined? Is there a graph or numerical summary of this assertion?

    __Answer:__ We sincerely thank the reviewer for pointing out the oversight in our initial submission regarding the quantification data. In response to this valuable feedback, we have now included the quantification of neurons co-expressing SIFaR24F06 and Crz in the optic lobe (OL) within Figure 6E. This addition ensures that the figure is complete and provides the necessary numerical support for our observations.

Comment 10. In lines 709-711: "Our experience suggests that the relative mating duration differences between naïve and experienced condition and singly reared are always consistent; however, both absolute values and the magnitude of the difference in each strain can vary. So, we always include internal controls for each treatment as suggested by previous studies." I had trouble understanding this section of methods. What is done with the data from the internal controls?

   __Answer:__ We appreciate the reviewer's attention to the methodology of our study, particularly regarding the use of internal controls in our mating duration assays. As referenced in our cited work by Bretman et al. (2011) (Bretman *et al*, 2011), our internal control strategy involves a comparison of mating durations between males that have been presented with specific sensory cues and those that have not. This approach includes assessing both males that have been exposed to signals and those that have not, which serves as an internal control for each experimental setup. The purpose of this design is to isolate the effects of our manipulations from other potential confounding factors. In response to the reviewer's comments, we have provided a more detailed description of our mating duration assay in the Methods section. We have also expanded our explanation to clarify how this internal control mechanism ensures that any observed differences in mating duration are attributable to the experimental manipulations and not to extraneous variables. This additional information should provide a clearer understanding of our methodology and the rationale behind our experimental design.

Comment 11. Could the authors comment on why the brain GRASP signal is so different in Figures 3A and 4A? I realize that different versions of GRASP were used in these experiments, but I would expect broad agreement between the different approaches.

   __Answer: __We appreciate the reviewer’s insight. GRASP and t-GRASP are similar technologies that can clearly show the synaptic connection between neurons. GRASP technology was first generated and performed in *C. elegens* (Feinberg *et al*, 2008b). In 2018, the researchers developed a targeted GFP Reconstitution Across Synaptic Partners method, t-GRASP, which resulted in a strong preferential GRASP signal in synaptic regions.

   In our study, we utilized both techniques because of the limitations of the chromosomes where GAL4 and lexA lines located. We also found that during data processing, our method could clearly distinguish the changes in GRASP and t-GRASP signals across three different conditions (naïve, single, and exp.). Therefore, we do not have a particular preference for one technique over the other; both methods are applicable to our experiment.

   The genotype we used in Figure 3A is *SIFa2A-lexA, GAL424F06; lexAop-nSyb-spGFP1-10, UAS-CD4-spGFP11*, where the synaptic transmission occurs from *SIFa2A-lexA *to* GAL424F06*. In Figure 4A, the genotype we used is *GAL4SIFa.PT, lexASIFaR-2A; lexAop-2-post-t-GRASP, UAS-pre-t-GRASP*, where the synaptic transmission occurs from SIFa. PT to SIFaR-2A.

   In our back-to-back submission paper, “Peptidergic neurons with extensive branching orchestrate the internal states and energy balance of male *Drosophila melanogaster,*” (Kim *et al*, 2024) we identified that *SIFa2A* can label posterior-ventral SIFa neurons (SIFaVP), which can only project to ellipsoid body and fan-shaped body. Combining the GRASP technique, Figure 3A cannot show a strong signal as in Figure 4A. We’ve shown in Figure 1G that *SIFaR-2A *covers almost the whole CNS in *Drosophila*. Thus, the synaptic transmission from SIFa. PT (label 4 SIFa neurons) to SIFaR-2A shows a strong signal under the use of the t-GRASP technique. In this case, the GRASP signals in Figure 3A and Figure 4A are so different because of the usage of different GRASP techniques and different fly lines. We appreciate the reviewer's attention to the clarity of our presentation. In response to the comments, we have taken the opportunity to meticulously revise the figure legends to ensure that the differences are explicitly highlighted and easily understood by the readers.

__ __

Reviewer #2

Major concerns: Comment 1.* It is highly interesting that the duration of mating behavior is dependent on external and motivational factors. In fact, that provides an elegant way to study which neuronal mechanisms orchestrate these factors. However, it remains elusive why the authors link the differentially motivated durations of mating behavior to the psychological concept of interval timing. This distracts from the actually interesting neurobiology, and is not necessary to make the study interesting. *

* * Answer: We are grateful for the opportunity provided by the reviewer to elaborate on our rationale for utilizing the mating duration of male fruit flies as an exemplary genetic model for studying interval timing. At the outset, we would like to acknowledge that mating duration has gained recognition as a valuable genetic model for interval timing, as evidenced by the NIH-NIGMS R01 grant awarded to Michael Crickmore. This grant, which can be reviewed at the provided link (https://grantome.com/grant/NIH/R01-GM134222-01), underscores the significance of this model. Crickmore and colleagues have described in the grant's abstract that "mating duration in Drosophila offers a powerful system for exploring changes in motivation over time as behavioral goals are achieved," and it has the potential to provide "the first mechanistic description of a neuronal interval timing system."

   In light of this, we have incorporated our rationale into the INTRODUCTION section of our manuscript, as detailed below. We believe that our argumentation, supported by the grant's emphasis on the topic, will not only address the reviewer's concerns but also demonstrate to the broader scientific community the significance of the fruit fly's mating duration as a model for interval timing. This concept has been a cornerstone in the historical development of neuroscientific understanding of time perception. We hope that our expanded discussion will effectively convey the potential of the fruit fly mating duration as a genetic model to offer profound insights into the neural mechanisms underlying interval timing, a concept of enduring importance in the field of neuroscience.

"The dimension of time is the fundamental basis for an animal's survival. Being able to estimate and control the time between events is crucial for all everyday activities (RICHELLE & LEJEUNE, 1980). The perception of time in the seconds-to-hours range, referred to as ‘interval timing’, is involved in foraging, decision making, and learning via activation of cortico-striatal circuits in mammals (Golombek et al, 2014). Interval timing requires entirely different neural mechanisms from millisecond or circadian timing (Meck et al, 2012; Merchant et al, 2012; Buhusi & Meck, 2005). There is abundant psychological research on time perception because it is a universal cognitive dimension of experience and behavioral plasticity. Despite decades of research, the genetic and neural substrates of temporal information processing have not been well established except for the molecular bases of circadian timing (Buhusi et al, 2009; Tucci et al, 2014). Thus, a simple genetic model system to study interval timing is required. Considering that the mating duration in fruit flies, which averages approximately 20 minutes, is well within the range addressed by interval timing mechanisms, this behavioral parameter provides a relevant context for examining the neural circuits that modulate the Drosophila's perception of time intervals. Such an investigation necessitates an understanding of the extensive neural and behavioral plasticity underlying interval timing (Thornquist et al, 2020; Gautham et al, 2024; Crickmore & Vosshall, 2013)."

Comment 2.* In figure 4 A and 4K, fluorescence microscopy images of brains and ventral nerve chords are shown, one illustrating GRASP experiments, and one showing CaLexA experiments. The extreme difference between the differentially treated flies (bright fluorescence versus almost no fluorescence) is - in its drastic form- surprising. Online access to the original confocal microscopy images (raw data) might help to convince the reader that these illustrations do not reflect the most drastic "representative" examples out of a series of brain stainings. *

* * Answer: We sincerely appreciate the reviewer's thoughtful suggestion to enhance the accessibility of our microscopy images for readers who may be interested. In response to this valuable feedback, we have compiled all of our quantified image files into zip format and included them as Supplementary Information 2 and 3. We believe that this additional material will be beneficial for readers seeking a more in-depth view of our data.

Comment 3. In particular for behavioral experiments, genetic controls should always be conducted. That is, both the heterozygous Gal4-line as well as the heterozygous UAS-line should be used as controls. This is laborious, but important.

   __Answer:__ We sincerely appreciate the reviewer's critical feedback regarding the genetic controls in our study. We acknowledge the importance of this aspect and wish to clarify that we have indeed conducted a substantial number of genetic control experiments for both LMD and SMD behaviors. It is worth noting that much of this data has been previously published in other works. Recognizing the interest from another reviewer on the same topic, we have chosen to reiterate our response here for clarity and convenience. Our comprehensive approach to genetic controls ensures the robustness of our findings, and we believe that the published data further substantiates the reliability of our experimental procedures.

   We sincerely thank the reviewer for insightful comments regarding the absence of traditional genetic controls in our study of LMD and SMD behaviors. We acknowledge the importance of such controls and wish to clarify our rationale for not including them in the current investigation. The primary reason for not incorporating all genetic control lines is that we have previously assessed the LMD and SMD behaviors of GAL4/+ and UAS/+ strains in our earlier studies. Our past experiences have consistently shown that 100% of the genetic control flies for both GAL4 and UAS exhibit normal LMD and SMD behaviors. Given these findings, we deemed the inclusion of additional genetic controls to be non-essential for the present study, particularly in the context of extensive screening efforts. However, in accordance with the reviewer's recommendation, we conducted genetic validation experiments on novel genetic crosses, including SIFaR-RNAi/+, CrzR-RNAi/+, and GAL4NP5270/+, and incorporated the results in the supplementary figures (Supplementary information 1). We have made the necessary modifications and additions to the manuscript as below.

"Given those genetic controls, as evidenced by consistent exhibition of normal LMD and SMD behaviors (Supplemental information. 1), the observed reduction in SIFaR expression, driven by elavc155, is deemed sufficient to induce significant disruptions in LMD and SMD behaviors."

We understand the value of providing a clear rationale for our methodology choices. To this end, we have added a detailed explanation in the "MATERIALS AND METHODS" section and the figure legends of Figure 1. This clarification aims to assist readers in understanding our decision to omit traditional controls, as outlined below.

"__Mating Duration Assays for Successful Copulation__The mating duration assay in this study has been reported (Kim et al. 2012; Kim et al. 2013; Lee et al. 2023). To enhance the efficiency of the mating duration assay, we utilized the Df(1)Exel6234 (DF here after) genetic modified fly line in this study, which harbors a deletion of a specific genomic region that includes the sex peptide receptor (SPR) (Parks et al. 2004; Yapici et al. 2008). Previous studies have demonstrated that virgin females of this line exhibit increased receptivity to males (Yapici et al. 2008). We conducted a comparative analysis between the virgin females of this line and the CS virgin females and found that both groups induced SMD. Consequently, we have elected to employ virgin females from this modified line in all subsequent studies. For group reared (naïve) males, 40 males from the same strain were placed into a vial with food for 5 days. For single reared males, males of the same strain were collected individually and placed into vials with food for 5 days. For experienced males, 40 males from the same strain were placed into a vial with food for 4 days then 80 DF virgin females were introduced into vials for last 1 day before assay. 40 DF virgin females were collected from bottles and placed into a vial for 5 days. These females provide both sexually experienced partners and mating partners for mating duration assays. At the fifth day after eclosion, males of the appropriate strain and DF virgin females were mildly anaesthetized by CO2. After placing a single female into the mating chamber, we inserted a transparent film then placed a single male to the other side of the film in each chamber. After allowing for 1 h of recovery in the mating chamber in 25℃ incubators, we removed the transparent film and recorded the mating activities. Only those males that succeeded to mate within 1 h were included for analyses. Initiation and completion of copulation were recorded with an accuracy of 10 sec, and total mating duration was calculated for each couple. Genetic controls with GAL4/+ or UAS/+ lines were omitted from supplementary figures, as prior data confirm their consistent exhibition of normal LMD and SMD behaviors (Kim et al. 2012; Kim et al. 2013; Lee et al. 2023; Huang et al. 2024; Zhang et al. 2024). Hence, genetic controls for LMD and SMD behaviors were incorporated exclusively when assessing novel fly strains that had not previously been examined. In essence, internal controls were predominantly employed in the experiments, as LMD and SMD behaviors exhibit enhanced statistical significance when internally controlled. Within the LMD assay, both group and single conditions function reciprocally as internal controls. A significant distinction between the naïve and single conditions implies that the experimental manipulation does not affect LMD. Conversely, the lack of a significant discrepancy suggests that the manipulation does influence LMD. In the context of SMD experiments, the naïve condition (equivalent to the group condition in the LMD assay) and sexually experienced males act as mutual internal controls for one another. A statistically significant divergence between naïve and experienced males indicates that the experimental procedure does not alter SMD. Conversely, the absence of a statistically significant difference suggests that the manipulation does impact SMD. Hence, we incorporated supplementary genetic control experiments solely if they deemed indispensable for testing. All assays were performed from noon to 4 PM. We conducted blinded studies for every test."

Minor comments: Comment 4.* Line 75: word missing ("...including FEEDING-RELATED BEHAVIOR, courtship, ..."). *

   __Answer:__ We appreciate your vigilance in identifying this error. We have made the necessary correction to ensure the accuracy of our manuscript.*

*

Comment 5.* Line 120: word missing ("SIFaR expression in adult neurons BUT not glia..."). *

   __Answer:__ We appreciate your careful review and attention to detail. Thank you for bringing this to our notice. We have made the necessary corrections to address the error.*

*

Comment 6.* I find the figures often to be quite overloaded, and anatomical details often very small (e.g., figure 7A). *

* * Answer: We appreciate the constructive critique on the layout of our data presentation. Following your insightful recommendation, we have revised the manuscript to enhance clarity. Specifically, we have resized the diagram to be more compact and have also increased the spacing between the panels for better readability.

__ __

Reviewer #3

Major Comments Comment 1.* Are the key conclusions convincing? The key conclusions are intriguing but require more robust data to be fully convincing. While the study presents compelling evidence for the involvement of SIFa and SIFaR in mating behaviors, additional experiments are needed to firmly establish the proposed mechanisms. *

* * Answer: We are deeply grateful for the insightful and constructive feedback provided by the reviewer on the SIFa-to-SIFaR signaling pathway. We are particularly encouraged by the reviewer's agreement with our findings that support the role of SIFa and SIFaR in regulating mating duration. We concur with the reviewer's suggestion that additional experiments and mechanistic insights are essential to substantiate our conclusions. To this end, we have conducted and included several new experiments, particularly GCaMP data, in the main figures (Figure 6 and S6). Our focus has been intensified on the SIFa-to-Crz signaling, given Crz's established role in controlling mating duration behavior. Below is a summary of the additional experiments we have incorporated:

1. We have repositioned the SIFa-Crz GCaMP data related to the VNC to Figures 6L-O to ensure that the main text highlights our primary findings.
2. Our more detailed analysis has identified two cells in the Super Intermediate Protocerebrum (SIP) regions that co-express Crz and SIFaR24F06, along with OL cells (Figure 6D-E).
3. To provide a complete view of the signaling dynamics, we have included GCaMP data from the brain region in the main figure (Figure 6P-R and Supplementary Figure S6N-P).
4. Through GCaMP calcium imaging to assess SIFa-to-Crz signaling, we found that calcium levels in Crz+/SIFaR+ SIP neurons consistently decreased with SIFa activation (Figure 6P-R). Conversely, calcium signals in Crz+/SIFaR+ OL neurons increased with SIFa activation, mirroring the pattern seen in Crz+ AG neurons in the VNC (Figure 6M-O and Supplementary Figure S6N-P).

5. A synthesis of these results is presented in Figure 6S, and we have elaborated on these findings in the manuscript with a detailed description, as detailed below. "To elucidate the direct response of Crz neurons to the activity of SIFa neurons, we conducted live calcium (Ca2+) imaging in the Super Intermediate Protocererbrum (SIP), OL and AG region of the VNC, where Crz neurons are situated (Fig. 6D, Fig. S6M). Upon optogenetic stimulation of SIFa neurons, we observed a significant increase in the activity of Crz in OL and AG region (Fig. 6L-O, Fig. S6N-P), evidenced by a sustained elevation in intracellular Ca2+ levels that persisted in a high level before gradually declining to baseline levels, where the cells in top region of the SIP exhibit consistently drop down after stimulated the SIFa neurons (Fig. 6P-R). These calcium level changes were in contrast to the control group (without all-trans retinal, ATR) (Fig. 6L-R, Fig. S6N-P). These findings confirm that Crz neurons in OL and AG are activated in response to SIFa neuronal activity, but the activity of Crz neurons in SIP are inhibited by the activition of SIFa neuron, reinforcing their role as postsynaptic effectors in the neural circuitry governed by SIFa neurons. Moreover, these results provide empirical support for the hypothesis that SIFa-SIFaR/Crz-CrzR long-range neuropeptide relay underlies the neuronal activity-based measurement of interval timing."

   We are truly grateful for the reviewer's perceptive recommendations concerning the possible mechanisms of LMD and SMD behaviors. In light of this constructive feedback, we have enhanced our discussion to encompass a theoretical framework on the potential role of neuropeptide relays in mediating context-dependent adjustments of synaptic plasticity and calcium signaling within specific neuronal populations. This supplementary perspective is designed to elucidate the intricate dynamics involved, as further elaborated in the updated manuscript.

"Employing two distinct yet comparable models of interval timing behavior, LMD and SMD, we demonstrated that differential SIFa to SIFaR signaling is capable of modulating context-dependent behavioral responses. Synaptic strengths between SIFa and SIFaR neurons was notably enhanced in group-reared naive males. However, these synaptic strengths specifically diminished in the OL, CB, and AG when males were singly reared, with a particular decrease in the AG region when males were sexually experienced (Fig. 4A-J). Intriguingly, overall calcium signaling within SIFaR24F06 neurons was significantly reduced in group-reared naive males, yet these signals surged dramatically in the OL with social isolation and in the AG with sexual experience (Fig. 4K-T). These calcium signals, as reported by the transcriptional calcium reporter CaLexA, were corroborated by GCaMP live imaging in both the AG and OL regions (Fig. 6L-O and Fig. S6N-P), indicating a close association between elevated calcium levels and LMD and SMD behaviors. The modulation of context-dependent synaptic plasticity and calcium dynamics by the SIFa neuropeptide through a single SIFaR receptor raises the question of how a single receptor can elicit such diverse responses. Recent neuroscientific studies in Drosophila have shown that individual neurons can produce multiple neurotransmitters and that neuropeptides are often colocalized with small molecule neurotransmitters (Nässel 2018,Deng 2019,Croset 2018,Kondo 2020). Consistent with this, we have previously reported that SIFa neurons utilize a variety of neurotransmitters, including glutamate, dopamine, and tyramine (Kim 2024). Therefore, we propose that the SIFa-SIFaR-Crz-CrzR neuropeptidergic relay circuitry may interact with different neurotransmitters in distinct neuronal subpopulations to regulate context-dependent behaviors. Supporting this hypothesis, glutamate, known to function as an inhibitory neurotransmitter in the olfactory pathway of Drosophila (Liu 2013), may be one such candidate. We speculate that neuropeptide cotransmission could underlie the mechanisms facilitating these complex, context-dependent behavioral patterns. Further research is warranted to elucidate how such cotransmission contributes to the intricate behavioral repertoire of the fly."

• *

Comment 2. Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? The authors should qualify certain claims as preliminary or speculative. Specifically, the proposed SIFa-SIFaR/Crz-CrzR neuropeptide relay pathway is only investigated via imaging approach. More experiments using behavioral tests are needed to confirm that Crz relays the SIFa signaling pathway. For example, Crz-Gal4>UAS-SIFaR RNAi should be done to show that SIFaR+ Crz+ cells are necessary for LMD and SMD.

   __Answer:__ We are grateful for the reviewer's constructive suggestion regarding the need to provide additional behavioral assays using RNAi knockdown to substantiate the SIFa-SIFaR/Crz-CrzR neuropeptide relay. Following the reviewer's advice, we have conducted experiments involving SIFaR24F06/Crz-RNAi and Crz-GAL4/SIFaR-RNAi. The outcomes of these experiments have been detailed and are now presented in a clear and comprehensive manner.

   To further aid in the understanding of our results, we have also included a summary diagram in Figure 6S, which illustrates the key findings from these assays. This visual representation is intended to provide a concise overview of the data and to highlight the significance of the SIFa-SIFaR/Crz-CrzR neuropeptide relay in the context of our study.

Comment 3.* Would additional experiments be essential to support the claims of the paper? Yes, additional experiments are essential. Detailed molecular and imaging studies are needed to support claims about synaptic reorganization. For example: ○ More controls are needed for RNAi and Gal80ts experiments, such as Gal4-only control, RNAi-only control, etc. *

   __Answer:__ We sincerely appreciate the reviewer's critical feedback regarding the genetic controls in our study. We acknowledge the importance of this aspect and wish to clarify that we have indeed conducted a substantial number of genetic control experiments for both LMD and SMD behaviors. It is worth noting that much of this data has been previously published in other works. Recognizing the interest from another reviewer on the same topic, we have chosen to reiterate our response here for clarity and convenience. Our comprehensive approach to genetic controls ensures the robustness of our findings, and we believe that the published data further substantiates the reliability of our experimental procedures.

   We sincerely thank the reviewer for insightful comments regarding the absence of traditional genetic controls in our study of LMD and SMD behaviors. We acknowledge the importance of such controls and wish to clarify our rationale for not including them in the current investigation. The primary reason for not incorporating all genetic control lines is that we have previously assessed the LMD and SMD behaviors of GAL4/+ and UAS/+ strains in our earlier studies. Our past experiences have consistently shown that 100% of the genetic control flies for both GAL4 and UAS exhibit normal LMD and SMD behaviors. Given these findings, we deemed the inclusion of additional genetic controls to be non-essential for the present study, particularly in the context of extensive screening efforts. However, in accordance with the reviewer's recommendation, we conducted genetic validation experiments on novel genetic crosses, including SIFaR-RNAi/+, CrzR-RNAi/+, and GAL4NP5270/+, and incorporated the results in the supplementary figures (Supplementary information 1). We have made the necessary modifications and additions to the manuscript as below.

"Given those genetic controls, as evidenced by consistent exhibition of normal LMD and SMD behaviors (Supplemental information 1), the observed reduction in SIFaR expression, driven by elavc155, is deemed sufficient to induce significant disruptions in LMD and SMD behaviors."

We understand the value of providing a clear rationale for our methodology choices. To this end, we have added a detailed explanation in the "MATERIALS AND METHODS" section and the figure legends of Figure 1. This clarification aims to assist readers in understanding our decision to omit traditional controls, as outlined below.

"__Mating Duration Assays for Successful Copulation__The mating duration assay in this study has been reported (Kim et al. 2012; Kim et al. 2013; Lee et al. 2023). To enhance the efficiency of the mating duration assay, we utilized the Df(1)Exel6234 (DF here after) genetic modified fly line in this study, which harbors a deletion of a specific genomic region that includes the sex peptide receptor (SPR) (Parks et al. 2004; Yapici et al. 2008). Previous studies have demonstrated that virgin females of this line exhibit increased receptivity to males (Yapici et al. 2008). We conducted a comparative analysis between the virgin females of this line and the CS virgin females and found that both groups induced SMD. Consequently, we have elected to employ virgin females from this modified line in all subsequent studies. For group reared (naïve) males, 40 males from the same strain were placed into a vial with food for 5 days. For single reared males, males of the same strain were collected individually and placed into vials with food for 5 days. For experienced males, 40 males from the same strain were placed into a vial with food for 4 days then 80 DF virgin females were introduced into vials for last 1 day before assay. 40 DF virgin females were collected from bottles and placed into a vial for 5 days. These females provide both sexually experienced partners and mating partners for mating duration assays. At the fifth day after eclosion, males of the appropriate strain and DF virgin females were mildly anaesthetized by CO2. After placing a single female into the mating chamber, we inserted a transparent film then placed a single male to the other side of the film in each chamber. After allowing for 1 h of recovery in the mating chamber in 25℃ incubators, we removed the transparent film and recorded the mating activities. Only those males that succeeded to mate within 1 h were included for analyses. Initiation and completion of copulation were recorded with an accuracy of 10 sec, and total mating duration was calculated for each couple. Genetic controls with GAL4/+ or UAS/+ lines were omitted from supplementary figures, as prior data confirm their consistent exhibition of normal LMD and SMD behaviors (Kim et al. 2012; Kim et al. 2013; Lee et al. 2023; Huang et al. 2024; Zhang et al. 2024). Hence, genetic controls for LMD and SMD behaviors were incorporated exclusively when assessing novel fly strains that had not previously been examined. In essence, internal controls were predominantly employed in the experiments, as LMD and SMD behaviors exhibit enhanced statistical significance when internally controlled. Within the LMD assay, both group and single conditions function reciprocally as internal controls. A significant distinction between the naïve and single conditions implies that the experimental manipulation does not affect LMD. Conversely, the lack of a significant discrepancy suggests that the manipulation does influence LMD. In the context of SMD experiments, the naïve condition (equivalent to the group condition in the LMD assay) and sexually experienced males act as mutual internal controls for one another. A statistically significant divergence between naïve and experienced males indicates that the experimental procedure does not alter SMD. Conversely, the absence of a statistically significant difference suggests that the manipulation does impact SMD. Hence, we incorporated supplementary genetic control experiments solely if they deemed indispensable for testing. All assays were performed from noon to 4 PM. We conducted blinded studies for every test."

*○ Using synaptic markers and high-resolution imaging to observe synaptic changes directly. *

   __Answer:__ We sincerely appreciate the reviewer's constructive suggestion to provide high-resolution imaging for a more direct observation of synaptic changes. While we have already included high-resolution imaging data showcasing postsynaptic and presynaptic alterations using Denmark and syt.eGFP (Figure S3), GRASP (Figure 3A-D), and tGRASP (Figure 4A-J), we recognize the value of further elucidation. Consequently, we have conducted additional experiments to examine the presynaptic changes in SIFaR24F06 neurons under varying social contexts, as presented in Figure 5A-G. We are confident that the comprehensive dataset we have now provided, which includes these new findings, will not only address the reviewer's concerns but also effectively convey to the readers the dynamic and critical nature of SIFa-SIFaR synaptic changes in modulating interval timing behaviors.

*○ Electrophysiological recordings from neurons expressing SIFa and SIFaR to analyze their functional connectivity and activity patterns. *

   __Answer:__ We sincerely appreciate the reviewer's constructive suggestions regarding the inclusion of electrophysiological recordings from neurons expressing SIFa and SIFaR to analyze functional connectivity and activity patterns. In response to this valuable feedback, we have conducted *in vivo* calcium imaging using the GCaMP indicator. The results have been incorporated into our manuscript, demonstrating SIFa-SIFaR connectivity and alterations in activity patterns (Figure 5H-L), as well as SIFa-Crz connectivity and changes in activity patterns (Figure 6 and Figure S6). We are confident that these additional data provide compelling evidence supporting the notion that the SIFa-SIFaR/Crz-CrzR neuropeptide relay circuits are robustly interconnected and exhibit activity changes in concert with the observed neuronal modifications.

• *

Comment 4.* Are the suggested experiments realistic in terms of time and resources? The suggested experiments are realistic but will require considerable time and resources. Detailed molecular interaction studies, imaging synaptic plasticity, and electrophysiological recordings could take several months to over a year, depending on the complexity and availability of necessary equipment and expertise. The cost would be moderate to high, involving expenses for reagents, imaging equipment, and animal husbandry for maintaining Drosophila stocks. *

* * Answer: We are grateful for the reviewers' understanding and support for our additional analysis in the revision experiments. While we have already conducted a multitude of experiments pertinent to this manuscript, we are well-positioned to provide a comprehensive revision of the data within a relatively short timeframe.

Comment 5. Are the data and the methods presented in such a way that they can be reproduced? The methods are generally described in detail, allowing for potential reproducibility. However, more precise documentation of certain experimental conditions, such as the timing and conditions of RNAi induction and temperature controls, is necessary. The methods about imaging analysis are too detailed. The exact steps about how to use ImageJ should be removed.

* * Answer: We sincerely appreciate the reviewer's meticulous comments regarding the omission of certain methodological details in our manuscript. In response, we have now included a detailed description of the temperature control procedures for conditional RNAi induction in the "Fly Stocks and Husbandry" section, as detailed below.

"For temperature-controlled experiments, including those utilizing the temperature-sensitive tub-GAL80ts driver, the flies were initially crossed and maintained at a constant temperature of 22℃ within an incubator. The temperature shift was initiated post-eclosion. Once the flies had emerged, they were transferred to an incubator set at an elevated temperature of 29℃ for a defined period, after which the experimental protocols were carried out. Wild-type flies were Canton-S (CS)."

   We appreciate the reviewer's guidance on refining our manuscript. In response to the suggestion, we have streamlined the image analysis methods section, removing excessive details to present the information in a more concise and clear manner as below.

"Quantitative analysis of fluorescence intensity

To ascertain calcium levels and synaptic intensity from microscopic images, we dissected and imaged five-day-old flies of various social conditions and genotypes under uniform conditions. The GFP signal in the brains and VNCs was amplified through immunostaining with chicken anti-GFP primary antibody. Image analysis was conducted using ImageJ software. For the quantification of fluorescence intensities, an investigator, blinded to the fly's genotype, thresholded the sum of all pixel intensities within a sub-stack to optimize the signal-to-noise ratio, following established methods (Feinberg 2008). The total fluorescent area or region of interest (ROI) was then quantified using ImageJ, as previously reported. For CaLexA signal quantification, we adhered to protocols detailed by Kayser et al. (Kayser et al, 2014), which involve measuring the ROI's GFP-labeled area by summing pixel values across the image stack. This method assumes that changes in the GFP-labeled area are indicative of alterations in the CaLexA signal, reflecting synaptic activity. ROI intensities were background-corrected by measuring and subtracting the fluorescent intensity from a non-specific adjacent area, as per Kayser et al. (Kayser et al, 2014). For the analysis of GRASP or tGRASP signals, a sub-stack encompassing all synaptic puncta was thresholded by a genotype-blinded investigator to achieve the optimal signal-to-noise ratio. The fluorescence area or ROI for each region was quantified using ImageJ, employing a similar approach to that used for CaLexA quantification (Feinberg 2008)."

Comment 6. Are the experiments adequately replicated and statistical analysis adequate? Most figures in the manuscript need to be re-plotted. The right y-axis "Difference between means" is not necessary, if not confusing. The image panels are too small to see, while the quantification of overlapping cells are unnecessarily large. The figures are too crowded with labels and texts, which makes it extremely difficult to comprehend the data.

   __Answer:__ We appreciate the reviewer's suggestion to refine our figures, and we have indeed reformatted them to provide clearer presentation and improved readability. Regarding the removal of dot blot membranes (DBMs), we have given this considerable thought. While we understand the recommendation, we have chosen to retain the DBMs in our manuscript. Our decision is based on the fact that our analysis encompasses not only traditional t-tests but also incorporates estimation statistics, which have been demonstrated to be effective for biological data analysis (Claridge-Chang & Assam, 2016). The inclusion of DBMs is essential for the accurate interpretation of these estimation statistics, ensuring a comprehensive representation of our findings.

Minor Comments Comment 7. Specific experimental issues that are easily addressable. Clarify the timing of RNAi induction and provide more detailed figure legends for better understanding and reproducibility.

   __Answer:__ We sincerely appreciate the reviewer's suggestion aimed at enhancing our manuscript. As previously addressed in our response to __*Comment 5*__, we have incorporated additional details regarding the timing of RNAi induction within the Methods section. Furthermore, we have expanded upon the figure legends to provide a clearer understanding of our findings, ensuring that the content is accessible to a broader readership.

* Comment 8. Are prior studies referenced appropriately? Yes. *

__ Answer:__ We are grateful for the reviewer's acknowledgment that our references have been appropriately included and integrated into the manuscript.

* Comment 9. Are the text and figures clear and accurate? The text is generally clear, but the figures need re-work. See comment above. *

• *Answer: We appreciate the feedback from the reviewers regarding the clarity of our figures. In response to other reviewers' concerns about the figures appearing too crowded, we have carefully revised the layout of all figures to ensure they are more spacious and aesthetically improved for better readability and visual appeal.

* Comment 10. Suggestions to improve the presentation of data and conclusions. Use smaller fonts in the bar plots and make the plots smaller. Enlarge the imaging panels and let the pictures tell the story. *

* * Answer: We sincerely appreciate the reviewer's constructive suggestion. In response, we have revised the figures by enlarging the images and adjusting the font sizes in the bar plots to enhance readability and clarity.

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Kim WJ, Jan LY & Jan YN (2012) Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals. Nat Neurosci 15: 876–883

Kim WJ, Jan LY & Jan YN (2013) A PDF/NPF Neuropeptide Signaling Circuitry of Male Drosophila melanogaster Controls Rival-Induced Prolonged Mating. Neuron 80: 1190–1205

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Matell MS (2014) Neurobiology of Interval Timing. Adv Exp Med Biol: 209–234

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Reviewer #3 (Public review):

Summary:

Schmidt et al., aimed to provide an extremely comprehensive demonstration of the influence cardiac electromagnetic fields have on the relationship between age and the aperiodic slope measured from electroencephalographic (EEG) and magnetoencephalographic (MEG) data.

Strengths:

Schmidt et al., used a multiverse approach to show that the cardiac influence on this relationship is considerable, by testing a wide range of different analysis parameters (including extensive testing of different frequency ranges assessed to determine the aperiodic fit), algorithms (including different artifact reduction approaches and different aperiodic fitting algorithms), and multiple large datasets to provide conclusions that are robust to the vast majority of potential experimental variations.

The study showed that across these different analytical variations, the cardiac contribution to aperiodic activity measured using EEG and MEG is considerable, and likely influences the relationship between aperiodic activity and age to a greater extent than the influence of neural activity.

Their findings have significant implications for all future research that aims to assess aperiodic neural activity, suggesting control for the influence of cardiac fields is essential.

Weaknesses:

Figure 4I: The regressions explained here seem to contain a very large number of potential predictors. Based on the way it is currently written, I'm assuming it includes all sensors for both the ECG component and ECG rejected conditions?

I'm not sure about the logic of taking a complete signal, decomposing it with ICA to separate out the ECG and non-ECG signals, then including these latent contributions to the full signal back into the same regression model. It seems that there could be some circularity or redundancy in doing so. Can the authors provide a justification for why this is a valid approach?

I'm not sure whether there is good evidence or rationale to support the statement in the discussion that the presence of the ECG signal in reference electrodes makes it more difficult to isolate independent ECG components. The ICA algorithm will still function to detect common voltage shifts from the ECG as statistically independent from other voltage shifts, even if they're spread across all electrodes due to the referencing montage. I would suggest there are other reasons why the ICA might lead to imperfect separation of the ECG component (assumption of the same number of source components as sensors, non-Gaussian assumption, assumption of independence of source activities).

The inclusion of only 32 channels in the EEG data might also have reduced the performance of ICA, increasing the chances of imperfect component separation and the mixing of cardiac artifacts into the neural components, whereas the higher number of sensors in the MEG data would enable better component separation. This could explain the difference between EEG and MEG in the ability to clean the ECG artifact (and perhaps higher-density EEG recordings would not show the same issue).

In addition to the inability to effectively clean the ECG artifact from EEG data, ICA and other component subtraction methods have also all been shown to distort neural activity in periods that aren't affected by the artifact due to the ubiquitous issue of imperfect component separation (https://doi.org/10.1101/2024.06.06.597688). As such, component subtraction-based (as well as regression-based) removal of the cardiac artifact might also distort the neural contributions to the aperiodic signal, so even methods to adequately address the cardiac artifact might not solve the problem explained in the study. This poses an additional potential confound to the "M/EEG without ECG" conditions.

Literature Analysis, Page 23: was there a method applied to address studies that report reducing artifacts in general, but are not specific to a single type of artifact? For example, there are automated methods for cleaning EEG data that use ICLabel (a machine learning algorithm) to delete "artifact" components. Within these studies, the cardiac artifact will not be mentioned specifically, but is included under "artifacts".

Statistical inferences, page 23: as far as I can tell, no methods to control for multiple comparisons were implemented. Many of the statistical comparisons were not independent (or even overlapped with similar analyses in the full analysis space to a large extent), so I wouldn't expect strong multiple comparison controls. But addressing this point to some extent would be useful (or clarifying how it has already been addressed if I've missed something).

Methods:

Applying ICA components from 1Hz high pass filtered data back to the 0.1Hz filtered data leads to worse artifact cleaning performance, as the contribution of the artifact in the 0.1Hz to 1Hz frequency band is not addressed (see Bailey, N. W., Hill, A. T., Biabani, M., Murphy, O. W., Rogasch, N. C., McQueen, B., ... & Fitzgerald, P. B. (2023). RELAX part 2: A fully automated EEG data cleaning algorithm that is applicable to Event-Related-Potentials. Clinical Neurophysiology, result reported in the supplementary materials). This might explain some of the lower frequency slope results (which include a lower frequency limit <1Hz) in the EEG data - the EEG cleaning method is just not addressing the cardiac artifact in that frequency range (although it certainly wouldn't explain all of the results).

It looks like no methods were implemented to address muscle artifacts. These can affect the slope of EEG activity at higher frequencies. Perhaps the Riemannian Potato addressed these artifacts, but I suspect it wouldn't eliminate all muscle activity. As such, I would be concerned that remaining muscle artifacts affected some of the results, particularly those that included high frequency ranges in the aperiodic estimate. Perhaps if muscle activity were left in the EEG data, it could have disrupted the ability to detect a relationship between age and 1/f slope in a way that didn't disrupt the same relationship in the cardiac data (although I suspect it wouldn't reverse the overall conclusions given the number of converging results including in lower frequency bands). Is there a quick validity analysis the authors can implement to confirm muscle artifacts haven't negatively affected their results? I note that an analysis of head movement in the MEG is provided on page 32, but it would be more robust to show that removing ICA components reflecting muscle doesn't change the results. The results/conclusions of the following study might be useful for objectively detecting probable muscle artifact components: Fitzgibbon, S. P., DeLosAngeles, D., Lewis, T. W., Powers, D. M. W., Grummett, T. S., Whitham, E. M., ... & Pope, K. J. (2016). Automatic determination of EMG-contaminated components and validation of independent component analysis using EEG during pharmacologic paralysis. Clinical neurophysiology, 127(3), 1781-1793.

Me encanta esta imagen, al ver como van todos bien vestidos y atentos a salir a distraerse em ves de estar en casa deprimidos. Se le ve calma en la cara del muchacho, no como las otras imágenes. Y me gusta como la imagen se mira "retro". Se pudiera colgar en un salon o una Barberia.

The only speech that was deemed orthodox for women to use, was talking into thin air. By doing that it inherently silenced their voice while giving them the illusion of actually being heard. On the contrary, black male preachers were to be heard and remembered.

Rabid attacks were almost non-existent, yet the fear surrounding the wolf was as strong as ever.

Artigo 65º - Habitação e Urbanismo: "4. O Estado, as regiões autónomas e as autarquias locais definem as regras de ocupação, uso e transformação dos solos urbanos, designadamente através de instrumentos de planeamento, no quadro das leis respeitantes ao ordenamento do território e ao urbanismo, e procedem às expropriações dos solos que se revelem necessárias à satisfação de fins de utilidade pública urbanística.” Artigo 66º - Ambiente e qualidade de vida: "1. Todos têm direito a um ambiente de vida humano, sadio e ecologicamente equilibrado e o dever de o defender. 2. Para assegurar o direito ao ambiente, no quadro de um desenvolvimento sustentável, incumbe ao Estado, por meio de organismos próprios e com o envolvimento e a participação dos cidadãos: a) Prevenir e controlar a poluição e os seus efeitos e as formas prejudiciais de erosão; b) Ordenar e promover o ordenamento do território, tendo em vista uma correcta localização das actividades, um equilibrado desenvolvimento sócio-económico e a valorização da paisagem; c) Criar e desenvolver reservas e parques naturais e de recreio, bem como classificar e proteger paisagens e sítios, de modo a garantir a conservação da natureza e a preservação de valores culturais de interesse histórico ou artístico; d) Promover o aproveitamento racional dos recursos naturais, salvaguardando a sua capacidade de renovação e a estabilidade ecológica, com respeito pelo princípio da solidariedade entre gerações; e) Promover, em colaboração com as autarquias locais, a qualidade ambiental das povoações e da vida urbana, designadamente no plano arquitectónico e da protecção das zonas históricas; f) Promover a integração de objectivos ambientais nas várias políticas de âmbito sectorial; "

A Carta Europeia do Ordenamento do Território, adotada pela Conferência Europeia dos Ministros responsáveis pelo Ordenamento do Território do Conselho da Europa em 20 de maio de 1983, define ordenamento do território da seguinte forma:

“O ordenamento do território é a expressão social das políticas económica, social, cultural e ecológica de toda a sociedade. É simultaneamente uma disciplina científica, uma técnica administrativa e uma política, concebida como uma abordagem INTERDISCIPLINAR E GLOBAL (meu sublinhado) tendo em vista o desenvolvimento equilibrado das regiões e a organização física do espaço segundo um conceito orientador.”

Tendo em conta as duas dimensões do ordenamento do território – desenvolvimento regional / coesão territorial e organização física dos usos do solo -, partilhem a vossa definição pessoal de ordenamento do território.

Some recent studies have found lower incidences (e.g. 8%) in allogeneic HSCT recipients.

Invasive Aspergillosis in Allogeneic Hematopoietic Stem Cell Transplantation Recipients: A Large Cohort Single-Center Study. Yuliya A. Rogacheva, Vladislav V. Markelov, Marina O. Popova, et al. Blood. 2021; 138 (Suppl 1): doi.org/10.1182/blood-2021-153762.

foi em 2011

1 - competition breeds narcissism. This is a pillar o the illusion of freedom.

doing so w/o actually agreeing

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Reply to the reviewers

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Reviewer #1 (Evidence, reproducibility, and clarity (Required)): This is an interesting manuscript from the Jagannathan laboratory, which addresses the interaction proteome of two satellite DNA-binding proteins, D1 and Prod. To prevent a bias by different antibody affinities they use GFP-fusion proteins of D1 and Prod as baits and purified them using anti GFP nanobodies. They performed the purifications in three different tissues: embryo, ovary and GSC enriched testes. Across all experiments, they identified 500 proteins with surprisingly little overlap among tissues and between the two different baits. Based on the observed interaction of prod and D1 with members of the canonical piRNA pathway the authors hypothesized that both proteins might influence the expression of transposable elements. However, neither by specific RNAi alleles or mutants that lead to a down regulation of D1 and Prod in the gonadal soma nor in the germline did they find an effect on the repression of transposable elements. They also did not detect an effect of a removal of piRNA pathway proteins on satellite DNA clustering, which is mediated by Prod and D1. However, they do observe a mis-localisation of the piRNA biogenesis complex to an expanded satellite DNA in absence of D1, which presumably is the cause of a mis-regulation of transposable elements in the F2 generation.This is an interesting finding linking satellite DNA and transposable element regulation in the germline. However, I find the title profoundly misleading as the link between satellite DNA organization and transgenerational transposon repression in Drosophila has not been identified by multi-tissue proteomics but by a finding of the Brennecke lab that the piRNA biogenesis complex has a tendency to localise to satellite DNA when the localisation to the piRNA locus is impaired. Nevertheless, the investigation of the D1 and Prod interactome is interesting and might reveal insights into the pathways that drive the formation of centromeres in a tissue specific manner.

We thank the reviewer for the overall positive comments on our manuscript. As noted above, we have performed a substantial number of revision experiments and improved our text. We believe that our revised manuscript demonstrates a clear link between our proteomics data and the transposon repression. We would like to make three main points,

1. Our proteomics data identified that D1 and Prod co-purified transposon repression proteins in embryos. To test the functional significance of this association, we have used Drosophila genetics to generate flies lacking embryonic D1. In adult ovaries from these flies, we observe a striking elevation in transposon expression and Chk2-dependent gonadal atrophy. Along with the results from the control genotypes (F1 D1 mutant, F2 D1 het), our data clearly indicate that embryogenesis (and potentially early larval development) are a period when D1 establishes heritable TE silencing that can persist throughout development.
2. Based on the newly acquired RNA-seq and small RNA seq data, we have edited our title to more accurately reflect our data. Specifically, we have substituted the word 'transgenerational' with 'heritable', meaning that the presence of D1 during early development alone is sufficient to heritably repress TEs at later stages of development.
3. In addition, our RNA seq and small RNA seq experiments demonstrate that D1 makes a negligible contribution to piRNA biogenesis and TE repression in adults, despite the significant mislocalization of the RDC complex. In this regard, our results are substantially different from the recent Kipferl study from the Brennecke lab (Baumgartner et al. 2022).

   Major comments Unfortunately, the proteomic data sets are not very convincing. Not even the corresponding baits are detected in all assays. I wonder whether the extraction procedure really allows the authors to analyze all functionally relevant interactions of Prod and D1. It would be good to see a western blot or an MS analysis of the soluble nuclear extract they use for purification compared to the insoluble chromatin. It may well be that a large portion of Prod or D1 is lost in this early step. I also find the description of the proteomic results hard to follow as the authors mostly list which proteins the find as interactors of Prod and D1 without stating in which tissue or with what bait they could purify them (i.e. p7: Importantly, our hits included known chromocenter-associated or pericentromeric heterochromatin-associated proteins, such as Su(var)3-9[52], ADD1[23,24], HP5[23,24],mei-S332[53], Mes-4[23], Hmr[24,39,54], Lhr[24,39], and members of the chromosome passenger complex, such as borr and Incenp[55]). It would be interesting to at least discuss tissue specific interactions.

Out of six total AP-MS experiments in this manuscript (D1 x 3, Prod x 2 and Piwi), we observe a strong enrichment of the bait in 5/6 attempts (log2FC between 4-12). In the initial submission, the lack of a third high-quality biological replicate for the D1 testis sample meant that only the p-value (0.07) was not meeting the cutoff. To address this, we have repeated this experiment with an additional biological replicate (Fig. S2A), and our data now clearly show that D1 is significantly enriched in the testis sample.

As suggested by the reviewer, we have also assessed our lysis conditions (450mM NaCl and benzonase) and the solubilization of our baits post-lysis. In Fig. S1D, we have blotted equivalent fractions of the soluble supernatant and insoluble pellet from GFP-Piwi embryos and show that both GFP-Piwi and D1 are largely solubilized following lysis. In Fig. S1E, we also show that our IP protocol works efficiently.

GFP-Prod pulldown in embryos is the only instance in which we do not detect the bait by mass spectrometry. Here, one reason could be relatively low expression of GFP-Prod in comparison to GFP-D1 (Fig. S1E), which may lead to technical difficulties in detecting peptides corresponding to Prod. However, we note that Prod IP co-purified proteins such as Bocks that were previously suggested as Prod interactors from high-throughput studies (Giot et al. 2003; Guruharsha et al. 2011). In addition, Prod IP from embryos also co-purified proteins known to associate with chromocenters such as Hcs and Saf-B. Finally, the concordance between D1 and Prod co-purified proteins from embryo lysates (Fig. 2A, C) suggest that the Prod IP from embryos is of reasonable quality.

We also acknowledge the reviewer's comment that the description of the proteomic data was hard to follow. Therefore, we have revised our text to clearly indicate which bait pulled down which protein in which tissue (lines 148-156). We have also highlighted and discussed bait-specific and tissue-specific interactions in the text (lines 162-173).

Minor comments The authors may also want to provide a bit more information on the quantitation of the proteomic data such as how many values were derive from the match-between runs function and how many were imputed as this can severely distort the quantification.

Figure 1: Distribution of data after imputation in embryo (left), ovary (middle) and testis (right) datasets. Imputation is performed with random sampling from the 1% least intense values generated by a normal distribution.

To ensure the robustness of our data analysis, we considered only those proteins that were consistently identified in all replicates for at least one bait (GFP-D1, GFP-Prod or NLS-GFP). This approach resulted in a relative low number of missing values. However, it is also important to bear in mind that in an AP-MS experiment, the number of missing values is higher, as interactors are not identified in the control pulldown. Importantly, the imputation of missing values during the data analysis did not alter the normal distribution of the dataset (Fig. 1, this document). Detailed information regarding the imputed values is also provided (Table 1, this document). The coding script used for the data analysis is available in the PRIDE submission of the dataset (Table 2, this document). This information has been added to our methods section under data availability.

Table 1: ____Number of match-between-runs and imputations for embryo, ovary and testis datasets

Dataset

#match-between-runs

%match-between-runs

%imputation

Embryo

5541/27543

20.11%

8.36%

Ovary

1936/9530

20.30%

8.18%

Testis

1748/7168

24.39%

3.12%

Table 2: ____Access to the PRIDE submission of the datasets

Name

ID PRIDE

UN reviewer

PW reviewer

IP-MS of D1 from Testis tissue

PXD044026

reviewer_pxd044026@ebi.ac.uk

ydswDQVW

IP-MS of Piwi from Embryo tissue

PXD043237

reviewer_pxd043237@ebi.ac.uk

TMCoDsdx

IP-MS of Prod and D1 proteins from Ovary tissue

PXD043236

reviewer_pxd043236@ebi.ac.uk

VOHqPmaS

IP-MS of Prod and D1 proteins from Embryo tissue

PXD043234

reviewer_pxd043234@ebi.ac.uk

L77VXdvA

**Referee Cross-Commenting** I agree with the two other reviewers that the connection between the interactome and the transgenerational phenotype is unclear. This is also what I meant i my comment that the title is somewhat misleading. A systematic analysis of the D1 and Prod knock down effect on mRNAs and small Rnas would indeed be helpful to better understand the interesting effect.

As suggested by the reviewer, we have performed RNA seq and small RNA seq in control and D1 mutant ovaries (Fig. 4) to fully understand the contribution of D1 in piRNA biogenesis and TE repression. Briefly, the mislocalization of RDC complex in D1 mutant ovaries does not significantly affect TE-mapping piRNA biogenesis (Fig. 4C, E). In addition, loss of D1 does not substantially alter TE expression in the ovaries (Fig. 4B) or alter the expression of genes involved in TE repression (Fig. 4F). Along with the results presented in Fig. 5 and Fig. 6, our data clearly indicate that embryogenesis (and potentially early larval development) is a critical period during which D1 makes an important contribution to TE repression.

Reviewer #1 (Significance (Required)): Nevertheless, the investigation of the D1 and Prod interactome is interesting and might reveal insights into the pathways that drive the formation of centromeres in a tissue specific manner. It may be mostly interesting for the Drosophila community but could also be exiting for a broader audience interested in the connection of heterochromatin and its indirect effect on the regulation of transposable elements.

We thank the reviewer again for the helpful and constructive comments, which have enabled us to significantly improve our study. We are excited by the results from our study, which illuminate unappreciated aspects of transcriptional silencing in constitutive heterochromatin.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): Chavan et al. set out to enrich our compendium of pericentric heterochromatin-associated proteins - and to learn some new biology along the way. An ambitious AP-Mass baited with two DNA satellite-binding proteins (D1 and Prod), conducted across embryos, ovaries, and testes, yielded hundreds of candidate proteins putatively engaged at chromocenters. These proteins are enriched for a restricted number of biological pathways, including DNA repair and transposon regulation. To investigate the latter in greater depth, the authors examine D1 and prod mutants for transposon activity changes using reporter constructs for multiple elements. These reporter constructs revealed no transposon activation in the adult ovary, where many proteins identified in the mass spec experiments restrict transposons. However, the authors suggest that the D1 mutant ovaries do show disrupted localization of a key member of a transposon restriction pathway (Cuff), and infer that this mislocalization triggers a striking, transposon derepression phenotype in the F2 ovaries.

The dataset produced by the AP-Mass Spec offers chromosome biologists an unprecedented resource. The PCH is long-ignored chromosomal region that has historically received minimal attention; consequently, the pathways that regulate heterochromatin are understudied. Moreover, attempting to connect genome organization to transposon regulation is a new and fascinating area. I can easily envision this manuscript triggering a flurry of discovery; however, there is quite a lot of work to do before the data can fully support the claims.

We appreciate the reviewer taking the time to provide thoughtful comments and constructive suggestions to improve the manuscript. We believe that we have addressed all the comments raised to the significant advantage of our paper.

Major comments 1. The introduction requires quite a radical restructure to better highlight the A) importance of the work and B) limit information whose relevance is not clear early in the manuscript. A. Delineating who makes up heterochromatin is a long-standing problem in chromosome biology. This paper has huge potential to contribute to this field; however, it is not the first. Others are working on this problem in other systems, for example PMID:29272703. Moreover, we have some understanding of the distinct pathways that may impact heterochromatin in different tissues (e.g., piRNA biology in ovaries vs the soma). Also, the mutant phenotypes of prod and D1 are different. Fleshing out these three distinct points could help the reader understand what we know and what we don't know about heterochromatin composition and its special biology. Understanding where we are as a field will offer clear predictions about who the interactors might be that we expect to find. For example, given the dramatically different D1 and prod mutant phenotypes (and allele swap phenotypes), how might the interactors with these proteins differ? What do we know about heterochromatin formation differences in different tissues? And how might these differences impact heterochromatin composition?

The reviewer brings up a fair point and we have significantly reworked our introduction. We share the reviewer's opinion that improved knowledge of the constitutive heterochromatin proteome will reveal novel biology.

1. The attempt to offer background on the piRNA pathway and hybrid dysgenesis in the Introduction does not work. As a naïve reader, it was not clear why I was reading about these pathways - it is only explicable once the reader gets to the final third of the Results. Moreover, the reader will not retain this information until the TE results are presented many pages later. I strongly urge the authors to shunt the two TE restriction paragraphs to later in the manuscript. They are currently a major impediment to understanding the power of the experiment - which is to identify new proteins, pathways, and ultimately, biology that are currently obscure because we have so little handle on who makes up heterochromatin.

We agree with this suggestion. We have introduced the piRNA pathway in the results section (lines 205 - 216), right before this information is needed. We've also removed the details on hybrid dysgenesis, since our new data argues against a maternal effect from the D1 mutant.

The implications of the failure to rescue female fertility by the tagged versions of both D1 and Prod are not discussed. Consequently, the reader is left uneasy about how to interpret the data.

We understand this point raised by the reviewer. However, in our proteomics experiments, we have used GFP-D1 and GFP-Prod ovaries from ~1 day old females (line 579, methods). These ovaries are morphologically similar to the wild type (Fig. S1C) and their early germ cell development appears to be intact. Moreover, chromocenter formation in female GSCs is comparable to the wildtype (Fig. 1C-D). These data, along with the rescue of the viability of the Prod mutant (Fig. S1A-B), suggest that the presence of a GFP tag is not compromising D1 or Prod function in the early stages of germline development and is consistent with published and unpublished data from our lab. In addition, D1 and Prod from ovaries co-purify proteins such as Rfc38 (D1), Smn (D1), CG15107 (Prod), which have been identified in previous high-throughput screens (Guruharsha et al. 2011; Tang et al. 2023). For these reasons, we believe that GFP-D1 and GFP-Prod ovaries are a good starting point for the AP-MS experiment. We speculate that the failure to completely rescue female fertility may be due to improper expression levels of GFP-D1 or GFP-Prod flies at later stages of oogenesis, which are not present in ovaries from newly eclosed females and therefore unlikely to affect our proteomic data.

1. How were the significance cut-offs determined? Is the p-value reported the adjusted p-value? As a non-expert in AP-MS, I was surprised to find that the p-value, at least according to the Methods, was not adjusted based on the number of tests. This is particularly relevant given the large/unwieldy(?) number of proteins that were identified as signficant in this study. Moreover, the D1 hit in Piwi pull down is actually not significant according to their criteria of p We used a standard cutoff of log2FC>1 and p2FC and low p-value) since these are more likely to be bona fide interactors. Third, we have used string-DB for functional analyses where we can place our hits in existing protein-protein interaction networks. Using this approach, we detect that Prod (but not D1) pulls down multiple members of the RPA complex in the embryo (RPA2 and RpA-70, Fig. S2B) while embryonic D1 (but not Prod) pulls down multiple components of the origin recognition complex (Orc1, lat, Orc5, Orc6, Fig. S2C) and the condensin I complex (Cap-G, Cap-D2, barr, Fig. S2D). Altogether, these filtering strategies allow us to eliminate as many false positives as possible while making sure to minimize the loss of true hits through multiple testing correction.

How do we know if the lack of overlap across tissues is indeed germline- or soma-specialization rather than noise?

To address this part of the comment, we have amended our text (lines 162-173) as follows,

'We also observed a substantial overlap between D1- and Prod-associated proteins (yellow points in Fig. 2A, B, Table S1-3), with 61 hits pulled down by both baits (blue arrowheads, Fig. 2C) in embryos and ovaries. This observation is consistent with the fact that both D1 and Prod occupy sub-domains within the larger constitutive heterochromatin domain in nuclei. Surprisingly, only 12 proteins were co-purified by the same bait (D1 or Prod) across different tissues (magenta arrowheads, Fig. 2C, Table S1-3). In addition, only a few proteins such as an uncharacterized DnaJ-like chaperone, CG5504, were associated with both D1 and Prod in embryos and ovaries (Fig. 2D). One interpretation of these results is that the protein composition of chromocenters may be tailored to cell- and tissue-specific functions in Drosophila. However, we also note that the large number of unidentified peptides in AP-MS experiments means that more targeted experiments are required to validate whether certain proteins are indeed tissue-specific interactors of D1 and Prod.'

To make this inference, conducting some validation would be required. More generally, I was surprised to see no single interactor validated by reciprocal IP-Westerns to validate the Mass-Spec results, though I am admittedly only adjacent to this technique. Note that colocalization, to my mind, does not validate the AP-MS data - in fact, we would a priori predict that piRNA pathway members would co-localize with PCH given the enrichment of piRNA clusters there.

Here, we would point out that we have conducted multiple validation experiments with a specific focus on the functional significance of the associations between D1/Prod and TE repression proteins in embryos. While the reviewer suggests that piRNA pathway proteins may be expected to enrich at the pericentromeric heterochromatin, this is not always the case. For example, Piwi and Mael are present across the nucleus (pulled down by D1/Prod in embryos) while Cutoff, which is present adjacent to chromocenters in nurse cells, was not observed to interact with either D1 or Prod in the ovary samples.

Therefore, for Piwi, we performed a reciprocal AP-MS experiment in embryos due to the higher sensitivity of this method (GFP-Piwi AP-MS, Fig. 3B). Excitingly, this experiment revealed that four largely uncharacterized proteins (CG14715, CG10208, Ugt35D1 and Fit) were highly enriched in the D1, Prod and Piwi pulldowns and these proteins will be an interesting cohort for future studies on transposon repression in Drosophila (Fig. 3C).

Furthermore, we believe that determining the localization of proteins co-purified by D1/Prod is an important and orthogonal validation approach. For Sov, which is implicated in piRNA-dependent heterochromatin formation, we observe foci that are in close proximity to D1- and Prod-containing chromocenters (Fig. 3A).

As for suggestion to validate by IP-WBs, we would point out that chromocenters exhibit properties associated with phase separated biomolecular condensates. Based on the literature, these condensates tend to associate with other proteins/condensates through low affinity or transient interactions that are rarely preserved in IP-WBs, even if there are strong functional relationships. One example is the association between D1 and Prod, which do not pull each other down in an IP-WB (Jagannathan et al. 2019), even though D1 and Prod foci dynamically associate in the nucleus and mutually regulate each other's ability to cluster satellite DNA repeats (Jagannathan et al. 2019). Similarly, IP-WB using GFP-Piwi embryos did not reveal an interaction with D1 (Fig. S4B). However, our extensive functional validations (Figures 4-6) have revealed a critical role for D1 in heritable TE repression.

The AlphaFold2 data are very interesting but seem to lack of negative control. Is it possible to incorporate a dataset of proteins that are not predicted to interact physically to elevate the impact of the ones that you have focused on? Moreover, the structural modeling might suggest a competitive interaction between D1 and piRNAs for Piwi. Is this true? And even if not, how does the structural model contribute to your understanding for how D1 engages with the piRNA pathway? The Cuff mislocalization?

In the revised manuscript, we have generated more structural models using AlphaFold Multimer (AFM) for proteins (log2FC>2, p0.5 and ipTM>0.8), now elaborated in lines 175-177. Despite the extensive disorder in D1 and Prod, we identified 22 proteins, including Piwi, that yield interfaces with ipTM scores >0.5 (Table S4, Table S8). These hits are promising candidates to further understand D1 and Prod function in the future.

For the predicted model between Prod/D1 and Piwi (Fig. S4A), one conclusion could indeed be competition between D1/Prod and piRNAs for Piwi. Another possibility is that a transient interaction mediated by disordered regions on D1/Prod could recruit Piwi to satellite DNA-embedded TE loci in the pericentromeric heterochromatin. These types of interactions may be especially important in the early embryonic cycles, where repressive histone modifications such as H3K9me2/3 must be deposited at the correct loci for the first time. We suggest that mutating the disordered regions on D1 and Prod to potentially abrogate the interaction with Piwi will be important for future studies.

The absence of a TE signal in D1 and Prod mutant ovaries would be much more compelling if investigated more agnostically. The observation that not all TE reporter constructs show a striking signal in the F2 embryos makes me wonder if Burdock and gypsy are not regulated by these two proteins but possibly other TEs are. Alternatively, small RNA-seq would more directly address the question of whether D1 and Prod regulate TEs through the piRNA pathway.

We completely agree with this comment from the reviewer. We have performed RNA seq on D1 heterozygous (control) and D1 mutant ovaries in a chk26006 background. Since Chk2 arrests germ cell development in response to TE de-repression and DNA damage(Ghabrial and Schüpbach 1999; Moon et al. 2018), we reasoned that the chk2 mutant background would prevent developmental arrest of potential TE-expressing germ cells and we observed that both genotypes exhibited similar gonad morphology (Fig. 4A). From our analysis, we do not observe a significant effect on TE expression in the absence of D1, except for the LTR retrotransposon tirant (Fig. 4B). We also do not observe differential expression of TE repression genes (Fig. 4F).

We have complemented our RNA seq experiment with small RNA profiling from D1 heterozygous (control) and D1 mutant ovaries. Here, overall piRNA production and antisense piRNAs mapping to TEs were largely unperturbed (Fig. 4C-E).

Overall, our data is consistent with the TE reporter data (Fig. S7) and suggests that zygotic depletion of D1 does not have a prominent role in TE repression. However, we have uncovered that the presence of D1 during embryogenesis is critical for TE repression in adult gonads (Fig. 6, Fig. S9).

I had trouble understanding the significance of the Cuff mis-localization when D1 is depleted. Given Cuff's role in the piRNA pathway and close association with chromatin, what would the null hypothesis be for Cuff localization when a chromocenter is disrupted? What is the null expectation of % Cuff at chromocenter given that the chromocenter itself expands massively in size (Figure 4D). The relationship between these two factors seems rather indirect and indeed, the absence of Cuff in the AP would suggest this. The biggest surprise is the absence of TE phenotype in the ovary, given the Cuff mutant phenotype - but we can't rule out given the absence of a genome-wide analysis. I think that these data leave the reader unconvinced that the F2 phenotype is causally linked to Cuff mislocalization.

We apologize that this data was not more clearly represented. In a wild-type context, Cuff is distributed in a punctate manner across the nurse cell nuclei, with the puncta likely representing piRNA clusters (Fig. 5A-B). We find that a small fraction of Cuff (~5%) is present adjacent to the nurse cell chromocenter (inset, Fig. 5A and Fig. 5D). In the absence of D1, the nurse cell chromocenters increase ~3-4 fold in size. However, the null expectation is still that ~5% of total Cuff would be adjacent to the chromocenter, since the piRNA clusters are not expected to expand in size. In reality, we observe ~27% of total Cuff is mislocalized to chromocenters. Our data indicate that the satellite DNA repeats at the larger chromocenters must be more accessible to Cuff in the D1 mutant nurse cells. This observation is corroborated by the significant increase in piRNAs corresponding to the 1.688 satellite DNA repeat family (Fig. 4E).

The lack of TE expression in the F1 D1 mutant was indeed surprising based on the Cuff mislocalization but as the reviewers pointed out, we only analyzed two TE reporter constructs in the initial submission. In the revised manuscript, we have performed both RNA seq and small RNA seq. Surprisingly, our data reveal that the Cuff mislocalization does not significantly affect piRNA biogenesis (Fig. 4C, D) and piRNAs mapping to TEs. As a result, both TE repression (Fig. 4B) and fertility (Fig. 6D) are largely preserved in the absence of D1 in adult ovaries.

Finally, we thank the reviewer for their excellent suggestion to incorporate the F2 D1 heterozygote (Fig. S9) in our analysis! This important control has revealed that the maternal effect of the D1 mutant is negligible for gonad development and fertility (Fig. 6B-D). Rather, our data clearly emphasize embryogenesis (or early larval development) as a key period during which D1 can promote heritable TE repression. Essentially, D1 is not required during adulthood for TE repression if it was present in the early stages of development.

Apologies if I missed this, but Figure 5 shows the F2 D1 mutant ovaries only. Did you look at the TM6 ovaries as well? These ovaries should lack the maternally provisioned D1 (assuming that females are on the right side) but have the zygotic transcription.

As mentioned above, this was a great suggestion and we've now characterized this important control in the context of germline development and fertility, to the significant advantage of our paper.

Minor comments 9. Add line numbers for ease of reference

We apologize for this. Line numbers have been added in the full revision.

1. The function of satellite DNA itself is still quite controversial - I would recommend being a bit more careful here - the authors could refer instead to genomic regions enriched for satellite DNA are linked to xyz function (see Abstract line 2 and 7, for example.)

The abstract has been rewritten and does not directly implicate satellite DNA in a specific cellular function. However, we have taken the reviewer's suggestion in the introduction (line 57-58).

"Genetic conflicts" in the introduction needs more explanation.

This sentence has been removed from the introduction in the revised manuscript.

"In contrast" is not quite the right word. Maybe "However" instead (1st line second paragraph of Intro)

Done. Line 57 of the revised manuscript.

Results: what is the motivation for using GSC-enriched testis?

We use GSC-enriched testes for practical reasons. First, they contain a relatively uniform population of mitotically dividing germ cells unlike regular testes which contain a multitude of post-mitotic germ cells such as spermatocytes, spermatids and sperm. Second, GSC-enriched testes are much larger than normal testes and reduced the number of dissections that were needed for each replicate.

1. Clarify sentence about the 500 proteins in the Results section - it's not clear from context that this is the union of all experiments.

Done. Lines 145-149 in the revised manuscript.

The data reported are not the first to suggest that satellite DNA may have special DNA repair requirements. e.g., PMID: 25340780

We apologize if we gave the impression that we were making a novel claim. Specialized DNA repair requirements at repetitive sequences have indeed been previously hypothesized(Charlesworth et al. 1994) and studied and we have altered the text to better reflect this (lines 193-195). We have cited the study suggested by the reviewer as well as studies from the Chiolo(Chiolo et al. 2011; Ryu et al. 2015; Caridi et al. 2018) and Soutoglou(Mitrentsi et al. 2022) labs, which have also addressed this fascinating question.

Page 10: indicate-> indicates.

Done.

1. Page 14: revise for clarity: "investigate a context whether these interactions could not take place"

We've incorporated this suggestion in the revised text (lines 383-386).

1. Might be important to highlight the 500 interactions are both direct and indirect. "Interacting proteins" alone suggests direct interactions only.

Done. Lines 145-149.

The effect of the aub mutant on chromocenter foci did not seem modest to me - however, the bar graphs obscure the raw data - consider plotting all the data not just the mean and error?

Done. This data is now represented by a box-and-whisker plot (Fig. S5), which shows the distribution of the data.

Reviewer #2 (Significance (Required)):

The dataset produced by the AP-Mass Spec offers chromosome biologists an unprecedented resource. The PCH is long-ignored chromosomal region that has historically received minimal attention; consequently, the pathways that regulate heterochromatin are understudied. Moreover, attempting to connect genome organization to transposon regulation is a new and fascinating area. I can easily envision this manuscript triggering a flurry of discovery; however, there is quite a lot of work to do before the data can fully support the claims.

This manuscript represents a significant contribution to the field of chromosome biology.

We thank the reviewer for the positive evaluation of our manuscript, and we really appreciate the great suggestion for the F2 D1 heterozygote control! Overall, we believe that our manuscript is substantially improved with the addition of RNA seq, small RNA seq and important genetic controls. Moreover, we are excited by the potential of our study to open new doors in the study of pericentromeric heterochromatin.

Reviewer #3 (Evidence, reproducibility and clarity (Required)): In the manuscript entitled "Multi-tissue proteomics identifies a link between satellite DNA organization and transgenerational transposon repression", the authors used two satellite DNA-binding proteins, D1 and Prod, as baits to identify chromocenter-associated proteins through quantitative mass spectrometry. The proteomic analysis identified ~500 proteins across embryos, ovaries, and testes, including several piRNA pathways proteins. Depletion of D1 or Prod did not directly contribute to transposon repression in ovary. However, in the absence of maternal and zygotic D1, there was a dramatic increase of agametic ovaries and transgenerational transposon de-repression. Although the study provides a wealth of proteomic data, it lacks mechanistic insights into how satellite DNA organization influence the interactions with other proteins and their functional consequences.

We thank the reviewer for highlighting that this study will be a valuable resource for future studies on the composition and function of pericentromeric heterochromatin. Based on the reviewer's request for more mechanistic knowledge into how satellite DNA organization affects transposon repression, we have performed RNA seq and small RNA seq, added important genetic controls and significantly improved our text. In the revised manuscript, our data clearly demonstrate that embryogenesis (and potentially early larval development) is a critical time period when D1 contributes to heritable TE repression. Flies lacking D1 during embryogenesis exhibit TE expression in germ cells as adults, which is associated with Chk2-dependent gonadal atrophy. We are particularly excited by these data since TE loci are embedded in the satellite DNA-rich pericentromeric heterochromatin and our study demonstrates an important role for a satellite DNA-binding protein in TE repression.

Major____ comments 1. While the identification of numerous interactions is significant, it would be helpful to acknowledge that lots of these proteins were known to bind DNA or heterochromatin regions. To strengthen the study, I recommend conducting functional validation of the identified interactions, in addition to the predictions made by Alphfold 2.

We are happy to take this comment on board. In fact, we believe that the large number of DNA-binding and heterochromatin-associated proteins identified in this study are a sign of quality for the proteomic datasets. In the revised manuscript, we have highlighted known heterochromatin proteins co-purified by D1/Prod in lines 148-151 as well as proteins previously suggested to interact with D1/Prod from high-throughput studies in lines 153-156 (Guruharsha et al. 2011; Tang et al. 2023). In this study, we have focused on the previously unknown associations between D1/Prod and TE repression proteins and functionally validated these interactions as presented in Figures 3-6.

The observation of transgenerational de-repression is intriguing. However, to better support this finding, it would be better to provide a mechanistic explanation based on the data presented.

We appreciate this comment from the reviewer, which is similar to major comment #6 raised by reviewer #2. To generate mechanistic insight into the underlying cause of gonad atrophy in the F2 D1 mutant, we have performed RNA seq, small RNA seq and analyzed germline development and fertility in the F2 D1 heterozygous control (Fig. S9).

For the RNA seq, we used D1 heterozygous (control) and D1 mutant ovaries in a chk26006 background. Since Chk2 arrests germ cell development in response to TE de-repression and DNA damage(Ghabrial and Schüpbach 1999; Moon et al. 2018), we reasoned that the chk2 mutant background would prevent developmental arrest of potential TE-expressing germ cells and we observed that both genotypes exhibited similar gonad morphology (Fig. 4A). From our analysis, we do not observe a significant effect on TE expression in the absence of D1, except for the LTR retrotransposon tirant (Fig. 4B). We also do not observe differential expression of TE repression genes (Fig. 4F).

We have complemented our RNA seq experiment with small RNA profiling from D1 heterozygous (control) and D1 mutant ovaries. Here, overall piRNA production and antisense piRNAs mapping to TEs were largely unperturbed (Fig. 4C-E). Together, these data are consistent with the TE reporter data (Fig. S7) and suggests that zygotic depletion of D1 does not have a prominent role in TE repression.

However, we have uncovered that the presence of D1 during embryogenesis is critical for TE repression in adult gonads (Fig. 6, Fig. S9). Essentially, either only maternal deposited D1 (F1 D1 mutant) or only zygotically expressed D1 (F2 D1 het) were sufficient to ensure TE repression and fertility. In contrast, a lack of D1 during embryogenesis (F2 D1 mutant) led to elevated TE expression and Chk2-mediated gonadal atrophy.

Our results also explain why previous studies have not implicated either D1 or Prod in TE repression, since D1 likely persists during embryogenesis when using depletion approaches such as RNAi-mediated knockdown or F1 generation mutants.

Minor____ comments 3. Given the maternal effect of the D1 mutant, in Figure 4, I suggest analyzing not only nurse cells but also oocytes to gain a more comprehensive understanding.

We agree with the reviewer that this experiment can be informative. In the F2 D1 mutant ovaries, germ cell development does not proceed to a stage where oocytes are specified, thus limiting microscopy-based approaches. Nevertheless, we have gauged oocyte quality in all the genotypes using a fertility assay (Fig. 6D) since even ovaries that have a wild-type appearance can produce dysfunctional gametes. In this experiment, we observe that fertility is largely intact in the F1 D1 mutant and F2 D1 heterozygote strains, suggesting that it is the presence of D1 during embryogenesis (or early larval development) that is critical for heritable TE repression and proper oocyte development.

The conclusion that D1 and Prod do not directly contribute to the repression of transposons needs further analysis from RNA-seq data. Alternatively, the wording could be adjusted to indicate that D1 and Prod are not required for specific transposon silencing, such as Burdock and gypsy.

Agreed. We have performed RNA-seq in D1 heterozygous (control) and D1 mutant ovaries in a chk26006 background (Fig. 4A, B) as described above.

As D1 mutation affects Cuff nuclear localization, it would be insightful to sequence the piRNA in the ovaries.

Agreed. We have performed small RNA profiling from D1 heterozygous (control) and D1 mutant ovaries. Despite the significant mislocalization of the RDC complex, overall piRNA production and antisense piRNAs mapping to TEs were largely unaffected (Fig. 4C-E). However, we observed significant changes in piRNAs mapping to complex satellite DNA repeats (Fig. 4D), but these changes were not associated with a maternal effect on germline development or fertility (F2 D1 heterozygote, Fig. 6B-D).

**Referee Cross-Commenting**

I appreciate the valuable insights provided by the other two reviewers regarding this manuscript. I concur with their assessment that substantial improvements are needed before considering this manuscript for publication.

1. The proteomics data has the potential to be a valuable resource for other scientific community. However, I share the concerns raised by reviewer 1 about the current quality of the data sets. Addressing this, it will augment the manuscript with additional data to show the success of the precipitation. Moreover, as reviewer 2 and I suggested, additional co-IP validations of the IP-MS results are needed to enhance the reliability.

In the revised manuscript, we have performed multiple experiments to address the quality of the MS datasets based on comments raised by reviewer #1. They are as follows,

Out of six total AP-MS experiments in this manuscript (D1 x 3, Prod x 2 and Piwi), we observe a strong enrichment of the bait in 5/6 attempts (log2FC between 4-12, Fig. 2A, B, Fig. S2A, Table S1-S3, Table S7). In the D1 testis sample from the initial submission, the lack of a third biological replicate meant that only the p-value (0.07) was not meeting the cutoff. To address this, we have repeated this experiment with an additional biological replicate (Fig. S2A), and our data now clearly show that D1 is also significantly enriched in the testis sample.

As suggested by the reviewer #1, we have assessed our lysis conditions (450mM NaCl and benzonase) and the solubilization of our baits post-lysis. In Fig. S1D, we have blotted equivalent fractions of the soluble supernatant and insoluble pellet from GFP-Piwi embryos and show that both GFP-Piwi and D1 are largely solubilized following lysis. In Fig. S1E, we also show that our IP protocol works efficiently.

The only instance in which we do not detect the bait by mass spectrometry is for GFP-Prod pulldown in embryos. Here, one reason could be relatively low expression of GFP-Prod in comparison to GFP-D1 (Fig. S1E), which may lead to technical difficulties in detecting peptides corresponding to Prod. However, we note that Prod IP from embryos co-purified proteins such as Bocks that were previously suggested as Prod interactors from high-throughput studies (Giot et al. 2003; Guruharsha et al. 2011). In addition, Prod IP from embryos also co-purified proteins known to associate with chromocenters such as Hcs(Reyes-Carmona et al. 2011) and Saf-B(Huo et al. 2020). Finally, the concordance between D1 and Prod co-purified proteins from embryo lysates (Fig. 2A, C) suggest that the Prod IP from embryos is of reasonable quality.

As for the IP-WB validations, we would point out that chromocenters exhibit properties associated with phase separated biomolecular condensates. In our experience, these condensates tend to associate with other proteins/condensates through low affinity or transient interactions that are rarely preserved in IP-WBs, even if there are strong functional relationships. One example is the association between D1 and Prod, which do not pull each other down in an IP-WB (Jagannathan et al. 2019), even though D1 and Prod foci dynamically associate in the nucleus and mutually regulate each other's ability to cluster satellite DNA repeats (Jagannathan et al. 2019). Similarly, IP-WB using GFP-Piwi embryos did not reveal an interaction with D1 (Fig. S4B). However, our extensive functional validations (Figures 4-6) have revealed a critical role for D1 in heritable TE repression.

I agree with reviewer 2 that the present conclusion is not appropriate regarding D1 and Prod do not contribute to transposon silencing. As reviewer 2 and I suggested, the systematical analysis by using both mRNA-seq and small RNA-seq is required to draw the conclusion.

Agreed. We have performed RNA seq and small RNA seq as elaborated above. Our conclusions on the role of D1 in TE repression are now much stronger.

1. The transgenerational phenotype is an intriguing aspect of the study. I agree with reviewer 2 that the inclusion of TM6 ovaries would be a nice control. Further, it is hard to connect this phenotype with the interactions identified in this manuscript. It would be appreciated if the author could provide a mechanistic explanation.

We have significantly improved these aspects of our study in the revised manuscript. Through the analysis of germline development in the F2 D1 heterozygotes as suggested by reviewer #2, in addition to the recommended RNA seq and small RNA seq, we have now identified embryogenesis (and potentially early larval development) as a time period when D1 makes an important contribution to TE repression. Loss of D1 during embryogenesis leads to TE expression in adult germline cells, which is associated with Chk2-dependent gonadal atrophy. Taken together, we have pinpointed the specific contribution of a satellite DNA-binding protein to transposon repression.

Reviewer #3 (Significance (Required)):

Although this study successfully identified several interactions, the authors did not fully elucidate how these interactions contribute to the transgenerational silencing of transposons or ovary development.

We thank the reviewer for the thoughtful comments and overall positive evaluation of our study, especially the proteomic dataset. We are confident that the revised manuscript has improved our mechanistic understanding of the contribution made by a satellite DNA-binding protein in TE repression.

References

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Guruharsha KG, Rual JF, Zhai B, Mintseris J, Vaidya P, Vaidya N, Beekman C, Wong C, Rhee DY, Cenaj O, et al. 2011. A protein complex network of Drosophila melanogaster. Cell 147: 690-703.

Huo X, Ji L, Zhang Y, Lv P, Cao X, Wang Q, Yan Z, Dong S, Du D, Zhang F, et al. 2020. The Nuclear Matrix Protein SAFB Cooperates with Major Satellite RNAs to Stabilize Heterochromatin Architecture Partially through Phase Separation. Molecular Cell 77: 368-383.e7.

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Can add refunds w/o TPV also please? Link: https://razorpay.com/docs/api/payments/smart-collect/refund-payments/

pueden ser positivos o negativos

The imagery reminds me of devils/demons/hellish creatures? Especially since we've already seen some Christian notes/influences (Paragraph starting with "'God and his holiness.' This is what the Winnebago...")

I still believe religion was somehow founded through the use of hallucinogenics

This passage interests me because it highlights the deep regional differences among early human populations, suggesting they were far more distinct from each other than we are today. The idea that our ancestors might have appeared as different as "hobbits, giants, and elves" challenges my understanding of human diversity and evolution.

For the author, this timeline is based off of what exactly? The Theory of Evolution?

I found this interesting because there are so many different factors and examples that you could consider the "human revolution". A lot of people grew up with religious texts being the only bit of evidence to this early human life and are now making up different conclusions based on new evidence and documenting. With half being 'documented' and half being myths and stories, how do we know that this documented evidence, like early religious texts, are truthful?

Revisar los espacios verticales y horizontales del código, de manera que correspondan a las convenciones, en particular los que indican que código pertenece o está anidado dentro de otro conjunto de intrucciones.

Esta explicación valdría la pena agregar capturas de pantalla que muestren el resultado.

Hacinados significa pequeños. En este caso pequeños o reducidos centros de salud.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

[...] Overall the manuscript is well written, and the successful generation of the new endogenous Cac tags (Td-Tomato, Halo) and CaBeta, stj, and stolid genes with V5 tags will be powerful reagents for the field to enable new studies on calcium channels in synaptic structure, function, and plasticity. There are also some interesting, though not entirely unexpected, findings regarding how Brp and homeostatic plasticity modulate calcium channel abundance. However, a major concern is that the conclusions about how "molecular and organization diversity generate functional synaptic heterogeneity" are not really supported by the data presented in this study. In particular, the key fact that frames this study is that Cac levels are similar at Ib and Is active zones, but that Pr is higher at Is over Ib (which was previously known). While Pr can be influenced by myriad processes, the authors should have first assessed presynaptic calcium influx - if they had, they would have better framed the key questions in this study. As the authors reference from previous studies, calcium influx is at least two-fold higher per active zone at Is over Ib, and the authors likely know that this difference is more than sufficient to explain the difference in Pr at Is over Ib. Hence, there is no reason to invoke differences in "molecular and organization diversity" to explain the difference in Pr, and the authors offer no data to support that the differences in active zone structure at Is vs Ib are necessary for the differences in Pr. Indeed, the real question the authors should have investigated is why there are such differences in presynaptic calcium influx at Is over Ib despite having similar levels/abundance of Cac. This seems the real question, and is all that is needed to explain the Pr differences shown in Fig. 1. The other changes in active zone structure and organization at Is vs Ib may very well contribute to additional differences in Pr, but the authors have not shown this in the present study, and rely on other studies (such as calcium-SV coupling at Is vs Ib) to support an argument that is not necessitated by their data. At the end of this manuscript, the authors have found an interesting possibility that Stj levels are reduced at Is vs Ib, that might perhaps contribute to the difference in calcium influx. However, at present this remains speculative.

Overall, the authors have generated powerful reagents for the field to study calcium channels and how they are regulated, but draw conclusions about active zone structure and organization contributing to functional heterogeneity that are not strongly supported by the data presented.

Reviewer 1 raises an interesting question that we agree will form the basis of important studies. Here, we set out to address a different question, which we will work to better frame. While we and others had previously found a strong correlation between calcium channel abundance and synaptic release probability (Pr (Akbergenova et al., 2018; Gratz et al., 2019; Holderith et al., 2012; Nakamura et al., 2015; Sheng et al., 2012)), more recent studies found that calcium channel abundance does not necessarily predict synaptic strength (Aldahabi et al., 2022; Rebola et al., 2019). Our study explores this paradox and presents findings that provide an explanation: calcium channel abundance predicts Pr among individual synapses of either low-Pr type-Ib or high-Pr type-Is inputs where modulating channel number tunes synaptic strength, but does not predict Pr between the two inputs, indicating an inputspecific role for calcium channel abundance in promoting synaptic strength. Thus, we propose that calcium channel abundance predictably modulates synaptic strength among individual synapses of a single input or synapse subtype, which share similar molecular and spatial organization, but not between distinct inputs where the underlying organization of active zones differs. Consistently, in the mouse, calcium channel abundance correlates strongly with release probability specifically when assessed among homogeneous populations of connections (Aldahabi et al., 2022; Holderith et al., 2012; Nakamura et al., 2015; Rebola et al., 2019; Sheng et al., 2012).

As Reviewer 1 notes, the two-fold difference in calcium influx at type-Is synapses is certainly an important difference underlying three-fold higher Pr. However, growing evidence indicates that calcium influx alone, like calcium channel abundance, does not reliably predict synaptic strength between inputs. For example, Rebola et al. (2019) compared cerebellar synapses formed by granule and stellate cells and found that lower Pr granule synapses exhibit both higher calcium channel abundance and calcium influx. In another example, Aldahabi et al. (2023) demonstrate that even when calcium influx is greater at high-Pr synapses, it does not necessarily explain differences in synaptic strength between inputs. Studying excitatory hippocampal CA1 synapses onto distinct interneuronal targets, they found that raising calcium entry at low-Pr inputs to high-Pr synapse levels is not sufficient to increase synaptic strength to high-Pr synapse levels. Similarly, at the Drosophila NMJ, the finding that type-Ib synapses exhibit loose calcium channel-synaptic vesicle coupling whereas type-Is synapses exhibit tight coupling suggests factors beyond calcium influx also contribute to differences in Pr between the two inputs (He et al., 2023). Consistently, a two-fold increase in external calcium does not induce a three-fold increase in release at low-Pr type-Ib synapses (He et al., 2023). Thus, upon finding that calcium channel abundance is similar at type-Ib and -Is synapses, we focused on identifying differences beyond calcium channel abundance and calcium influx that might contribute their distinct synaptic strengths. We agree that these studies, ours included, cannot definitively determine the contribution of identified organizational differences to distinct release probabilities because it is not currently possible to specifically alter subsynaptic organization, and will ensure that our language is tempered accordingly. However, in addition to the studies cited above and our findings, recent work demonstrating that homeostatic potentiation of neurotransmitter release is accompanied by greater spatial compaction of multiple active zone proteins (Dannhauser et al., 2022; Mrestani et al., 2021) and decreased calcium channel mobility (Ghelani et al., 2023) provide support for the interpretation that subsynaptic organization is a key parameter for modulating Pr.

Reviewer #2 (Public Review):

The authors aim to investigate how voltage-gated calcium channel number, organization, and subunit composition lead to changes in synaptic activity at tonic and phasic motor neuron terminals, or type Is and Ib motor neurons in Drosophila. These neuron subtypes generate widely different physiological outputs, and many investigations have sought to understand the molecular underpinnings responsible for these differences. Additionally, these authors explore not only static differences that exist during the third-instar larval stage of development but also use a pharmacological approach to induce homeostatic plasticity to explore how these neuronal subtypes dynamically change the structural composition and organization of key synaptic proteins contributing to physiological plasticity. The Drosophila neuromuscular junction (NMJ) is glutamatergic, the main excitatory neurotransmitter in the human brain, so these findings not only expand our understanding of the molecular and physiological mechanisms responsible for differences in motor neuron subtype activity but also contribute to our understanding of how the human brain and nervous system functions.

The authors employ state-of-the-art tools and techniques such as single-molecule localization microscopy 3D STORM and create several novel transgenic animals using CRISPR to expand the molecular tools available for exploration of synaptic biology that will be of wide interest to the field. Additionally, the authors use a robust set of experimental approaches from active zone level resolution functional imaging from live preparations to electrophysiology and immunohistochemical analyses to explore and test their hypotheses. All data appear to be robustly acquired and analyzed using appropriate methodology. The authors make important advancements to our understanding of how the different motor neuron subtypes, phasic and tonic-like, exhibit widely varying electrical output despite the neuromuscular junctions having similar ultrastructural composition in the proteins of interest, voltage gated calcium channel cacophony (cac) and the scaffold protein Bruchpilot (brp). The authors reveal the ratio of brp:cac appears to be a critical determinant of release probability (Pr), and in particular, the packing density of VGCCs and availability of brp. Importantly, the authors demonstrate a brp-dependent increase in VGCC density following acute philanthotoxin perfusion (glutamate receptor inhibitor). This VGCC increase appears to be largely responsible for the presynaptic homeostatic plasticity (PHP) observable at the Drosophila NMJ. Lastly, the authors created several novel CRISPRtagged transgenic lines to visualize the spatial localization of VGCC subunits in Drosophila. Two of these lines, CaBV5-C and stjV5-N, express in motor neurons and in the nervous system, localize at the NMJ, and most strikingly, strongly correlate with Pr at tonic and phasic-like terminals.

(1) The few limitations in this study could be addressed with some commentary, a few minor follow-up analyses, or experiments. The authors use a postsynaptically expressed calcium indicator (mhcGal4>UAS -GCaMP) to calculate Pr, yet do not explore the contribution that glutamate receptors, or other postsynaptic contributors (e.g. components of the postsynaptic density, PSD) may contribute. A previous publication exploring tonic vs phasic-like activity at the drosophila NMJ revealed a dynamic role for GluRII (Aponte-Santiago et al, 2020). Could the speed of GluR accumulation account for differences between neuron subtypes?

We did observe that GCaMP signals are higher at type Is synapses, where synapses tend to form later but GluRs accumulate more rapidly upon innervation (Aponte-Santiago et al., 2020). However, because we are using our GCaMP indicator as a plus/minus readout of synaptic vesicle release at mature synapses, we do not expect differences in GluR accumulation to have a significant effect on our measures. Consistently, the difference in Pr we observe between type-Ib and -Is inputs (Fig. 1C) is similar to that previously reported (He et al., 2023; Lu et al., 2016; Newman et al., 2022).

(2) The observation that calcium channel density and brp:cac ratio as a critical determinant of Pr is an important one. However, it is surprising that this was not observed in previous investigations of cac intensity (of which there are many). Is this purely a technical limitation of other investigations, or are other possibilities feasible? Additionally, regarding VGCC-SV coupling, the authors conclude that this packing density increases their proximity to SVs and contributes to the steeper relationship between VGCCs and Pr at phasic type Is. Is it possible that brp or other AZ components could account for these differences. The authors possess the tools to address this directly by labeling vesicles with JanellaFluor646; a stronger signal should be present at Is boutons. Additionally, many different studies have used transmission electron microscopy to explore SVs location to AZs (t-bars) at the Drosophila NMJ.

To date, the molecular underpinnings of heterogeneity in synaptic strength have primarily been investigated among individual type-Ib synapses. However, a recent study investigating differences between type-Ib and -Is synapses also found that the Cac:Brp ratio is higher at type-Is synapses (He et al., 2023).

At this point, we do not know which active zone components are responsible for the organizational (Figs. 1, 2) and coupling (now demonstrated by He et al., 2023) differences between type-Ib and -Is synapses or what establishes the differences in active zone protein levels we observe (Figs. 3,6), although Brp likely plays a local role. We find that Brp is required for dynamically regulating calcium channel levels during homeostatic plasticity and plays distinct roles at type-Ib and -Is synapses (Figs. 3, 4). Brp regulates a number of proteins critical for the distribution of docked synaptic vesicles near T bars of type Ib active zones, including Unc13 (Bohme et al., 2016). Extending these studies to type-Is synapses will be of great interest.

(3) In reference to the contradictory observations that VGCC intensity does not always correlate with, or determine Pr. Previous investigations have also observed other AZ proteins or interactors (e.g. synaptotagmin mutants) critically control release, even when the correlation between cac and release remains constant while Pr dramatically precipitates.

This is an important point as a number of molecular and organizational differences between high- and low-Pr synapses certainly contribute to baseline functional differences. The other proteins we (Figs. 3,6) and others (Dannhauser et al., 2022; Ehmann et al., 2014; He et al., 2023; Jetti et al., 2023; Mrestani et al., 2021; Newman et al., 2022) have investigated are less abundant and/or more densely organized at type-Is synapses. Investigating additional active zone proteins, including synaptic proteins, and determining how these factors combine to yield increased synaptic strength are important next steps.

(4) To confirm the observations that lower brp levels results in a significantly higher cac:brp ratio at phasic-like synapses by organizing VGCCs; this argument could be made stronger by analyzing their existing data. By selecting a population of AZs in Ib boutons that endogenously express normal cac and lower brp levels, the Pr from these should be higher than those from within that population, but comparable to Is Pr. I believe the authors should also be able to correlate the cac:brp ratio with Pr from their data set generally; to determine if a strong correlation exists beyond their observation for cac correlation.

We do not have simultaneous measures of Pr and Cac and Brp abundance. However, our findings suggest that distinct Cac:Brp ratios at type Ib and Is inputs reflect underlying organizational differences that contribute to distinct release probabilities between the two synaptic subtypes. In contrast, within either synaptic subtype, release probability is positively correlated with both Cac and Brp levels. Thus, the mechanisms driving functional differences between synaptic subtypes are distinct from those driving functional heterogeneity within a subtype, so we do not expect Cac:Brp ratio to correlate with Pr among individual type-Ib synapses. We will work to clarify this point in the revised text.

(5) For the philanthotoxin induced changes in cac and brp localization underlying PHP, why do the authors not show cac accumulation after PhTx on live dissected preparations (i.e. in real time)? This also be an excellent opportunity to validate their brp:cac theory. Do the authors observe a dynamic change in brp:cac after 1, or 5 minutes; do Is boutons potentiate stronger due to proportional increases in cac and brp? Also regarding PhTx-induced PHP, their observations that stj and α2δ-3 are more abundant at Is synapses, suggests that they may also play a role in PhTx induced changes in cac. If either/both are overexpressed during PhTx, brp should increase while cac remains constant. These accessory proteins may determine cac incorporation at AZs.

As we have previously followed Cac accumulation in live dissected preparations and found that levels increase proportionally across individual synapses (Gratz et al., 2019), we did not attempt to repeat these challenging experiments at smaller type-Is synapses. We will reanalyze our data to investigate Cac:Brp ratio at individual active zones post PhTx. However, as noted above, we do not expect changes in the Cac:Brp ratio to correlate with Pr among individual synapses of single inputs as this measure reflects organization differences between inputs and PhTx induces an increase in the abundance of both proteins at both inputs.

Determining the effect of PhTx on Stj levels at type-Ib and -Is active zones is an excellent idea and might provide insight into how lower Stj levels correlate with higher Pr at type-Is synapses. While prior studies have demonstrated critical roles for Stj in regulating Cac accumulation during development and in promoting presynaptic homeostatic potentiation (Cunningham et al., 2022; Dickman et al., 2008; Kurshan et al., 2009; Ly et al., 2008; Wang et al., 2016), its regulation during PHP has not been investigated.

Taken together this study generates important data-driven, conceptional, and theoretical advancements in our understanding of the molecular underpinnings of different motor neurons, and our understanding of synaptic biology generally. The data are robust, thoroughly analyzed, appropriately depicted. This study not only generates novel findings but also generated novel molecular tools which will aid future investigations and investigators progress in this field.

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Reviewer #1 (Recommendations For The Authors): 

Major points: 

(1) A central question regarding VGCC differences at Is vs Ib active zones is why is calcium influx higher at Is active zones compared to Ib. Ideally, the authors would have started this study by showing correlations between Cac abundance, presynaptic calcium influx, and Pr at Is vs Ib active zones. If they had, they would likely find that Cac abundance scales with calcium influx and Pr within Is vs Ib, but that calcium influx is over two-fold enhanced at Is over Ib when normalized to the same Cac abundance. This is more than sufficient to explain the Pr differences, so the rest of the study should have focused on revealing why influx is different at Is over Ib despite an apparently similar level of Cac abundance. Then the examination of CaBeta, Stj, etc could have been used to help explain this conundrum. 

A lesson might be gleaned in how to structure this narrative from the Rebola 2019 study, which the authors cite and discuss at length. Similar to the current study, that paper started with two synapses ("strong" vs "weak") and sought to explain why they were so different in synaptic strength. First, they examined presynaptic calcium influx, and surprisingly found that the strong synapse had reduced calcium influx compared to the weak. Then the rest of the paper sought to explain why synaptic strength (Pr) was higher at the strong synapse despite reduced calcium influx. The authors do not use this logical flow and narrative in the present study, despite the focus being on how Cav2 channels contribute to strong vs weak synapses - and the primary function of Cav2 channels is to pass calcium at active zones to drive vesicle fusion. 

Although the authors did not show that presynaptic calcium influx is higher at Is vs Ib active zones in the current manuscript, other studies have previously established that calcium influx is two-fold higher at Is active zones vs Ib (as the authors cite). Rather than focusing so much on Pr at Is vs Ib active zones, which as the authors know can be influenced by myriad differences, it seems the more relevant parameter to study is simply to address presynaptic calcium influx at Is vs Ib, which is the primary function of Cac. Put more simply, if Cac levels are the same at Is vs Ib active zones, why is calcium influx at least two-fold higher at Is? 

It would therefore seem crucial for the authors to determine presynaptic calcium influx levels (ideally at individual AZs) to really understand how Cac intensity levels correlate with calcium influx. The authors instead map Pr at individual AZs, but as the authors know there are many variables that influence whether a SV releases in addition to calcium influx. There are a number of options for this kind of imaging in Drosophila, including genetically encoded calcium indicators targeted to active zones. But since several studies have previously established that influx is higher at Is active zones over Ib, this may not be necessary. That being said, there is a lot of value in quantitatively analyzing Cac/Stj/CaBeta abundance, calcium influx, and Pr together at individual active zones.

We appreciate the perspective that we could have focused on why Ca2+ influx is 2x greater at type Is active zones, which we agree is an important and interesting question. However, growing evidence indicates that Ca2+ influx alone, like Ca2+ channel abundance, does not reliably predict synaptic strength between inputs. So, here we focused instead on how other differences between synapses influence Pr and contribute to synaptic heterogeneity between and/or among synapses formed by strong and weak inputs. We have changed our title and framing to better reflect this focus. 

As Reviewer 1 notes, Rebola et al. (2019) found that lower Pr granule synapses exhibit higher Ca2+ influx (and Ca2+ channel abundance). In another example, Aldahabi et al. (2022) demonstrated that even when Ca2+ influx is greater at high-Pr synapses, it does not necessarily explain differences in synaptic strength as raising Ca2+ entry at low-Pr synapses to high-Pr synapse levels was not sufficient to increase synaptic strength to high-Pr input levels. Similar findings have been reported at tonic and phasic synapses of the Crayfish NMJ (Msghina, 1999).

Several lines of evidence argue that factors beyond Ca2+ influx also play important roles in establishing distinct release properties at the Drosophila NMJ. A recent study using using a botulinum transgene to isolate type Ib and Is synapses for electrophysiological analysis found that increasing external [Ca2+] from physiological levels (1.8 mM) to 3 mM or even 6 mM does not result in a 3-fold increase in EPSCs or quantal content at type Ib synapses despite the prediction that the increase would be even greater given the power dependence of release on between Ca2+ concentration (He et al., 2023). The authors further found that type Ib synapses are more sensitive than type Is synapses to the slow Ca2+ chelator EGTA, indicating looser Ca2+ channel-SV coupling. 

Consistently, we find that although VGCC levels are similar at the two inputs, their density is greater at type Is active zones (Figs. 1 and 2). Our findings also reveal additional molecular differences that may contribute to the observed differences in neurotransmitter release properties between the two inputs, including lower levels of the active zone protein Brp (Fig 3) and the auxiliary subunit α2δ-3/Stj (Fig. 6) at high Pr type Is inputs. In contrast, levels of each of these proteins positively correlate with synaptic strength among active zones of a single input, whether low- or high-Pr (Figs. 1, 3, 6). Similarly, levels of each of these proteins increase during homeostatic potentiation of neurotransmitter release (Figs. 4 and 7). Thus, we propose that two broad mechanisms contribute to synaptic diversity in the nervous system: (1) spatial organization and relative molecular content establish distinct average basal release probabilities that differ between inputs and (2) among individual synapses of distinct inputs, coordinated modulation of Ca2+ channel and active zone protein abundance independently tunes Pr. These intersecting mechanisms provide a framework for understanding the extensive and dynamic synaptic diversity observed across nervous systems.

(2) In addition to key points made above, it seems the authors should at least consider (if not experimentally test) what other differences might contribute to the higher calcium influx at Is over Ib:  

- Distinct splice isoforms of Cac (and/or Stj/Cabeta): The recent RNAseq analysis of gene expression at Is vs Ib motor neurons from Troy Littleton's group may inform this consideration? 

- Stj reduction at Is: Do channel studies in heterologous systems give any insight into VGCC channel function with and without a2d-3? Do Cav2 channels without a2d pass more calcium? This would then offer an obvious solution to the key conundrum underlying this study. 

These are excellent questions that we are actively pursuing. While there is no evidence of differentially expressed splice isoforms of Stj or Ca-β in the recent RNA-seq data from Jetti et al., 2023, subtle changes in Cac isoform usage were observed that may contribute to differences in Ca2+ influx. In heterologous systems, α2δ expression generally increases Ca2+ channel membrane insertion and  Ca2+ currents. However, in vivo α2δ’s can also mediate extracellular interactions that may modulate channel function. We address these points in greater detail in the revised discussion.  

(3) Assess Stj and CaBeta levels at AZs after PhTx: The successful generation of endogenously tagged Stj and CaBeta enables some relatively easy experiments that would be of interest, similar to what the authors present for Cac. Does Brp similarly control Stj and CaBeta at Is vs Ib compared to what they show for Cac? In addition, does homeostatic plasticity similarly change Stj and CaBeta at Is vs Ib compared to what the authors have shown for Cac? i.e., do they both similarly increase in intensity, by the same amount, as Cac? 

We agree and have included an analysis of α2δ-3/Stj levels following PhTx exposure (Fig. 7A-C). We have also investigated the regulation of Stj during chronic presynaptic homeostatic potentiation (Fig. 7D-F). In both cases, StjV5-N levels significantly increase at type Ib and Is active zones, consistent with our finding that among AZs of either type Ib or Is inputs, Stj levels correlate with Cac abundance and, thus, Pr. Together with our and others’ findings, this suggests that coordinated increases Ca2+ channel, auxiliary subunit,  and active zone protein abundance positively tunes synaptic strength at diverse synaptic subtypes.

Minor points: 

(1) Including line numbers would make reviewing/commenting easier. 

We apologize for this oversight and have added line numbers to the revised manuscript.

(2) Fig. 2I: It is not apparent what the mean cluster density is between Ib vs Is (as it is in Fig. 2F-H graphs). The mean and error bars should be included in 2I as it is in 2G. Same with Fig. 3C. 

Thank you for pointing this out. We have added error bars to the paired analysis in 2I as well as in 3C and 1C.

(3) Fig. 4 - it might make more sense to normalize Brp and Cac intensity as a percentage of baseline (PhTx at Is or Ib) rather than normalizing everything to control Ib. 

We have revised the graphs as suggested in Figure 4 and throughout.

(4) Page 5 bottom - REFS missing after Fig. 1E. 

Thank you for catching this. We have fixed it.

Reviewer #2 (Recommendations For The Authors): 

This reader found differentiating between low Pr sites (deep purple) and cac measurements (black) difficult in Fig 1B. You may consider depicting this differently. 

Thank you for this feedback. We have changed the color scheme to improve readability.

I found it difficult to discern the difference between experiments Fig 1E and Fig 1J. Why are individual dots distributed differently? 

The individual data points are the same as in 1E and 1F, but we have removed the individual NMJ dimensionality to combine all Is and Ib data points together along with best fit lines for comparison of their slopes. We have added text to the revised manuscript to clarify this.

Results section, second paragraph, add references, remove 'REF': We next investigated the correlation between Pr and VGCC levels and found that at type Is inputs, single-AZ Cac intensity positively correlates with Pr (Fig. 1E; REFS). 

Thank you. We have corrected this error.

Acidosis se define como cualquier trastorno que aumenta la ca entidad de acido en la sangre y puede clasificarse como respiratoria o metabólica (ácidos volátiles o no volátiles)

La acidosis metabolica primaria la acidemia estimula los centros respiratorios para aumentar la respiración con la consiguiente disminucion de la PCO2.

La compensación renal comienza >6-12 horas de acidemia o alcalemia sostenida. Acidosis respiratoria primaria los riñones aumentan la producción de bicarbonato

se refiere a algo o alguien que es descuidado, desordenado o que no presta atención a los detalles. Puede aplicarse a personas que no se esmeran en sus tareas o al resultado de un trabajo que se hizo sin cuidado.

Dal 1939 in poi venne resa obbligatoria la vaccinazione antidifterica ma nessuno dei curatori di questa voce su wikipedia ha mai fatto riferimento che sia il periodo antecedente che quello successivo al 1939 (1932-1940) venne costellato da eventi di numerosi bambini morti o resi invalidi in Italia proprio a causa dei sieri antidifterici. Il fatto più eclatante, la Strage di Gruaro, avvenne nel periodo aprile-maggio 1933, durante il quale 253 bambini residenti nel Comune di Gruaro (VE) vennero costretti con la forza di legge a vaccinarsi con questi sieri. Morirono in 28 e le inchieste in merito, depositate presso l'Archivio di Stato di Venezia, vennero fatte sparire in breve tempo. Nessuno ha mai reso onore alla memoria di questi bambini oggetto di infanticidio di Stato. Non fidatevi del racconto che wikipedia fa della Strage di Gruaro la cui giustificazione fu errata sperimentazione. Si trattò invece di "caccia" ai bambini per "provare" gli effetti e non di sperimentazione consensuale... Il vaccino venne somministrato ai bimbi dai tredici mesi agli otto anni, i decessi si verificarono a causa di paralisi ma non risulta alcuna indagine giudiziaria avviata per accertare le responsabilità Leggi di più qui: «Noi, bambini scelti come cavie». La verità sulla strage di Gruaro del '33

Assunto completamente privo di fondamento scientifico in quanto la responsabilità di coloro che decidono di non vaccinarsi non può essere dimostrata scientificamente poiché è falsa l'assunzione che l'immunità di gregge indotta dai vaccini protegge la popolazione...

Welcome back and in this demo lesson you're going to configure S3 cross-region replication.

So that's a replication of objects from one bucket to another bucket in different AWS regions.

And the scenario that we're going to be stepping through is where you as a systems engineer are looking to configure replication to allow disaster recovery of an S3 static website from one region to another AWS region.

So the first thing that I'll need you to do is to make sure that you're logged in to the general AWS account and you'll need to have the Northern Virginia region selected.

Assuming that's the case then go ahead and move to the S3 console because we're going to be creating our source and destination buckets.

Now if you see any notifications about updates to the UI or any additional functionality then just go ahead and close that down.

What we want you to do is to go ahead and click on create bucket.

Now we want to keep these names simple so that we can distinguish between source and destination bucket.

So for the source bucket we're going to start with source bucket and then we want you to use your initials in my case AC and then we want you to put a random number at the end and I'm going to attempt to use 1337.

For region for the source bucket let's use US - East - 1 and then scroll down past the block public access settings scroll down past bucket versioning and default encryption and then just create the bucket.

In my case it successfully created a bucket using this name and again because of the globally unique naming requirements of S3 buckets you need to make sure that you pick something different than me and different from other students.

Now the scenario is that we're using this bucket to host a static S3 website so we need to enable that functionality.

So go into source bucket, click on properties, scroll down all the way to the bottom and click on edit next to static website hosting and we need to enable static website hosting and for hosting type just make sure that you select host a static website.

Now we're going to use index.html for both the index document and for the error document.

So enter index.html in both of those boxes and once you've done that you can save those changes.

Now so that this bucket can host a static website we need to go to permissions and then we need to edit the block public access settings so click on edit and uncheck block all public access and once you've done so click on save changes.

You need to confirm that so follow the instructions and then confirm and this means that the bucket can be made public but in order to make it public we need to add a bucket policy.

So to edit the bucket policy scroll down below the block public access settings and then edit the bucket policy.

Now attached to this lesson is a demo files link.

I'll need you to click that link which will download a zip file.

Go ahead and extract that zip file which will create a folder and go inside that folder.

Containment in this folder are all the files which you'll need for this demo and inside that folder is a file called bucket_policy.json.

Go ahead and open that file so this is the file bucket_policy.

This is a bucket policy which allows any principal to use the star tile card to use the S3 get object action on this particular ARN.

Now this is a placeholder we need to update this placeholder so that it references any objects within our source bucket so I want you to copy this entire bucket policy into your clipboard, move back to the console, paste it in and once you've pasted it in go ahead and copy the bucket ARN for this bucket into your clipboard by clicking on this icon and then I want you to select the placeholder that you've just pasted in so straight after the first speech mark all the way up to before the forward slash so select this component of this placeholder ARN and paste it in the source bucket ARN so it should look like this.

It references ARN colon, ALUS colon, S3 and then colon, colon, colon and then the source bucket so whatever you've called your source bucket it should reference this so don't use what's on my screen, use your specific source bucket ARN and then you should have a forward slash star on the end.

That means that any anonymous or unauthenticated identity will be able to get any objects within this S3 bucket and that's what we want, we want this to be a public bucket.

So click on save changes to commit those changes and now this bucket is public, you get the warning under permissions overview that this bucket is public and under the bucket name at the top it will say publicly accessible.

Now once you've done that go back to the S3 console and we're going to create the destination bucket, we're going to follow exactly the same process so click on create bucket, this time we'll call it destination bucket and then again your initials and then ideally the same random number so in my case 1-3-3-7.

Go ahead and click on the region drop down and instead of picking US-EAST-1 this time we're going to use US-WEST-1 Now to save us some time we're going to uncheck this block all public access while we're creating the bucket so that we don't need to do that afterwards and we'll need to acknowledge that by checking this box, scroll all the way down to the bottom and then click on create bucket, then we're going to go into destination bucket, select properties, move down to the bottom and we'll need to enable static website hosting so click edit, enable, choose the option to host a static website and then just like before we're going to use index.html for both the index document and the error document so enter index.html in both of those boxes and then save those changes.

Then we'll need to go to permissions and edit the bucket policy so scroll down, click on edit for bucket policy you'll need to copy this template bucket policy into your clipboard, paste it into the policy box and again we need to replace this placeholder so copy the destination bucket ARN into your clipboard, select from after the speech mark through to before the forward slash paste that in and that will now reference any objects in the destination bucket and go ahead and click on save changes so now at this point we have the source and destination bucket, both of them are public, both of them are set to be static websites and neither of them have any objects inside that bucket.

The next step is to enable cross region replication from the source bucket through to the destination bucket so to do that click on the source bucket, click on the management tab, scroll down and we need to configure a replication rule so click on create replication rule, we're told that replication requires versioning to be enabled for the source bucket and we're given a convenient button to click to enable versioning on this bucket and that's what we're going to do so click on enable bucket versioning and this means this bucket can now be the source of this replication rule for replication rule name, I'm going to call it static website dr for disaster recovery we want it to be enabled straight away so make sure the status is set to enabled now we can limit this replication rule to have a certain scope, we can filter it based on prefix or tags and this allows us to replicate only part of this bucket, in our case we want to replicate the entire bucket so we're going to select the option that this rule applies to all objects in the bucket so check this option now that we've configured the source, so we've configured the bucket name, the source region and chosen the scope of this replication rule now we need to decide on the destination, now for the destination we can use a bucket within the same AWS account or we can specify a bucket within another AWS account, if we choose another AWS account then we need to worry about object ownership we need to make sure that the objects which are replicated into the destination account have the correct ownership because by default they will be owned by our account because we will be creating them we can specify as part of the replication rule that the destination account owns those objects but because we're using the same account that doesn't apply in this scenario so we're going to choose a bucket within this account, click on browse s3 and we're going to select the destination bucket that we just created so select the destination bucket that you created in the previous step and then click on choose path you're going to be informed that as well as replication requiring versioning on the source bucket it also is required on the destination bucket and again you're presented with a convenient button to enable versioning on this destination bucket so click on enable bucket versioning, just to confirm versioning is required on both the source and destination bucket when you're using s3 replication so that's the destination configured, we've picked the destination bucket and enabled versioning, so scroll down next we need to give this replication rule the permissions that it needs to interact with different AWS resources so this rule needs the ability to read from the source bucket and write those objects into the destination bucket so we need to give this replication rule an IAM role which provides those permissions so click in the choose IAM role drop down and select create new role you could select an existing one if you already had one which was preconfigured but for this demo lesson we've done so we're going to create a new role now you can enable the ability of s3 replication to support objects which are encrypted with AWS KMS you do have to select that option explicitly though when you're creating a replication rule and in our case we're not going to be replicating any encrypted objects using sse-kms so we can leave this unchecked I've talked about this in the theory lesson but you can also change the storage class as part of the replication process so generally you're replicating objects from a primary location or a source bucket through to a destination bucket and this is often part of disaster recovery scenarios where you often want cheaper storage at the destination end and so the default is to maintain the storage class that's used in the source bucket but you can override that and change the storage class which is used when storing those replicated objects in the destination bucket now we're not going to set that in this demonstration but you do have the option to do so now these are all relatively new features of s3 replication so s3 replication has been a feature available at AWS for some time but over time they've evolved and enhanced the feature sets available to the product so you're able to set replication time control or RTC and this imposes an SLA on the replication process so by enabling it you ensure that 99.99% of new objects are replicated within 15 minutes and this feature also provides access to replication metrics and notifications but do note that this does come at an additional cost because s3 versioning is used on both the source and destination buckets if you do delete an object in a source bucket then by default that deletion is not replicated through to the destination so by default s3 replication does not replicate delete markers and I've talked about delete markers elsewhere in the course by selecting this option you do replicate delete markers and that means that deletions are replicated from the source through to the destination for now though we're not going to select any of these just go ahead and click on save after a few moments you'll be presented with this option so the default s3 replication historically didn't replicate any objects which existed in a bucket before you enabled replication you're now offered the ability to replicate existing objects within a bucket so if you wanted to do this if you had a bucket with objects that already existed then you could replicate those as part of starting this process now our bucket is empty so we won't do this anyway so go ahead and select no do not replicate existing objects and click submit and at this point replication is enabled between our source bucket and our destination bucket so let's give it a try what we're going to do is click at the top here next to the source bucket name and this will take us to the top level of the source bucket and we're going to upload some objects so go ahead and click on upload and then click on add files at this point go ahead and locate the folder which you downloaded and extracted earlier in this demo lesson so the demo files folder so go inside that folder and inside here as well as the bucket policy template which you used earlier you'll see two folders website and website 2 I want you to expand the website and select both the aotm.jpg object and the index.html object select both of those and make sure you use the ones from the website folder not the website 2 folder and then click on open once they're selected for upload you need to scroll down to the bottom and click upload and now upload both of those objects to our source bucket once that's complete we can close down that dialog then I want you to click on properties we're going to move down to the bottom and we're going to copy the bucket website endpoint into our clipboard and then open that in a new tab so this is the source bucket website endpoint and this opens animalsforlife.org animal.monthformarch and this is a picture of my cat Winky so let's go back to the S3 console move to the top go back to the main S3 console and then move in to the destination bucket now don't be concerned if you see something slightly different at this point the timetaping for replicating objects from source to destination can vary wildly if you don't enable the replication SLA option in our case because these objects are relatively small they should be replicated fairly quickly now at this point I want you to go ahead and pause this video and wait until you see two objects in your destination bucket don't be alarmed if these objects take five or ten minutes to appear just wait pause the video wait for those objects to appear and then you're good to continue you'll see that we have both of the objects which we added to the source bucket they're both now stored in the destination bucket and that's because they've been replicated using S3 replication what we can do is go to properties, move all the way down to the bottom copy the destination bucket website endpoint into our clipboard and open that in a new tab so this is the destination static website endpoint and again we see the same website animalsforlife.org animal.monthformarch again my cat Winky so next we're going to move back to the main S3 console so click on Amazon S3 and then move back to the source bucket click on upload again and we're going to replace the objects that we just added so click on add files and then expand the website to folder and go ahead and upload both of these objects so we have the same name O T N dot JPEG and index dot HTML go ahead and upload both of these and make sure you're uploading to the source bucket so upload those you'll need to confirm that that'll take a few moments to complete once you see that the upload was successful you can close down this dialogue and then you should still have the tab open to the source bucket if you go to that tab and refresh you'll see now that we have animalsforlife.org animal.monthforapril and this is my cat Truffles if we go immediately to the destination bucket and hit refresh on this bucket you might still see the picture of Winky if the image doesn't change and you keep refreshing it just give it a few minutes because without the replication SLA then the time taken to replicate from source to destination can vary significantly in my case it took about three or four minutes of refreshers but now we see that the destination bucket has been updated with the new image again this is a picture of my cat Truffles so this has been a very simple example of cross region replication at this point I've got everything that I wanted to do in this demo lesson so all we need to do is to clean up the account and return it to the same state as it was at the start of the demo so close down both of these tabs and just return to the main S3 console select the destination bucket and click empty confirm this by following the instructions and then confirm click exit with the bucket still selected, delete the bucket again you need to follow the instructions to confirm deletion of this bucket so go ahead and do that and then we need to follow the same process for the source bucket so select it and empty it, confirm that, close down this dialogue and then delete the bucket and again you need to confirm that, follow those instructions and delete bucket then we need to click on services, move to the IAM console because remember S3 replication created an IAM role that was used before that replication so click on roles, locate the role which starts with S3 C R R and it will have the name of the source bucket as part of that role name so go ahead and select the correct role, make sure that you select the one which has the name of your source bucket and then delete that role and you need to confirm that process and that will delete the role and at this point that's the account back in the same state as it was at the start of this demo lesson so I hope you've enjoyed this demo and a simple example of how to use S3 replication specifically in this case cross region replication from a source to a destination bucket at this point that's everything that I wanted to do, I'll be talking about S3 replication elsewhere in the course in other associate courses and at the professional level so don't worry, you'll get plenty of opportunities to explore this specific ease of functionality available as part of S3 but at this point that's everything I wanted to do in this demo lesson go ahead, complete the video and when you're ready I'll look forward to you joining me in the next.

Author response:

We are grateful for the reviewers' acknowledgment of the originality of our manuscript and its potential importance in cancer treatment. We appreciate the reviewers' critiques on certain conclusions and thank them for their thorough feedback on the manuscript. In the revised version, we will provide a more detailed clarification of the previous data and methods, bolster the existing data, and present additional evidence in support of our hypothesis. Please find below our replies to particular concerns.

In brief, to address the comments from Reviewer 1, we will make the following revisions in the manuscript:

(1) To discuss the issues regarding the specificity of ATP5⍺ CAT-tailing, we will provide new patient-derived cell lines and tumor samples and investigate the CAT-tail modifications of nuclear genome-encoded mitochondrial proteins and changes in RQC proteins within them. We will endeavor to explore the nature of NEMF modifications in GSC cells (Fig. S1A).

(2) To enhance the quality of image data, we will substitute some images (such as Fig. 1E and 3A) with higher quality images.

(3) To further understand the influence of NEMF on cancer, the effects of NEMF overexpression in GSC cells will be evaluated through testing (e.g., Fig. 3D).

(4) To further explore changes in apoptosis, we will employ additional methods to detect apoptosis, including Annexin-PI FACS assays, caspase cleavage analysis, assessing BAX-BCL2 ratios, and monitoring cytochrome c release.

(5) To further confirm the effectiveness of the CAT-tailing-mitochondria mechanism in in vivo tumor models, we will utilize a Drosophila model to study the impact of the RQC pathway and CAT-tailing mechanism on tumor proliferation in vivo. The overactivation of the Notch signaling pathway in Drosophila can stimulate malignant proliferation of neural stem cells (NSCs) through both canonical (c-Myc mediated pathway) and non-canonical (PINK1-mitochondrial-mTORC2 pathway) pathways, leading to the development of a tumor-like phenotype in the larval brain. A recent publication in PNAS Nexus (Khaket et al., PNAS Nexus, 2024) discusses the impact of the RQC pathway on c-Myc. It is possible for us to analyze the alterations in CAT-tailing on mitochondrial proteins and mitochondrial membrane potential in this Notch model and study how the RQC pathway regulates them. Moreover, tumor implantation experiments will be carried out using immunodeficient mice. Our goal is to conduct a comparative analysis of the growth of control and NEMF KD glioblastoma cell lines in animal models, alongside performing essential biochemical analyses.

Reference:

Khaket, T. P., et al. (2024). Ribosome stalling during c-myc translation presents actionable cancer cell vulnerability. PNAS nexus, 3(8), pgae321.

To address the comments from Reviewer 2, we will make the following revisions in the manuscript:

(1) The concerns raised by the reviewer regarding the authenticity of the ATP5a CAT-tail modification are duly noted. Critical control experiments will be incorporated into our study, including NEMF knockout (or NFACT domain mutants) and cycloheximide treatment, alongside other methodologies. The results of these experiments will include placements such as Fig. 1B, 1C, S3A, and S3B to improve comprehension of the CAT-tail modification on ATP5⍺.

(2) We thank the reviewer for reminding us to consider the differences between the artificial tail and the endogenous CAT-tail. A recently published study (Khan et al., 2024) provides a thorough analysis of the components of the CAT-tail. Our approach to addressing this issue involves emphasizing the use of the artificial CAT-tail sequence and adopting a more measured tone in the revised version. Additionally, we will induce the endogenous ATP5⍺-CAT-tail by express ATP5⍺-K20-non-stop in cells to validate their function in glioblastoma cells.

(3) Moreover, we aim to examine the impact of different amino acid compositions in the ATP5⍺ c-terminus extension, such as the poly (Gly-Ser) repeats noted by the reviewer, on both mitochondrial function and glioblastoma biology in our revision. By comparing the results obtained from ATP5⍺-CAT-tails with different compositions, it is anticipated that more definitive conclusions can be drawn.

(4) Additional minor revisions will be implemented to the text in accordance with the feedback given by the reviewer.

Reference:

Khan, D., Vinayak, A. A., Sitron, C. S., & Brandman, O. (2024). Mechanochemical forces regulate the composition and function of CAT tails. bioRxiv, 2024-08.