Reviewer #3 (Public review):

Summary:

The authors used the model organism Drosophila melanogaster to show that the neurotrophin Toll-6 and its ligands, DNT-2 and kek-6, play a role in maintaining the number of dopaminergic neurons and modulating their synaptic connectivity. This supports previous findings on the structural plasticity of dopaminergic neurons and suggests a molecular mechanism underlying this plasticity.

Strengths:

The experiments are overall very well designed and conclusive. Methods are in general state-of-the-art, the sample sizes are sufficient, the statistical analyses are sound, and all necessary controls are in place. The data interpretation is straightforward, and the relevant literature is taken into consideration. Overall, the manuscript is solid and presents novel, interesting, and important findings.

Weaknesses:

There are three technical weaknesses that could perhaps be improved.

First, the model of reciprocal, inhibitory feedback loops (Figure 2F) is speculative. On the one hand, glutamate can act in flies as an excitatory or inhibitory transmitter (line 157), and either situation can be the case here. On the other hand, it is not clear how an increase or decrease in cAMP level translates into transmitter release. One can only conclude that two types of neurons potentially influence each other.

Second, the quantification of bouton volumes (no y-axis label in Figure 5 C and D!) and dendrite complexity are not convincingly laid out. Here, the reader expects fine-grained anatomical characterizations of the structures under investigation, and a method to precisely quantify the lengths and branching patterns of individual dendritic arborizations as well as the volume of individual axonal boutons.

Third, Figure 1C shows two neurons with the goal of demonstrating between-neuron variability. It is not convincingly demonstrated that the two neurons are actually of the very same type of neuron in different flies or two completely different neurons.

Author response:

Reviewer #1 (Public review):

Summary:

Sun et al. are interested in how experience can shape the brain and specifically investigate the plasticity of the Toll-6 receptor-expressing dopaminergic neurons (DANs). To learn more about the role of Toll-6 in the DANs, the authors examine the expression of the Toll-6 receptor ligand, DNT-2. They show that DNT-2 expressing cells connect with DANs and that loss of function of DNT-2 in these cells reduces the number of PAM DANs, while overexpression causes alterations in dendrite complexity. Finally, the authors show that alterations in the levels of DNT-2 and Toll-6 can impact DAN-driven behaviors such as climbing, arena locomotion, and learning and long-term memory.

Strengths:

The authors methodically test which neurotransmitters are expressed by the 4 prominent DNT-2 expressing neurons and show that they are glutamatergic. They also use Trans-Tango and Bac-TRACE to examine the connectivity of the DNT-2 neurons to the dopaminergic circuit and show that DNT-2 neurons receive dopaminergic inputs and output to a variety of neurons including MB Kenyon cells, DAL neurons, and possibly DANS.

We are very pleased that Reviewer 1 found our connectivity analysis a strength.

Weaknesses:

(1) To identify the DNT-2 neurons, the authors use CRISPR to generate a new DN2-GAL4. They note that they identified at least 12 DNT-2 plus neurons. In Supplementary Figure 1A, the DNT-2-GAL4 driver was used to express a UAS-histoneYFP nuclear marker. From these figures, it looks like DNT-2-GAL4 is labeling more than 12 neurons. Is there glial expression?

Indeed, we claimed that DNT-2 is expressed in at least 12 neurons (see line 141, page 6 of original manuscript), which means more than 12 could be found. The membrane tethered reporters we used – UAS-FlyBow1.1, UASmcD8-RFP, UAS-MCFO, as well as UAS-DenMark:UASsyd-1GFP – gave a consistent and reproducible pattern. However, with DNT-2GAL4>UAS-Histone-YFP more nuclei were detected that were not revealed by the other reporters. We have found also with other GAL4 lines that the patterns produced by different reporters can vary. This could be due to the signal strength (eg His-YFP is very strong) and perdurance of the reporter (e.g. the turnover of His-YFP may be slower than that of the other fusion proteins).

We did not test for glial expression, as it was not directly related to the question addressed in this work.

(2) In Figure 2C the authors show that DNT-2 upregulation leads to an increase in TH levels using q-RT-PCR from whole heads. However, in Figure 3H they also show that DNT-2 overexpression also causes an increase in the number of TH neurons. It is unclear whether TH RNA increases due to expression/cell or the number of TH neurons in the head.

Figure 3H shows that over-expression of DNT-2 FL increased the number of Dcp1+ apoptotic cells in the brain, but not significantly (p=0.0939). The ability of full-length neurotrophins to induce apoptosis and cleaved neurotrophins promote cell survival is well documented in mammals. We had previously shown that DNT-2 is naturally cleaved, and that over-expression of DNT-2 does not induce apoptosis in the various contexts tested before (McIlroy et al 2013 Nature Neuroscience; Foldi et al 2017 J Cell Biol; Ulian-Benitez et al 2017 PLoS Genetics). Similarly, throughout this work we did not find DNT-2FL to induce apoptosis.

Instead, in Figure 3G we show that over-expression of DNT-2FL causes a mild yet statistically significant increase in the number of TH+ cells. This is an important finding that supports the plastic regulation of PAM cell number. We thank the Reviewer for highlighting this point, as we had forgotten to add the significance star in the graph. In this context, we cannot rule out the possibility that the increase in TH mRNA observed when we over-express DNT-2FL could not be due to an increase in cell number instead. Unfortunately, it is not possible for us to separate these two processes at this time. Either way, the result would still be the same: an increase in dopamine production when DNT-2 levels rise.

(3) DNT-2 is also known as Spz5 and has been shown to activate Toll-6 receptors in glia (McLaughlin et al., 2019), resulting in the phagocytosis of apoptotic neurons. In addition, the knockdown of DNT-2/Spz5 throughout development causes an increase in apoptotic debris in the brain, which can lead to neurodegeneration. Indeed Figure 3H shows that an adult-specific knockdown of DNT-2 using DNT2-GAL4 causes an increase in Dcp1 signal in many neurons and not just TH neurons.

Indeed, we did find Dcp1+ cells in TH-negative cells too (although not widely throughout the brain). This is not surprising, as DNT-2 neurons have large arborisations that can reach a wide range of targets; DNT-2 is secreted, and could reach beyond its immediate targets; Toll-6 is expressed in a vast number of cells in the brain; DNT-2 can bind promiscuously at least also Toll-7 and other Keks, which are also expressed in the adult brain (Foldi et al 2017 J Cell Biology; Ulian-Benitez et al 2017 PLoS Genetics; Li et al 2020 eLife). Together with the findings by McLaughlin et al 2019, our findings further support the notion that DNT-2 is a neuroprotective factor in the adult brain. It will be interesting to find out what other neuron types DNT-2 maintains.

We would like to thank Reviewer 1 for their positive comments on our work and their interesting and valuable feedback.

Reviewer #2 (Public review):

This paper examines how structural plasticity in neural circuits, particularly in dopaminergic systems, is regulated by Drosophila neurotrophin-2 (DNT-2) and its receptors, Toll-6 and Kek-6. The authors show that these molecules are critical for modulating circuit structure and dopaminergic neuron survival, synaptogenesis, and connectivity. They show that loss of DNT-2 or Toll-6 function leads to loss of dopaminergic neurons, dendritic arborization, and synaptic impairment, whereas overexpression of DNT-2 increases dendritic complexity and synaptogenesis. In addition, DNT-2 and Toll-6 modulate dopamine-dependent behaviors, including locomotion and long-term memory, suggesting a link between DNT-2 signaling, structural plasticity, and behavior.

A major strength of this study is the impressive cellular resolution achieved. By focusing on specific dopaminergic neurons, such as the PAM and PPL1 clusters, and using a range of molecular markers, the authors were able to clearly visualize intricate details of synapse formation, dendritic complexity, and axonal targeting within defined circuits. Given the critical role of dopaminergic pathways in learning and memory, this approach provides a good opportunity to explore the role of DNT-2, Toll-6, and Kek-6 in experience-dependent structural plasticity. However, despite the promise in the abstract and introduction of the paper, the study falls short of establishing a direct causal link between neurotrophin signaling and experience-induced plasticity.

Simply put, this study does not provide strong evidence that experience-induced structural plasticity requires DNT-2 signaling. To support this idea, it would be necessary to observe experience-induced structural changes and demonstrate that downregulation of DNT-2 signaling prevents these changes. The closest attempt to address this in this study was the artificial activation of DNT-2 neurons using TrpA1, which resulted in overgrowth of axonal arbors and an increase in synaptic sites in both DNT-2 and PAM neurons. However, this activation method is quite artificial, and the authors did not test whether the observed structural changes were dependent on DNT-2 signaling. Although they also showed that overexpression of DNT-2FL in DNT-2 neurons promotes synaptogenesis, this phenotype was not fully consistent with the TrpA1 activation results (Figures 5C and D).

In conclusion, this study demonstrates that DNT-2 and its receptors play a role in regulating the structure of dopaminergic circuits in the adult fly brain. However, it does not provide convincing evidence for a causal link between DNT-2 signaling and experience-dependent structural plasticity within these circuits.

We would like to thank Reviewer 2 for their very positive assessment of our approach to investigate structural circuit plasticity. We are delighted that this Reviewer found our cellular resolution impressive. We are also very pleased that Reviewer 2 found that our work demonstrates that DNT-2 and its receptors regulate the structure of dopaminergic circuits in the adult fly brain. This is already a very important finding that contributes to demonstrating that, rather than being hardwired, the adult fly brain is plastic, like the mammalian brain.

We are very pleased that this Reviewer acknowledges that this work provides a good opportunity to explore the role of DNT-2, Toll-6, and Kek-6 in experience-dependent structural plasticity. We provide a molecular mechanism and proof of principle, and we demonstrate a direct link between the function of DNT-2 and its receptors in circuit plasticity, and a suggestive link to neuronal activity. Finding out the direct link to lived experience is a big task, beyond the scope of this manuscript, and we will be testing this with future projects. Nevertheless, it is important to place our findings within this context, as it opens opportunities for discovery by the neuroscience community.

We would like to thank Reviewer 2 for the positive and thoughtful evaluation of our work, and for their feedback.

Reviewer #3 (Public review):

Summary:

The authors used the model organism Drosophila melanogaster to show that the neurotrophin Toll-6 and its ligands, DNT-2 and kek-6, play a role in maintaining the number of dopaminergic neurons and modulating their synaptic connectivity. This supports previous findings on the structural plasticity of dopaminergic neurons and suggests a molecular mechanism underlying this plasticity.

Strengths:

The experiments are overall very well designed and conclusive. Methods are in general state-of-the-art, the sample sizes are sufficient, the statistical analyses are sound, and all necessary controls are in place. The data interpretation is straightforward, and the relevant literature is taken into consideration. Overall, the manuscript is solid and presents novel, interesting, and important findings.

We are delighted that Reviewer 3 found our work solid, novel, interesting and with important findings. We are also very pleased that this Reviewer found that all necessary controls have been carried out.

Weaknesses:

There are three technical weaknesses that could perhaps be improved.

First, the model of reciprocal, inhibitory feedback loops (Figure 2F) is speculative. On the one hand, glutamate can act in flies as an excitatory or inhibitory transmitter (line 157), and either situation can be the case here. On the other hand, it is not clear how an increase or decrease in cAMP level translates into transmitter release. One can only conclude that two types of neurons potentially influence each other.

Thank you for pointing out that glutamate can be inhibitory. In mammals, the neurotrophin BDNF has an important function in glutamatergic synapses, thus we were intrigued by a potential evolutionary conservation. Our evidence that DNT-2A neurons could be excitatory is indirect, yet supportive: exciting DNT-2 neurons with optogenetics resulted in an increase in GCaMP in PAMs (data not shown); over-expression of DNT-2 in DNT-2 neurons increased TH mRNA levels; optogenetic activation of DNT-2 neurons results in the Dop2R-dependent downregulation of cAMP levels in DNT-2 neurons. Dop2R signals in response to dopamine, which would be released only if dopaminergic neurons had been excited. Accordingly, glutamate released from DNT-2 neurons would have been rather unlikely to inhibit DANs.

cAMP is a second messenger that enables the activation of PKA. PKA phosphorylates many target proteins, amongst which are various channels. This includes the voltage gated calcium channels located at the synapse, whose phosphorylation increases their opening probability. Thus, a rise in cAMP could facilitate neurotransmitter release, and a downregulation would have the opposite effect. Other targets of PKA include CREB, leading to changes in gene expression. Conceivably, a decrease in PKA activity could result in the downregulation of DNT-2 expression in DNT-2 neurons. This negative feedback loop would restore the homeostatic relationship between DNT-2 and dopamine levels.

Our data indeed demonstrate that DNT-2 and PAM neurons influence each other, not potentially, but really. We have provided data that: DNT-2 and PAMs are connected through circuitry; that the DNT-2 receptors Toll-6 and kek-6 are expressed in DANs, including in PAMs; that alterations in the levels of DNT-2 (both loss and gain of function) and loss of function for the DNT-2 receptors Toll-6 and Kek-6 alter PAM cell number, alter PAM dendritic complexity and alter synaptogenesis in PAMs; alterations in the levels of DNT-2, Toll-6 and kek-6 in adult flies alters dopamine dependent behaviours of climbing, locomotion in an arena and learning and long-term memory. These data firmly demonstrate that the two neuron types DNT-2 and PAMs influence each other.

We have also shown that over-expression of DNT-2 in DNT-2 neurons increases TH mRNA levels, whereas activation of DNT-2 neurons decreases cAMP levels in DNT-2 neurons in a dopamine/Dop2R-dependent manner. These data show a functional interaction between DNT-2 and PAM neurons.

Second, the quantification of bouton volumes (no y-axis label in Figure 5 C and D!) and dendrite complexity are not convincingly laid out. Here, the reader expects fine-grained anatomical characterizations of the structures under investigation, and a method to precisely quantify the lengths and branching patterns of individual dendritic arborizations as well as the volume of individual axonal boutons.

Figure 5C, D do contain Y-axis labels, all our graphs in main manuscript and in supplementary files contain Y-axis labels.

In fact, we did use a method to precisely quantify the lengths and branching patterns of individual dendritic arborisations, volume of individual boutons and bouton counting. These analyses were carried out using Imaris software. For dendritic branching patterns, the “Filament Autodetect” function was used. Here, dendrites were analysed by tracing semi-automatically each dendrite branch (ie manual correction of segmentation errors) to reconstruct the segmented dendrite in volume. From this segmented dendrite, Imaris provides measurements of total dendrite volume, number and length of dendrite branches, terminal points, etc. For bouton size and number, we used the Imaris “Spot” function. Here, a threshold is set to exclude small dots (eg of background) that do not correspond to synapses/boutons. All samples and genotypes are treated with the same threshold, thus the analysis is objective and large sample sizes can be analysed effectively. We have already provided a description of the use of Imaris in the methods section.

Third, Figure 1C shows two neurons with the goal of demonstrating between-neuron variability. It is not convincingly demonstrated that the two neurons are actually of the very same type of neuron in different flies or two completely different neurons.

We thank Reviewer 3 for raising this interesting point. It is not possible to prove which of the four DNT-2A neurons per hemibrain, which we visualised with DNT-2>MCFO, were the same neurons in every individual brain we looked at. This is because in every brain we have looked at, the soma of the neurons were not located in exactly the same location. Furthermore, the arborisation patterns are also different and unique, for each individual brain. Thus, there is natural variability in the position of the soma and in the arborisation patterns. Such variability presumably results from the combination of developmental and activity-dependent plasticity.

We would like to thank Reviewer 3 for the very positive evaluation of our work and the interesting and valuable feedback.

Author response:

Public Reviews: 

Reviewer #1 (Public review): 

Summary: 

Here the authors present their evidence linking the mitochondrial uniporter (MCU-1) and olfactory adaptation in C. elegans. They clearly demonstrate a behavioral defect of mcu-1 mutants in adaptation over 60 minutes and present evidence that this gene functions in the AWC primary sensory neurons at, or close to, the time of adaptation. 

Strengths: 

The paper is very well organized and their approach to unpacking the role of mcu-1 mutants in olfactory adaptation is very reasonable. The authors lean into diverse techniques including behavior, genetics, and pharmacological manipulation in order to flesh out their model for how MCU-1 functions in AWC neurons with respect to olfaction. 

Weaknesses: 

I would like to see the authors strengthen the link between mitochondrial calcium and olfactory adaptation. The authors present some gCaMP data in Figure 5 but it is unclear to me why this tool is not better utilized to explore the mechanism of MCU-1 activity. I think this is very important as the title of the paper states that "mitochondrial calcium modulates.." behavior in AWC and so it would be nice to see more evidence to support this direct connection. I would also like to see the authors place their findings into a model based on previous findings and perhaps examine whether mcu-1 is required for EGL-4 nuclear translocation, which would be straightforward to examine. 

We agree that observing calcium levels inside the mitochondria would conclusively demonstrate that mitochondria calcium directly impacts neuropeptide secretion and behavior. We will try to do this with a mitochondrially targeted calcium indicator. We will also better integrate our findings to existing models in the literature, such as EGL-4 nuclear localization in AWC in response to prolonged odor exposure. Thank you for your comments.

Reviewer #2 (Public review): 

Summary: 

In their manuscript, "Mitochondrial calcium modulates odor-mediated behavioural plasticity in C. elegans", Lee et al. aim to link a mitochondrial calcium transporter to higher-order neuronal functions that mediate memory and aversive learning behaviours. The authors characterise the role of the mitochondrial calcium uniporter, and a specific subunit of this complex, MCU-1, within a single chemosensory neuron (AWCOFF) during aversive odor learning in the nematode. By genetically manipulating mcu-1 as well as using pharmacological activators and blockers of MCU activity, the study presents compelling evidence that the activity of this individual mitochondrial ion transporter in AWCOFF is sufficient to drive animal behaviour through aversive memory formation. The authors show that perturbations to mcu-1 and MCU activity prevent aversive learning to several chemical odors associated with food absence. The authors propose a model, experimentally validated at several steps, whereby an increase in MCU activity during odor conditioning stimulates mitochondrial calcium influx and an increase in mitochondrial reactive oxygen species (mtROS) production, triggering the release of the neuropeptide NLP-1 from AWC, all of which are required to mediate future avoidance behaviour of the chemical odor. 

Strengths: 

Overall, the authors provided robust evidence that mitochondrial function, mediated through MCU activity, contributes to behavioural plasticity. They also demonstrated that ectopic MCU activation or mtROS during odor exposure could accelerate learning. This is quite profound, as it highlights the importance of mitochondrial function in complex neuronal processes beyond their general roles in the development and maintenance of neurons through energy homeostasis and biosynthesis, amongst their other cell-non-specific roles. 

Weaknesses: 

While the manuscript is generally robust, there are some concerns that should be addressed to improve the strength of the proposed model: 

(1) Throughout the manuscript, it is implied that MCU activation caused by odor conditioning changes mitochondrial calcium levels. However, there is no direct experimental evidence of this. For example, the authors write on p.10 "This shows that H2O2 production occurs downstream of MCU activation and calcium influx into the mitochondria", and on p. 11, the statement that prolonged exposure to odors causes calcium influx. Because this is a key element of the proposed model, experimental evidence would be required to support it. 

We are planning to measure mitochondrial calcium levels directly by using a mitochondrially targeted calcium indicator. We agree that this is a key element of our model.

(2) Some controls missing, e.g. a heat-shock-only control in WT and mcu-1 (non-transgenic) background in Figure 1h is required to ensure the heat-shock stress does not interfere with odor learning. 

We will conduct the experiments again with necessary controls.

(3) Lee et al propose that mcu-1 is required at the adult stage to accomplish odor learning because inducing mcu-1 expression at larval stages did not rescue the phenotype of mcu-1 mutants during adulthood. However, the requirement of MCU for odor learning was narrowed down to a 15' window at the end of odor conditioning (Figure 5c). Is it possible that MCU-1 protein levels decline after larval induction so that MCU-1 is no longer present during adulthood when odor conditioning is performed? 

Yes, we also noted that the early induction of MCU-1 is not effective to restore learning, and hypothesized that MCU-1 protein may be subject to high turnover. It may be that MCU-1 induced during larval stages no longer exist by the time odor conditioning is performed, although we have not confirmed this. We had a brief sentence noting this in the discussion section, but we will discuss this a little further in the revision. Thank you.

(4) There is a limited learning effect observable after 30 minutes, and a very pronounced effect in all animals after 90 minutes. The authors very carefully dissect the learning mechanism at 60 minutes of exposure and distinguish processes that are relevant at 60 minutes from those important at 30 minutes. Some explanation or speculation as to why the processes crucial at the 60-minute mark are redundant at 90 minutes of exposure would be important. 

I think this is in line with Reviewer #1’s comments that we should discuss our findings more in relation to existing models in the literature. We will do this in our revision.

(5) Given the presumably ubiquitous function of mcu-1/MCU in mitochondrial calcium homeostasis, it is remarkable that its perturbation impacts only a very specific neuronal process in AWC at a very specific time. The authors should elaborate on this surprising aspect of their discovery in the discussion. 

We will discuss the implication further in our revised manuscript.

(6) Associated with the above comment, it remains possible that mcu-1 is required in coelomocytes for their ability to absorb NLP-1::Venus (Figure 3B), and the AWC-specific role of mcu-1 for this phenotype should be determined. 

To confirm that mcu-1 is not required for coelomocyte uptake, we can stimulate NLP-1:Venus secretion in mcu-1 worms by adding H2O2, then observe whether Venus is observed in the coelomocytes. We will include this in our revised manuscript. Thank you for your comments.

Reviewer #3 (Public review): 

Summary: 

This manuscript reports a role for the mitochondrial calcium uniporter gene (mcu-1) in regulating associative learning behavior in C. elegans. This regulation occurs by mcu-1-dependent secretion of the neuropeptide NLP-1 from the sensory neuron AWC. The authors report a post-developmental role for mcu-1 in AWC to promote learning. The authors further show that odor conditioning leads to increases in NLP-1 secretion from AWC, and that interfering with mcu-1 function reduces NLP-1 secretion. Finally, the authors show that NLP-1 secretion increases when ROS levels in AWC are genetically or pharmacologically elevated. The authors propose that mitochondrial calcium entry through MCU-1 in response to odor conditioning leads to the generation of ROS and the subsequent increase in neuropeptide secretion to promote conditioned behavior. 

Strengths: 

(1) The authors show convincingly that genetically or pharmacologically manipulating MCU function impacts chemotaxis in a conditioned learning paradigm. 

(2) The demonstration that the secretion of a specific neuropeptide can be up-regulated by MCU, ROS and odor conditioning is an important and interesting advance that addresses mechanisms by which neuropeptide secretion can be regulated in vivo. 

Weaknesses: 

(1) The authors conclusion that mcu-1 functions in the AWC-on neuron is not adequately supported by their rescue experiments. The promoter they use for rescue drives expression in a number of additional neurons including AWC-on, that themselves are implicated in adaptation, leaving open the possibility that mcu-1 may function non-autonomously instead of autonomously in AWC to regulate this behavior. 

We recognized this as well, and we now have a promoter construct more specific to AWCON (str-2). Using this more specific promoter, we will confirm that the role of mcu-1 is indeed AWCON-specific in our revised manuscript.

(2) The authors conclude MCU promotes neuropeptide release from AWC by controlling calcium entry into mitochondria, but they did not directly examine the effects of altered MCU function on calcium dynamics either in mitochondria or in the soma, even though they conducted calcium imaging experiments in AWC of wild type animals. Examination of calcium entry in mitochondria would be a direct test of their model.

We agree. As we stated above for reviewer #1 and #2, we will include results from the mitochondrial calcium data in our revised manuscript.

(3) The authors' conclusion that mitochondrial-derived ROS produced by MCU activation drives neuropeptide release does not appear to be experimentally supported. A major weakness of this paper is that experiments addressing whether mcu-1 activity indeed produces ROS are not included, leaving unanswered the question of whether MCU is the endogenous source of ROS that drives neuropeptide secretion.

We can confirm this using mitochondrially targeted redox indicator roGFP, and we will be sure to include the data in the revised manuscript. Thank you for your comments.

eLife Assessment

This study presents important findings that will allow for a better understanding of the role of mitochondria in behaviours of C. elegans. There is convincing evidence that mutants in a subunit of the mitochondrial calcium uniporter (MCU-1) show defects in olfactory adaptation and this gene regulates neuropeptide secretion and allows for behavioural modulation in C. elegans. However, the evidence that mitochondrial calcium modulates odour-based behaviour in C. elegans is incomplete. This claim would require support from calcium imaging in conditioned WT and mcu-1 animals. This work would be of interest to labs working on behaviours across phyla.

Reviewer #1 (Public review):

Summary:

Here the authors present their evidence linking the mitochondrial uniporter (MCU-1) and olfactory adaptation in C. elegans. They clearly demonstrate a behavioral defect of mcu-1 mutants in adaptation over 60 minutes and present evidence that this gene functions in the AWC primary sensory neurons at, or close to, the time of adaptation.

Strengths:

The paper is very well organized and their approach to unpacking the role of mcu-1 mutants in olfactory adaptation is very reasonable. The authors lean into diverse techniques including behavior, genetics, and pharmacological manipulation in order to flesh out their model for how MCU-1 functions in AWC neurons with respect to olfaction.

Weaknesses:

I would like to see the authors strengthen the link between mitochondrial calcium and olfactory adaptation. The authors present some gCaMP data in Figure 5 but it is unclear to me why this tool is not better utilized to explore the mechanism of MCU-1 activity. I think this is very important as the title of the paper states that "mitochondrial calcium modulates.." behavior in AWC and so it would be nice to see more evidence to support this direct connection. I would also like to see the authors place their findings into a model based on previous findings and perhaps examine whether mcu-1 is required for EGL-4 nuclear translocation, which would be straightforward to examine.

Reviewer #2 (Public review):

Summary:

In their manuscript, "Mitochondrial calcium modulates odor-mediated behavioural plasticity in C. elegans", Lee et al. aim to link a mitochondrial calcium transporter to higher-order neuronal functions that mediate memory and aversive learning behaviours. The authors characterise the role of the mitochondrial calcium uniporter, and a specific subunit of this complex, MCU-1, within a single chemosensory neuron (AWCOFF) during aversive odor learning in the nematode. By genetically manipulating mcu-1 as well as using pharmacological activators and blockers of MCU activity, the study presents compelling evidence that the activity of this individual mitochondrial ion transporter in AWCOFF is sufficient to drive animal behaviour through aversive memory formation. The authors show that perturbations to mcu-1 and MCU activity prevent aversive learning to several chemical odors associated with food absence. The authors propose a model, experimentally validated at several steps, whereby an increase in MCU activity during odor conditioning stimulates mitochondrial calcium influx and an increase in mitochondrial reactive oxygen species (mtROS) production, triggering the release of the neuropeptide NLP-1 from AWC, all of which are required to mediate future avoidance behaviour of the chemical odor.

Strengths:

Overall, the authors provided robust evidence that mitochondrial function, mediated through MCU activity, contributes to behavioural plasticity. They also demonstrated that ectopic MCU activation or mtROS during odor exposure could accelerate learning. This is quite profound, as it highlights the importance of mitochondrial function in complex neuronal processes beyond their general roles in the development and maintenance of neurons through energy homeostasis and biosynthesis, amongst their other cell-non-specific roles.

Weaknesses:

While the manuscript is generally robust, there are some concerns that should be addressed to improve the strength of the proposed model:

(1) Throughout the manuscript, it is implied that MCU activation caused by odor conditioning changes mitochondrial calcium levels. However, there is no direct experimental evidence of this. For example, the authors write on p.10 "This shows that H2O2 production occurs downstream of MCU activation and calcium influx into the mitochondria", and on p. 11, the statement that prolonged exposure to odors causes calcium influx. Because this is a key element of the proposed model, experimental evidence would be required to support it.

(2) Some controls missing, e.g. a heat-shock-only control in WT and mcu-1 (non-transgenic) background in Figure 1h is required to ensure the heat-shock stress does not interfere with odor learning.

(3) Lee et al propose that mcu-1 is required at the adult stage to accomplish odor learning because inducing mcu-1 expression at larval stages did not rescue the phenotype of mcu-1 mutants during adulthood. However, the requirement of MCU for odor learning was narrowed down to a 15' window at the end of odor conditioning (Figure 5c). Is it possible that MCU-1 protein levels decline after larval induction so that MCU-1 is no longer present during adulthood when odor conditioning is performed?

(4) There is a limited learning effect observable after 30 minutes, and a very pronounced effect in all animals after 90 minutes. The authors very carefully dissect the learning mechanism at 60 minutes of exposure and distinguish processes that are relevant at 60 minutes from those important at 30 minutes. Some explanation or speculation as to why the processes crucial at the 60-minute mark are redundant at 90 minutes of exposure would be important.

(5) Given the presumably ubiquitous function of mcu-1/MCU in mitochondrial calcium homeostasis, it is remarkable that its perturbation impacts only a very specific neuronal process in AWC at a very specific time. The authors should elaborate on this surprising aspect of their discovery in the discussion.

(6) Associated with the above comment, it remains possible that mcu-1 is required in coelomocytes for their ability to absorb NLP-1::Venus (Figure 3B), and the AWC-specific role of mcu-1 for this phenotype should be determined.

Reviewer #3 (Public review):

Summary:

This manuscript reports a role for the mitochondrial calcium uniporter gene (mcu-1) in regulating associative learning behavior in C. elegans. This regulation occurs by mcu-1-dependent secretion of the neuropeptide NLP-1 from the sensory neuron AWC. The authors report a post-developmental role for mcu-1 in AWC to promote learning. The authors further show that odor conditioning leads to increases in NLP-1 secretion from AWC, and that interfering with mcu-1 function reduces NLP-1 secretion. Finally, the authors show that NLP-1 secretion increases when ROS levels in AWC are genetically or pharmacologically elevated. The authors propose that mitochondrial calcium entry through MCU-1 in response to odor conditioning leads to the generation of ROS and the subsequent increase in neuropeptide secretion to promote conditioned behavior.

Strengths:

(1) The authors show convincingly that genetically or pharmacologically manipulating MCU function impacts chemotaxis in a conditioned learning paradigm.

(2) The demonstration that the secretion of a specific neuropeptide can be up-regulated by MCU, ROS and odor conditioning is an important and interesting advance that addresses mechanisms by which neuropeptide secretion can be regulated in vivo.

Weaknesses:

(1) The authors conclusion that mcu-1 functions in the AWC-on neuron is not adequately supported by their rescue experiments. The promoter they use for rescue drives expression in a number of additional neurons including AWC-on, that themselves are implicated in adaptation, leaving open the possibility that mcu-1 may function non-autonomously instead of autonomously in AWC to regulate this behavior.

(2) The authors conclude MCU promotes neuropeptide release from AWC by controlling calcium entry into mitochondria, but they did not directly examine the effects of altered MCU function on calcium dynamics either in mitochondria or in the soma, even though they conducted calcium imaging experiments in AWC of wild type animals. Examination of calcium entry in mitochondria would be a direct test of their model.

(3) The authors' conclusion that mitochondrial-derived ROS produced by MCU activation drives neuropeptide release does not appear to be experimentally supported. A major weakness of this paper is that experiments addressing whether mcu-1 activity indeed produces ROS are not included, leaving unanswered the question of whether MCU is the endogenous source of ROS that drives neuropeptide secretion.

eLife Assessment

This manuscript presents a valuable minimal model of habituation which is quantified by information theoretic measures. The results here could be of use in interpreting habituation behavior in a range of biological systems. However, the evidence presented is incomplete and would benefit from more rigorous approaches and a fuller accounting of the hallmarks of habituation.

Reviewer #1 (Public review):

Summary:

The manuscript by Nicoletti et al. presents a minimal model of habituation, a basic form of non-associative learning, addressing both from dynamical and information theory aspects of how habituation can be realized. The authors identify that negative feedback provided with a slow storage mechanism is sufficient to explain habituation.

Strengths:

The authors combine the identification of the dynamical mechanism with information-theoretic measures to determine the onset of habituation and provide a description of how the system can gain maximum information about the environment.

Weaknesses:

I have several main concerns/questions about the proposed model for habituation and its plausibility. In general, habituation does not only refer to a decrease in the responsiveness upon repeated stimulation but as Thompson and Spencer discussed in Psych. Rev. 73, 16-43 (1966), there are 10 main characteristics of habituation, including (i) spontaneous recovery when the stimulus is withheld after response decrement; dependence on the frequency of stimulation such that (ii) more frequent stimulation results in more rapid and/or more pronounced response decrement and more rapid spontaneous recovery; (iii) within a stimulus modality, the less intense the stimulus, the more rapid and/or more pronounced the behavioral response decrement; (iv) the effects of repeated stimulation may continue to accumulate even after the response has reached an asymptotic level (which may or may not be zero, or no response). This effect of stimulation beyond asymptotic levels can alter subsequent behavior, for example, by delaying the onset of spontaneous recovery.

These are only a subset of the conditions that have been experimentally observed and therefore a mechanistic model of habituation, in my understanding, should capture the majority of these features and/or discuss the absence of such features from the proposed model.

Furthermore, the habituated response in steady-state is approximately 20% less than the initial response, which seems to be achieved already after 3-4 pulses, the subsequent change in response amplitude seems to be negligible, although the authors however state "after a large number of inputs, the system reaches a time-periodic steady-state". How do the authors justify these minimal decreases in the response amplitude? Does this come from the model parametrization and is there a parameter range where more pronounced habituation responses can be observed?

The same is true for the information content (Figure 2f) - already at the first pulse, IU, H ~ 0.7 and only negligibly increases afterwards. In my understanding, during learning, the mutual information between the input and the internal state increases over time and the system extracts from these predictions about its responses. In the model presented by the authors, it seems the system already carries information about the environment which hardly changes with repeated stimulus presentation. The complexity of the signal is also limited, and it is very hard to clarify from the presented results, whether the proposed model can actually explain basic features of habituation, as mentioned above.<br /> Additionally, there have been two recent models on habituation and I strongly suggest that the authors discuss their work in relation to recent works (bioRxiv 2024.08.04.606534; arXiv:2407.18204).

Reviewer #2 (Public review):

In this study, the authors aim to investigate habituation, the phenomenon of increasing reduction in activity following repeated stimuli, in the context of its information-theoretic advantage. To this end, they consider a highly simplified three-species reaction network where habituation is encoded by a slow memory variable that suppresses the receptor and therefore the readout activity. Using analytical and numerical methods, they show that in their model the information gain, the difference between the mutual information between the signal and readout after and before habituation, is maximal for intermediate habituation strength. Furthermore, they demonstrate that the Pareto front corresponds to an optimization strategy that maximizes the mutual information between signal and readout in the steady state, minimizes some form of dissipation, and also exhibits similar intermediate habituation strength. Finally, they briefly compare predictions of their model to whole-brain recordings of zebrafish larvae under visual stimulation.

The author's simplified model might serve as a solid starting point for understanding habituation in different biological contexts as the model is simple enough to allow for some analytic understanding but at the same time exhibits all basic properties of habituation in sensory systems. Furthermore, the author's finding of maximal information gain for intermediate habituation strength via an optimization principle is, in general, interesting. However, the following points remain unclear or are weakly explained:

(1) Is it unclear what the meaning of the finding of maximal information gain for intermediate habituation strength is for biological systems? Why is information gain as defined in the paper a relevant quantity for an organism/cell? For instance, why is a system with low mutual information after the first stimulus and intermediate mutual information after habituation better than one with consistently intermediate mutual information? Or, in other words, couldn't the system try to maximize the mutual information acquired over the whole time series, e.g., the time series mutual information between the stimulus and readout?

(2) The model is very similar to (or a simplification of previous models) for adaptation in living systems, e.g., for adaptation in chemotaxis via activity-dependent methylation and demethylation. This should be made clearer.

(3) It remains unclear why this optimization principle is the most relevant one. While it makes sense to maximize the mutual information between stimulus and readout, there are various choices for what kind of dissipation is minimized. Why was \delta Q_R chosen and not, for instance, \dot{\Sigma}_int or the sum of both? How would the results change in that case? And how different are the results if the mutual information is not calculated for the strong stimulation input statistics but for the background one?

(4) The comparison to the experimental data is not too strong of an argument in favor of the model. Is the agreement between the model and the experimental data surprising? What other behavior in the PCA space could one have expected in the data? Shouldn't the 1st PC mostly reflect the "features", by construction, and other variability should be due to progressively reduced activity levels?

Reviewer #3 (Public review):

The authors use a generic model framework to study the emergence of habituation and its functional role from information-theoretic and energetic perspectives. Their model features a receptor, readout molecules, and a storage unit, and as such, can be applied to a wide range of biological systems. Through theoretical studies, the authors find that habituation (reduction in average activity) upon exposure to repeated stimuli should occur at intermediate degrees to achieve maximal information gain. Parameter regimes that enable these properties also result in low dissipation, suggesting that intermediate habituation is advantageous both energetically and for the purpose of retaining information about the environment.

A major strength of the work is the generality of the studied model. The presence of three units (receptor, readout, storage) operating at different time scales and executing negative feedback can be found in many domains of biology, with representative examples well discussed by the authors (e.g. Figure 1b). A key takeaway demonstrated by the authors that has wide relevance is that large information gain and large habituation cannot be attained simultaneously. When energetic considerations are accounted for, large information gain and intermediate habituation appear to be a favorable combination.

While the generic approach of coarse-graining most biological detail is appealing and the results are of broad relevance, some aspects of the conducted studies, the problem setup, and the writing lack clarity and should be addressed:

(1) The abstract can be further sharpened. Specifically, the "functional role" mentioned at the end can be made more explicit, as it was done in the second-to-last paragraph of the Introduction section ("its functional advantages in terms of information gain and energy dissipation"). In addition, the abstract mentions the testing against experimental measurements of neural responses but does not specify the main takeaways. I suggest the authors briefly describe the main conclusions of their experimental study in the abstract.

(2) Several clarifications are needed on the treatment of energy dissipation.<br /> - When substituting the rates in Eq. (1) into the definition of δQ_R above Eq. (10), "σ" does not appear on the right-hand side. Does this mean that one of the rates in the lower pathway must include σ in its definition? Please clarify.<br /> - I understand that the production of storage molecules has an associated cost σ and hence contributes to dissipation. The dependence of receptor dissipation on , however, is not fully clear. If the environment were static and the memory block was absent, the term with would still contribute to dissipation. What would be the nature of this dissipation?<br /> - Similarly, in Eq. (9) the authors use the ratio of the rates Γ_{s → s+1} and Γ_{s+1 → s} in their expression for internal dissipation. The first-rate corresponds to the synthesis reaction of memory molecules, while the second corresponds to a degradation reaction. Since the second reaction is not the microscopic reverse of the first, what would be the physical interpretation of the log of their ratio? Since the authors already use σ as the energy cost per storage unit, why not use σ times the rate of producing S as a metric for the dissipation rate?

(3) Impact of the pre-stimulus state. The plots in Figure 2 suggest that the environment was static before the application of repeated stimuli. Can the authors comment on the impact of the pre-stimulus state on the degree of habituation and its optimality properties? Specifically, would the conclusions stay the same if the prior environment had stochastic but aperiodic dynamics?

(4) Clarification about the memory requirement for habituation. Figure 4 and the associated section argue for the essential role that the storage mechanism plays in habituation. Indeed, Figure 4a shows that the degree of habituation decreases with decreasing memory. The graph also shows that in the limit of vanishingly small Δ⟨S⟩, the system can still exhibit a finite degree of habituation. Can the authors explain this limiting behavior; specifically, why does habituation not vanish in the limit Δ⟨S⟩ -> 0?

Author response:

Reviewer #1 (Public review):

Summary:

The manuscript by Nicoletti et al. presents a minimal model of habituation, a basic form of non-associative learning, addressing both from dynamical and information theory aspects of how habituation can be realized. The authors identify that negative feedback provided with a slow storage mechanism is sufficient to explain habituation.

Strengths:

The authors combine the identification of the dynamical mechanism with information-theoretic measures to determine the onset of habituation and provide a description of how the system can gain maximum information about the environment.

We thank the reviewer for highlighting the strength of our work.

Weaknesses:

I have several main concerns/questions about the proposed model for habituation and its plausibility. In general, habituation does not only refer to a decrease in the responsiveness upon repeated stimulation but as Thompson and Spencer discussed in Psych. Rev. 73, 16-43 (1966), there are 10 main characteristics of habituation, including (i) spontaneous recovery when the stimulus is withheld after response decrement; dependence on the frequency of stimulation such that (ii) more frequent stimulation results in more rapid and/or more pronounced response decrement and more rapid spontaneous recovery; (iii) within a stimulus modality, the less intense the stimulus, the more rapid and/or more pronounced the behavioral response decrement; (iv) the effects of repeated stimulation may continue to accumulate even after the response has reached an asymptotic level (which may or may not be zero, or no response). This effect of stimulation beyond asymptotic levels can alter subsequent behavior, for example, by delaying the onset of spontaneous recovery.

These are only a subset of the conditions that have been experimentally observed and therefore a mechanistic model of habituation, in my understanding, should capture the majority of these features and/or discuss the absence of such features from the proposed model.

We are really grateful to the reviewer for pointing out these aspects of habituation that we overlooked in the previous version of our manuscript. Indeed, our model is able to capture most of these 10 observed behaviors, specifically: 1) habituation; 2) spontaneous recovery; 3) potentiation of habituation; 4) frequency sensitivity; and 5) intensity sensitivity. Here, we are following the same terminology employed in bioRxiv 2024.08.04.606534, the paper highlighted by the referee. Regarding the hallmark 6) subliminal accumulation, we also believe that our model can capture it as well, but more analyses are needed to substantiate this claim. We will include the discussion of these points in the revised version.

Notably, in line with the discussion in bioRxiv 2024.08.04.606534, we also think that feature 10) long-term habituation, is ambiguous and its appearance might be simply related to the other features discussed above. In the revised version, we will detail our take on this aspect in relation to the presented model.

All other hallmarks require the presence of multiple stimuli and, as a consequence, they cannot be observed within our model, but are interesting lines of research for future investigations. We believe that this addition will help clarify the validity of the model and the relevance of our result, consequently improving the quality of our manuscript.

Furthermore, the habituated response in steady-state is approximately 20% less than the initial response, which seems to be achieved already after 3-4 pulses, the subsequent change in response amplitude seems to be negligible, although the authors however state "after a large number of inputs, the system reaches a time-periodic steady-state". How do the authors justify these minimal decreases in the response amplitude? Does this come from the model parametrization and is there a parameter range where more pronounced habituation responses can be observed?

The referee is correct, but this is solely a consequence of the specific set of parameters we selected. We made this choice solely for visualization purposes. In the next version, when different emerging behaviors characterizing habituation are discussed, we will also present a set of parameters for which habituation can be better appreciated, justifying our new choice.

We stated that the time-periodic steady-state is reached “after a large number of stimuli” from a mathematical perspective. However, by using a habituation threshold, as defined in bioRxiv 2024.08.04.606534 for example, we can say that the system is habituated after a few stimuli for the set of parameters selected in the first version of the manuscript. We will also discuss this aspect in the Supplemental Material of the revised version, as it will also be important to appreciate the hallmarks of habituation listed above.

The same is true for the information content (Figure 2f) - already at the first pulse, IU, H ~ 0.7 and only negligibly increases afterwards. In my understanding, during learning, the mutual information between the input and the internal state increases over time and the system extracts from these predictions about its responses. In the model presented by the authors, it seems the system already carries information about the environment which hardly changes with repeated stimulus presentation. The complexity of the signal is also limited, and it is very hard to clarify from the presented results, whether the proposed model can actually explain basic features of habituation, as mentioned above.

The point about information is more subtle. We can definitely choose a set of parameters for which the information gain is higher and we will show it in the Supplemental Material of the revised version. However, as the reviewer correctly points out, it is difficult to give an interpretation of the specific value of I_U,H for such a minimal model.

We also remark that, since the readout population and the receptor both undergo a fast dynamics (with appropriate timescales as discussed in the text), we are not observing the transient gain of information associated with the first stimulus and, as such, the mutual information presents a discontinuous behavior resembling the dynamics of the readout.

Additionally, there have been two recent models on habituation and I strongly suggest that the authors discuss their work in relation to recent works (bioRxiv 2024.08.04.606534; arXiv:2407.18204).

We thank the reviewer for pointing out these relevant references. We will discuss analogies and differences in the revised version of the main text. The main difference is the fact that information-theoretic aspects of habituation are not discussed in the presented references, while the idea of this work is to elucidate exactly the interplay between information gain and habituation dynamics.

Reviewer #2 (Public review):

In this study, the authors aim to investigate habituation, the phenomenon of increasing reduction in activity following repeated stimuli, in the context of its information-theoretic advantage. To this end, they consider a highly simplified three-species reaction network where habituation is encoded by a slow memory variable that suppresses the receptor and therefore the readout activity. Using analytical and numerical methods, they show that in their model the information gain, the difference between the mutual information between the signal and readout after and before habituation, is maximal for intermediate habituation strength. Furthermore, they demonstrate that the Pareto front corresponds to an optimization strategy that maximizes the mutual information between signal and readout in the steady state, minimizes some form of dissipation, and also exhibits similar intermediate habituation strength. Finally, they briefly compare predictions of their model to whole-brain recordings of zebrafish larvae under visual stimulation.

The author's simplified model might serve as a solid starting point for understanding habituation in different biological contexts as the model is simple enough to allow for some analytic understanding but at the same time exhibits all basic properties of habituation in sensory systems. Furthermore, the author's finding of maximal information gain for intermediate habituation strength via an optimization principle is, in general, interesting. However, the following points remain unclear or are weakly explained:

We thank the reviewer for deeming our work interesting and for considering it a solid starting point for understanding habituation in biological systems.

(1) Is it unclear what the meaning of the finding of maximal information gain for intermediate habituation strength is for biological systems? Why is information gain as defined in the paper a relevant quantity for an organism/cell? For instance, why is a system with low mutual information after the first stimulus and intermediate mutual information after habituation better than one with consistently intermediate mutual information? Or, in other words, couldn't the system try to maximize the mutual information acquired over the whole time series, e.g., the time series mutual information between the stimulus and readout?

This is an important and delicate aspect to discuss. We considered the mutual information with a prolonged stimulation when building the Pareto front, by maximizing this quantity while minimizing the dissipation. The observation that the Pareto front lies in the vicinity of the maximum of the information gain hints at the fact that reducing the information gain by increasing the mutual information at each stimulation will require more energy. However, we did not thoroughly explore this aspect by considering all sources of dissipation and the fact that habituation is, anyway, a dynamical phenomenon. In the revised version, we will clarify this point, extending our analyses.

We would like to add that, from a naive perspective, while the first stimulation will necessarily trigger a certain mutual information, multiple observations of the same stimulus have to reflect into accumulated infor

mation that consequently drives the onset of observed dynamical behaviors, such as habituation.

(2) The model is very similar to (or a simplification of previous models) for adaptation in living systems, e.g., for adaptation in chemotaxis via activity-dependent methylation and demethylation. This should be made clearer.

We apologize for having missed this point. Our choice has been motivated by the fact that we wanted to avoid any confusion between the usual definition of (perfect) adaptation and habituation. At any rate, we will add this clarification in the revised version.

(3) It remains unclear why this optimization principle is the most relevant one. While it makes sense to maximize the mutual information between stimulus and readout, there are various choices for what kind of dissipation is minimized. Why was \delta Q_R chosen and not, for instance, \dot{\Sigma}_int or the sum of both? How would the results change in that case? And how different are the results if the mutual information is not calculated for the strong stimulation input statistics but for the background one?

We thank the referee for giving us the opportunity to deepen this aspect of the manuscript. We decided to minimize \delta Q_R since this dissipation is unavoidable. In fact, considering the existence of two different pathways implementing sensing and feedback, the presence of any input will result in a dissipation produced by the receptor. This energy consumption is reflected in \delta Q_R. Conversely, the dissipation associated with the storage is always zero in the limit of a fast memory. However, we know that such a limit is pathological and leads to no habituation. As a consequence, in the revised version we will discuss other choices for our optimization approach, along with their potentialities and limitations.

The dependence of the Pareto front on the stimulus strength is shown in the Supplemental Material, but not in relation to habituation and information gain. We will strengthen this part in the revised version of the manuscript, elaborating more on the connection between optimality, information gain, and dynamical behavior.

(4) The comparison to the experimental data is not too strong of an argument in favor of the model. Is the agreement between the model and the experimental data surprising? What other behavior in the PCA space could one have expected in the data? Shouldn't the 1st PC mostly reflect the "features", by construction, and other variability should be due to progressively reduced activity levels?

The agreement between data and model is not surprising - we agree on this - since the data exhibit habituation. However, the fact that, without any explicit biological details, our minimal model is able to capture the features of a complex neural system just by looking at the PCs is non-trivial. The 1st PC only reflects the feature that captures most of the variance of the data and, as such, it is difficult to have a-priori expectations on what it should represent. Depending on the behavior of higher-order PCs, we may include them in the revised version if any interesting results arise.

Reviewer #3 (Public review):

The authors use a generic model framework to study the emergence of habituation and its functional role from information-theoretic and energetic perspectives. Their model features a receptor, readout molecules, and a storage unit, and as such, can be applied to a wide range of biological systems. Through theoretical studies, the authors find that habituation (reduction in average activity) upon exposure to repeated stimuli should occur at intermediate degrees to achieve maximal information gain. Parameter regimes that enable these properties also result in low dissipation, suggesting that intermediate habituation is advantageous both energetically and for the purpose of retaining information about the environment.

A major strength of the work is the generality of the studied model. The presence of three units (receptor, readout, storage) operating at different time scales and executing negative feedback can be found in many domains of biology, with representative examples well discussed by the authors (e.g. Figure 1b). A key takeaway demonstrated by the authors that has wide relevance is that large information gain and large habituation cannot be attained simultaneously. When energetic considerations are accounted for, large information gain and intermediate habituation appear to be a favorable combination.

We thank the referee for this positive assessment of our work and its generality.

While the generic approach of coarse-graining most biological detail is appealing and the results are of broad relevance, some aspects of the conducted studies, the problem setup, and the writing lack clarity and should be addressed:

(1) The abstract can be further sharpened. Specifically, the "functional role" mentioned at the end can be made more explicit, as it was done in the second-to-last paragraph of the Introduction section ("its functional advantages in terms of information gain and energy dissipation"). In addition, the abstract mentions the testing against experimental measurements of neural responses but does not specify the main takeaways. I suggest the authors briefly describe the main conclusions of their experimental study in the abstract.

We thank the referee for this suggestion. The revised version will present a modified abstract in line with the reviewer’s proposal.

(2) Several clarifications are needed on the treatment of energy dissipation.

- When substituting the rates in Eq. (1) into the definition of δQ_R above Eq. (10), "σ" does not appear on the right-hand side. Does this mean that one of the rates in the lower pathway must include σ in its definition? Please clarify.

We apologize to the referee for this typo. Indeed, \sigma sets the energy scale of the feedback and, as such, it appears in the energetic driving given by the feedback on the receptor, i.e., together with \kappa in Eq. (1). We will fix this issue in the revised version. Moreover, we will check the entire manuscript to be sure that all formulas are consistent.

- I understand that the production of storage molecules has an associated cost σ and hence contributes to dissipation. The dependence of receptor dissipation on <H>, however, is not fully clear. If the environment were static and the memory block was absent, the term with <H> would still contribute to dissipation. What would be the nature of this dissipation?

In the spirit of building a paradigmatic minimal model with a thermodynamic meaning, we considered H to act as an external thermodynamic driving. Since this driving acts on a different pathway with respect to the one affected by the storage, the receptor is driven out of equilibrium by its presence. By eliminating the memory block, we would also be necessarily eliminating the presence of the pathway associated with the storage effect (“internal pathway” in the manuscript). In this case, the receptor is a 2-state, 1-pathway system and, as such, it always satisfies an effective detailed balance. As a consequence, the definition of \delta Q_R reported in the manuscript does not hold anymore and the receptor does not exhibit any dissipation. Our choice to model two different pathways has been biologically motivated. We will make this crucial aspect clearer in the revised manuscript.

- Similarly, in Eq. (9) the authors use the ratio of the rates Γ_{s → s+1} and Γ_{s+1 → s} in their expression for internal dissipation. The first-rate corresponds to the synthesis reaction of memory molecules, while the second corresponds to a degradation reaction. Since the second reaction is not the microscopic reverse of the first, what would be the physical interpretation of the log of their ratio? Since the authors already use σ as the energy cost per storage unit, why not use σ times the rate of producing S as a metric for the dissipation rate?

In the current version of the manuscript, we employed the scheme of a controlled birth and death process to model the coupled process of readout and storage production. Since we are not dealing with a detailed biochemical underlying network, we used this coarse-grained description to capture the main features of the dynamics. In this sense, the considered reactions produce and destroy a molecule from a certain pool even if they are controlled in different ways by the readout. However, we completely agree with the point of view of the referee and will analyze our results following their suggestion.

(3) Impact of the pre-stimulus state. The plots in Figure 2 suggest that the environment was static before the application of repeated stimuli. Can the authors comment on the impact of the pre-stimulus state on the degree of habituation and its optimality properties? Specifically, would the conclusions stay the same if the prior environment had stochastic but aperiodic dynamics?

The initial stimulus is indeed stochastic with an average constant in time. Model response depends on the pre-stimulus level, since it also sets the stationary storage concentration before the first “strong” stimulation arrives. This dependence is not crucial for our result but deserves proper discussion, as the referee correctly pointed out. We will clarify this point in the revised version of this study.

(4) Clarification about the memory requirement for habituation. Figure 4 and the associated section argue for the essential role that the storage mechanism plays in habituation. Indeed, Figure 4a shows that the degree of habituation decreases with decreasing memory. The graph also shows that in the limit of vanishingly small Δ⟨S⟩, the system can still exhibit a finite degree of habituation. Can the authors explain this limiting behavior; specifically, why does habituation not vanish in the limit Δ⟨S⟩ -> 0?

We apologize for the lack of clarity here. Actually, Δ⟨S⟩ is not strictly zero, but equal to 0.15% at the final point. However, due to rounding this appears as 0% in the plot, and we will fix it in the revised version. Let us note that the fact that Δ⟨S⟩ is small signals a nonlinear dependence of Δ⟨U⟩ from Δ⟨S⟩, but no contradiction. We will clarify this aspect in the revised version.

Did you note a higher rate of "non-recovery" on the vaccinated animals? I'm wondering whether you'd hypothesize that vaccination against the EVs could be the cause of increased itching or irritation that alerts the animals to the ticks.

Would you mind sharing your rationale for isolating EVs at this time point? Do you know how EV cargo or composition might vary depending on the feeding time point when you harvest?

Curious if you know anything about the composition of these vesicles relative to each other? Are they carrying very distinct cargo?

Poverty is not only related to lack of material resources but also involves social shame. In school, students are taught that being poor is seen as inferior, leading to a feeling of being lesser than those who are not poor. This internalized classism demonstrates how economic status can have a detrimental impact on an individual's sense of self and self-esteem.

The author describes harsh living conditions, emphasizing the lack of basic amenities like reliable heating, air conditioning, and even a telephone. These are things normal families take for granted, illustrating how poverty can shape one’s daily existence through limited access to necessities. The necessities that the author's family did not have access to were proper climate control, safety, and communication.

This mindset that children born into poverty have conveys the internal struggle with class-based exclusion and insecurity. Their self-consciousness stems from repeated verbal and nonverbal cues that allowed for feelings of inadequacy due to poverty during their school years. Now, standing in front of students as a teacher, the narrator returns to the “scene of the crime,” reflecting on how deeply these formative experiences of social exclusion shaped their sense of self-worth.

This is how every low-income student must feel when their possessions can't measure up to their peers because of their family's inability to afford what other children's families could. It conveys the author's disheartened tone through his painful memories that served as a reminder of his family's financial limitations.

I agree with the author, as I do believe children who are born into poverty possess an innate resourcefulness that typical children are not forced to develop. This is due to the lack of resources available to them, which leads to a mentality of making the best out of what you have.

I wonder what different strategies can be implemented to combat the increase of US children in low-income situations. If there are less and less low-wage jobs available, but the correlation with low wage jobs and increased childbearing is evident, would providing improved access to birth control be one way to combat this rising issue?

It's important to note that there can be many different types of poverty but situational poverty seems especially relevant considering the recent hurricanes that thrust many residents of Florida into chaos.

subjectivity crucial to feminist perspectives but not the aim of anthro

Palestinian-American anthropologist. She is the Joseph L. Buttenweiser Professor of Social Science in the Department of Anthropology at Columbia

应为 ISO/IEC 42001，下同——所有使用标准全称时，均应体现制定标准的两个组织。 should be ISO/IEC, and this comment should apply to all the products involving the standards developed by JTC1.

eLife Assessment

The paper describes a novel approach for inferring features of synaptic networks from recordings of individual cells within the network. The paper will be a valuable contribution to those studying central pattern generators, including those involved in respiration. However, the theoretical approach to drawing inferences regarding the underlying synaptic currents is incomplete as it relies on unsupported simplifying assumptions.

Reviewer #1 (Public review):

Summary:

The paper develops a phase method to obtain the excitatory and inhibitory afferents to certain neuron populations in the brainstem. The inferred contributions are then compared to the results of voltage clamp and current clamp experiments measuring the synaptic contributions to post-I, aug-E, and ramp-I neurons.

Strengths:

The electrophysiology part of the paper is sound and reports novel features with respect to earlier work by JC Smith et al 2012, Paton et al 2022 (and others) who have mapped circuits of the respiratory central pattern generator. Measurements on ramp-I neurons, late-I neurons, and two types of post-I neurons in Figure 2 besides measurements of synaptic inputs to these neurons in Figure 5 are to my knowledge new.

Weaknesses:

The phase method for inferring synaptic conductances fails to convince. The method rests on many layers of assumptions and the inferred connections in Figure 4 remain speculative. To be convincing, such a method ought to be tested first on a model CPG with known connectivity to assess how good it is at inferring known connections back from the analysis of spatio-temporal oscillations. For biological data, once the network connectivity has been inferred as claimed, the straightforward validation is to reconstruct the experimental oscillations (Figure 2) noting that Rybak et al (Rybak, Paton Schwaber J. Neurophysiol. 77, 1994 (1997)) have already derived models for the respiratory neurons.

The transformation from time to phase space, unlike in the Kuramoto model, is not justified here (Line 94) and is wrong. The underpinning idea that "the synaptic conductances depend on the cycle phase and not on time explicitly" is flawed because synapses have characteristic decay times and delays to response which remain fixed when the period of network oscillations increases. Synaptic properties depend on time and not on phase in the network. One major consequence relevant to the present identification of excitatory or inhibitory behaviour, is that it cannot account for change in the behaviour of inhibitory synapses - from inhibitory to excitatory action - when the inhibitory decay time becomes commensurable to the period of network oscillations (Wang & Buzsaki Journal of Neuroscience 16, 6402 (1996), van Vreeswijk et al. J. Comp. Neuroscience 1,313 (1994), Borgers and Kopell Neural Comput. 15, 2003). In addition, even small delays in the inhibitory synapse response relative to the pre-synaptic action potential also produce in-phase synchronization (Chauhan et al., Sci. Rep. 8, 11431 (2018); Borgers and Kopell, Neural Comput. 15, 509 (2003)). The present assumptions are way too simplistic because you cannot account for these commensurability effects with a single parameter like the network phase. There is therefore little confidence that this model can reliably distinguish excitatory from inhibitory synapses when their dynamic properties are not properly taken into account.

Line 82, Equation 1 makes extremely crude assumptions that the displacement current (CdV/dt) is negligible and that the ion channel currents are all negligible. Vm(t) is also not defined. The assumption that the activation/inactivation times of all ion channels are small compared to the 10-20ms decay time of synaptic currents is not true in general. Same for the displacement current. The leak conductance is typically g~0.05-0.09ms/cm^2 while C~1uF/cm^2. Therefore the ratio C/g leak is in the 10-20ms range - the same as the typical docking neurotransmitter time in synapses.

Models of brainstem CPG circuits have been known to exist for decades: JC Smith et al 2012, Paton et al 2022, Bellingham Clin. Exp. Pharm. And Physiol. 25, 847 (1998); Rubin et al., J. Neurophysiol. 101, 2146 (2009) among others. The present paper does not discuss existing knowledge on respiratory networks and gives the impression of reinventing the wheel from scratch. How will this paper add to existing knowledge?

Reviewer #2 (Public review):

Summary:

By measuring intracellular changes in membrane voltage from a single neuron of the medulla the authors describe a method for determining the balance of excitatory and inhibitory synaptic drive onto a single neuron within this important brain region.

Strengths:

This approach could be valuable in describing the microcircuits that generate rhythms within this respiratory control centre. This method could more generally be used to enable microcircuits to be studied without the need for time-consuming anatomical tracing or other more involved electrophysiological techniques.

Weaknesses:

This approach involves assuming the reversal potential that is associated with the different permeant ions that underlie the excitation and inhibition as well as the application of Ohms law to estimate the contribution of excitation and inhibitory conductance. My first concern is that this approach relies on a linear I-V relationship between the measured voltage and the estimated reversal potential. However, open rectification is a feature of any I-V relationship generated by asymmetric distributions of ions (see the GHK current equation) and will therefore be a particular issue for the inhibition resulting from asymmetrical Cl- ion gradients across GABA-A receptors. The mixed cation conductance that underlies most synaptic excitation will also generate a non-linear I-V relationship due to the inward rectification associated with the polyamine block of AMPA receptors. Could the authors please speculate what impact these non-linearities could have on results obtained using their approach?

This approach has similarities to earlier studies undertaken in the visual cortex that estimated the excitatory and inhibitory synaptic conductance changes that contributed to membrane voltage changes during receptive field stimulation. However, these approaches also involved the recording of transmembrane current changes during visual stimulation that were undertaken in voltage-clamp at various command voltages to estimate the underlying conductance changes. Molkov et al have attempted to essentially deconvolve the underlying conductance changes without this information and I am concerned that this simply may not be possible. The current balance equation (1) cited in this study is based on the parallel conductance model developed by Hodgkin & Huxley. However, one key element of the HH equations is the inclusion of an estimate of the capacitive current generated due to the change in voltage across the membrane capacitance. I would always consider this to be the most important motivation for the development of the voltage-clamp technique in the 1930's. Indeed, without subtraction of the membrane capacitance, it is not possible to isolate the transmembrane current in the way that previous studies have done. In the current study, I feel it is important that the voltage change due to capacitive currents is taken into consideration in some way before the contribution of the underlying conductance changes are inferred.

Studies using acute slicing preparations to examine circuit effects have often been limited to the study of small microcircuits - especially feedforward and feedback interneuron circuits. It is widely accepted that any information gained from this approach will always be compromised by the absence of patterned afferent input from outside the brain region being studied. In this study, descending control from the Pons and the neocortex will not be contributing much to the synaptic drive and ascending information from respiratory muscles will also be absent completely. This may not have been such a major concern if this study was limited to demonstrating the feasibility of a methodological approach. However, this limitation does need to be considered when using an approach of this type to speculate on the prevalence of specific circuit motifs within the medulla (Figure 4). Therefore, I would argue that some discussion of this limitation should be included in this manuscript.

eLife Assessment

The study by Power and colleagues is important as elucidating the dynamic immune responses to photoreceptor damage in vivo potentiates future work in the field to better understand the disease process. However the evidence supporting the authors' claims is incomplete. The current manuscript would further benefit from validating their conclusion with additional supporting data from earlier time points (6 to 12 hours), additional markers to characterize neutrophils, more n numbers to strengthen the analysis, and evaluation of immune responses in mice with a stronger laser ablation, as well as further evidence to distinguish resident microglia vs. infiltrating macrophages due to the BRB breakdown. The authors should reorganize the article to make it easier and more straightforward to follow.

Reviewer #1 (Public review):

Summary:

The authors aimed to investigate the interaction between tissue-resident immune cells (microglia) and circulating systemic neutrophils in response to acute, focal retinal injury. They induced retinal lesions using 488 nm light to ablate photoreceptor (PR) outer segments, then utilized various imaging techniques (AOSLO, SLO, and OCT) to study the dynamics of fluorescent microglia and neutrophils in mice over time. Their findings revealed that while microglia showed a dynamic response and migrated to the injury site within a day, neutrophils were not recruited to the area despite being nearby. Post-mortem confocal microscopy confirmed these in vivo results. The study concluded that microglial activation does not recruit neutrophils in response to acute, focal photoreceptor loss, a scenario common in many retinal diseases.

Strengths:

The primary strength of this manuscript lies in the techniques employed.

In this study, the authors utilized advanced Adaptive Optics Scanning Laser Ophthalmoscopy (AOSLO) to document immune cell interactions in the retina accurately. AOSLO's micron-level resolution and enhanced contrast, achieved through near-infrared (NIR) light and phase-contrast techniques, allowed visualization of individual immune cells without extrinsic dyes. This method combined confocal reflectance, phase-contrast, and fluorescence modalities to reveal various cell types simultaneously. Confocal AOSLO tracked cellular changes with less than 6 μm axial resolution, while phase-contrast AOSLO provided detailed views of vascular walls, blood cells, and immune cells. Fluorescence imaging enabled the study of labeled cells and dyes throughout the retina. These techniques, integrated with conventional histology and Optical Coherence Tomography (OCT), offered a comprehensive platform to visualize immune cell dynamics during retinal inflammation and injury.

Weaknesses:

One significant weakness of the manuscript is the use of Cx3cr1GFP mice to specifically track GFP-expressing microglia. While this model is valuable for identifying resident phagocytic cells when the blood-retinal barrier (BRB) is intact, it is important to note that recruited macrophages also express the same marker following BRB breakdown. This overlap complicates the interpretation of results and makes it difficult to distinguish between the contributions of microglia and infiltrating macrophages, a point that is not addressed in the manuscript.

Another major concern is the time point chosen for analyzing the neutrophil response. The authors assess neutrophil activity 24 hours after injury, which may be too late to capture the initial inflammatory response. This delayed assessment could overlook crucial early dynamics that occur shortly after injury, potentially impacting the overall findings and conclusions of the study.

Reviewer #2 (Public review):

Summary:

This study uses in vivo multimodal high-resolution imaging to track how microglia and neutrophils respond to light-induced retinal injury from soon after injury to 2 months post-injury. The in vivo imaging finding was subsequently verified by an ex vivo study. The results suggest that despite the highly active microglia at the injury site, neutrophils were not recruited in response to acute light-induced retinal injury.

Strengths:

An extremely thorough examination of the cellular-level immune activity at the injury site. In vivo imaging observations being verified using ex vivo techniques is a strong plus.

Weaknesses:

This paper is extremely long, and in the perspective of this reviewer, needs to be better organized.

Study weakness: though the finding prompts more questions and future studies, the findings discussed in this paper are potentially important for us to understand how the immune cells respond differently to different severity levels of injury.

Reviewer #3 (Public review):

Summary:

This work investigated the immune response in the murine retina after focal laser lesions. These lesions are made with close to 2 orders of magnitude lower laser power than the more prevalent choroidal neovascularization model of laser ablation. Histology and OCT together show that the laser insult is localized to the photoreceptors and spares the inner retina, the vasculature, and the pigment epithelium. As early as 1-day after injury, a loss of cell bodies in the outer nuclear layer is observed. This is accompanied by strong microglial proliferation at the site of injury in the outer retina where microglia do not typically reside. The injury did not seem to result in the extravasation of neutrophils from the capillary network constituting one of the main findings of the paper. The demonstrated paradigm of studying the immune response and potentially retinal remodeling in the future in vivo is valuable and would appeal to a broad audience in visual neuroscience. However, there are some issues with the conclusions drawn from the data and analysis that can be addressed to further bolster the manuscript.

Strengths:

Adaptive optics imaging of the murine retina is cutting edge and enables non-destructive visualization of fluorescently labeled cells in the milieu of retinal injury. As may be obvious, this in vivo approach is beneficial for studying fast and dynamic immune processes on a local time scale - minutes and hours, and also for the longer days-to-months follow-up of retinal remodeling as demonstrated in the article. In certain cases, the in vivo findings are corroborated with histology.

The analysis is sound and accompanied by stunning video and static imagery. A few different sets of mouse models are used, (a) two different mouse lines, each with a fluorescent tag for neutrophils and microglia, (b) two different models of inflammation - endotoxin-induced uveitis (EAU) and laser ablation are used to study differences in the immune interaction.

One of the major advances in this article is the development of the laser ablation model for 'mild' retinal damage as an alternative to the more severe neovascularization models. While not directly shown in the article, this model would potentially allow for controlling the size, depth, and severity of the laser injury opening interesting avenues for future study.

Weaknesses:

(1) It is unclear based on the current data/study to what extent the mild laser damage phenotype is generalizable to disease phenotypes. The outer nuclear cell loss of 28% and a complete recovery in 2 months would seem quite mild, thus the generalizability in terms of immune-mediated response in the face of retinal remodeling is not certain, specifically whether the key finding regarding the lack of neutrophil recruitment will be maintained with a stronger laser ablation.

(2) Mice numbers and associated statistics are insufficient to draw strong conclusions in the paper on the activity of neutrophils, some examples are below :

a) 2 catchup mice and 2 positive control EAU mice are used to draw inferences about immune-mediated activity in response to injury. If the goal was to show 'feasibility' of imaging these mouse models for the purposes of tracking specific cell type behavior, the case is sufficiently made and already published by the authors earlier. It is possible that a larger sample size would alter the conclusion.

b) There are only 2 examples of extravasated neutrophils in the entire article, shown in the positive control EAU model. With the rare extravasation events of these cells and their high-speed motility, the chance of observing their exit from the vasculature is likely low overall, therefore the general conclusions made about their recruitment or lack thereof are not justified by these limited examples shown.

c) In Figure 3, the 3-day time point post laser injury shows an 18% reduction in the density of ONL nuclei (p-value of 0.17 compared to baseline). In the case of neutrophils, it is noted that "Control locations (n = 2 mice, 4 z-stacks) had 15 {plus minus} 8 neutrophils per sq.mm of retina whereas lesioned locations (n = 2 mice, 4 z-stacks) had 23 {plus minus} 5 neutrophils per sq.mm of retina (Figure 10b). The difference between control and lesioned groups was not statistically significant (p = 0.19)." These data both come from histology. While the p-values - 0.17 and 0.19 - are similar, in the first case a reduction in ONL cell density is concluded while in the latter, no difference in neutrophil density is inferred in the lesioned case compared to control. Why is there a difference in the interpretation where the same statistical test and methodology are used in both cases? Besides this statistical nuance, is there an alternate possibility that there is an increased, albeit statistically insignificant, concentration of circulating neutrophils in the lesioned model? The increase is nearly 50% (15 {plus minus} 8 vs. 23 {plus minus} 5 neutrophils per sq.mm) and the reader may wonder if a larger animal number might skew the statistic towards significance.

(2) The conclusions on the relative activity of neutrophils and microglia come from separate animals. The reader may wonder why simultaneous imaging of microglia and neutrophils is not shown in either the EAU mice or the fluorescently labeled catchup mice where the non-labeled cell type could possibly be imaged with phase-contrast as has been shown by the authors previously. One might suspect that the microglia dynamics are not substantially altered in these mice compared to the CX3CR1-GFP mice subjected to laser lesions, but for future applicability of this paradigm of in vivo imaging assessment of the laser damage model, including documenting the repeatability of the laser damage model and the immune cell behavior, acquiring these data in the same animals would be critical.

(3) Along the same lines as above, the phase contrast ONL images at time points from 3-day to 2-month post laser injury are not shown and the absence of this data is not addressed. This missing data pertains only to the in vivo imaging mice model but are conducted in histology that adequately conveys the time-course of cell loss in the ONL. It is suggested that the reason be elaborated for the exclusion of this data and the simultaneous imaging of microglia and neutrophils mentioned above. Also, it would be valuable to further qualify and check the claims in the Discussion that "ex vivo analysis confirms in vivo findings" and "Microglial/neutrophil discrimination using label-free phase contrast"

eLife Assessment

This useful study combines multiplexed RNA-FISH with downstream analyses and modelling to describe novel dendritic mRNA distribution and behavioural features. Although the downstream analysis pipeline is novel, the results from this study are as of yet incomplete. Further inclusion of key missing controls, further work to better assess the physiological relevance, or additional modelling to expand their conclusions would make this work of greater interest to RNA biologists.

Reviewer #1 (Public review):

Summary:

Characterizing the molecular and spatial organization of dendritically localized RNAs is an important endeavor as the authors nicely articulate in their abstract and introduction. In particular, identifying patterns of mRNA distribution and colocalization between groups of RNAs could characterize new mechanisms of transport and/or reveal new functional relationships between RNAs. However, it's not clear to me how much the current study addresses those gaps in knowledge. The manuscript by Kim et al uses 8 overlapping combinations of 3-color fluorescence in situ hybridization to characterize the spatial distributions and pairwise colocalizations of six previously uncharacterized dendritically localized RNAs in cultured neurons (15 DIV). The strength of the work is in the graph-based analyses of individual RNA distances from the soma, but the conclusions reached, that spatial distributions vary per dendritic RNA, has been well known since early 2000s (as reviewed in Schuman and Steward, 2001 & 2003), but paradoxically the authors show that dendritic length can account for these differences. It's not clear to me the significance of the spatial distribution relationship with dendritic morphology as distinct spatial distribution patterns (i.e. proximal expression then drop off) have been clearly shown in intact circuits with homogeneity in dendrite length governed by neuropil laminae. The colocalization results are intriguing but as currently presented they lack sufficient control analyses and contextualization to be compelling. In general, the results of the manuscript are potentially interesting but unnecessarily difficult to follow both in text and figure presentation.

Major comments:

The authors state that their data expand upon our understanding of dendritic RNA spatial distributions by adding high-resolution data for six newly characterized dendritic RNAs. While this is true, without including data for a well-known/previously characterized RNA, it makes it difficult for the reader to contextualize how these new data on six dendritic RNAs fit in with our understanding of the dendritic RNAs with well-described spatial distributions and colocalization analyses (Camk2a, Actb, Map1b, etc). For example, how do we interpret the 7-fold higher colocalization values between RNAs in this manuscript compared to the results of Batish et al (as referred to in the paper)-is it because these RNAs are fundamentally different, or is it because of other experimental factors/conditions? The spatial distribution patterns described in this manuscript differ from those of Fonkeu et al, but an alternative explanation is that Fonkeu et al modeled based on Camk2a, not the six genes studied here. Is it possible that these six RNAs have similar distribution patterns (as shown) whereby dendritic morphology impacts distribution more than individual differences but inclusion of dendritic RNAs with demonstrably different distributions (Camk2a/distal localization vs Map2/proximal localization) would alter the results?

Reviewer #2 (Public review):

In the manuscript by Kim et al titled, "Characterizing the Spatial Distribution of Dendritic RNA at Single Molecule Resolution," the authors perform multiplex single-molecule FISH in cultured neurons, along with analysis and modeling, to show the spatial features, including differing mRNA densities between soma and dendrites, dendritic length-related distributions and clustering, of multiple mRNAs in dendrites. Although the clustering analyses and modeling are intriguing and offer previously underappreciated spatial association within and across mRNA molecules, the data is difficult to interpret and the conclusions lack novelty in their current form. There is a need for a stronger rationale as to why the methodology employed in the manuscript is better suited to characterize the clustering of mRNA in dendrites compared to previously published works and how such clustering or declustering can affect dendritic/neuronal function.

(1) Validation of mRNA labeling, detection, and quantification is necessary. Single-molecule fluorescence in situ hybridization (smFISH) is the gold standard to detect RNA inside cells. The method utilizes multiple fluorescent probes (~48) designed to hybridize along a single RNA, resulting in a population of diffraction-limited fluorescent puncta with varying intensities. A histogram of cytoplasmic smFISH puncta intensities should reveal a normally distributed population with a single major peak, where the upper and lower tails indicate the maximum probe binding and the lower detection limit, respectively. Once single molecule detection (and limits) have been established, smFISH should be performed for each gene individually to obtain ground truth of detection under identical experimentally-defined conditions using the same fluorophore. Total RNA counts from different probe combinations (Figure S1A) or total mRNA density (Figure 2A) is not sufficient to inform individual gene labeling efficiency or detection. It is difficult to interpret whether observed variabilities across different probe combinations are of significance. For example, the mRNA densities of Adap2 and Dtx3L in soma seem to vary even after normalization with the pixel area (Figure 2A).

Absolute counts and normalized counts for each gene detected should be included in the results or in supplementary data/table to provide the reader with a reference point for evaluation.

As a control, it is recommended to perform smFISH against beta-actin or aCaMKII, which are the two most abundant mRNA in dendrites, and serve as internal validation that the technique, detection, and quantification are consistent with previously published works.

(2) The rationale for single dendrite selection is unclear. To suggest that dendrite length, as a feature of dendritic morphology, may affect mRNA localization in dendrites, the authors manually selected segments of dendrites that have no branching or overlap, 'biased for shorter dendrites,' resulting in a subset of dendritic segments that changes mRNA distribution in raw distances (Figure S3A) into the normalized distance (Figure 4A). As a result, the distribution appears to convert from a monotonic- or exponential-decay to a more even distribution of mRNA (plateau). The rationale for this normalization is unclear, as manual curation of dendritic segments can incorporate experimenter bias. Moreover, the inclusion of short dendritic segments can stretch out their mRNA distributions following distance normalization which can give the appearance of an even distribution of mRNAs when aggregated.

Next, the authors use pairwise Jensen-Shannon distance cluster analysis to identify 4 different patterns of clustering among mRNAs. Although the patterns are quite intriguing, the distributions of mRNA clusters were i) difficult to interpret and ii) compared to Fonkeu et al (2019) protein distribution is not a sufficient explanation for the observed clustering. For example, the clustering patterns (C1-4) are quite striking and even if the authors' analyses were an improvement in identifying mRNA clustering in dendrites, the authors need to provide better justification or modeling on what role such clustering can play on dendritic function or cellular physiology. This is important and necessary as the authors are suggesting that their analysis is different from mRNA distributions previously observed or modeled by Buxbaum et al (2014) and Fonkeu et al (2019), respectively.<br /> Of note, the identity-independent and dendritic length-dependent aspect of spatial distributions of mRNAs is striking (Figure S3E-F, Figure 4), and this length-related feature is one of the major take-home points in the first part of the manuscript. However, it is evident that some mRNAs (e.g. Adap2 and Dtx3L) or probe combinations (e.g. Colec12-Adap2-Nsmf) disproportionally make up the mRNA distribution clusters (Figure 4D and Figure S3F). It seems plausible that the copy numbers of mRNAs can differentially affect clusters' distribution patterns. Appropriate statistical tests among the cluster groups, therefore, will help to strengthen the interpretation of the results provided in the supplementary figures (Figures S3E and S3F).

(3) It is not clear how Figure 5 GradCAM analysis helps the point that the authors put forth in previous sections or forthcoming sections. Unless this section and figure are more effectively linked to the general theme of the paper - the morphological features as a determinant of mRNA distribution or clustering of mRNA molecules, it may be included in the supplementary figure section.

(4) Clustering of mRNA remains an exception rather than the rule. From their high-resolution triple smFISH data, the authors make some interesting findings regarding colocalization in dendrites. Among the six genes tested, the authors found higher incidents of colocalization between pair-wise genes (up to 23%) than previously reported (5-10%). Also, they report higher levels of colocalization within the same gene (17-23%) than previously reported (5-10%). First, to better evaluate this increased colocalization efficiency overall, the histograms of smFISH puncta intensity are necessary (as stated in 1) to determine whether a second peak is present in the population. Second, even though 23% is higher than previously reported, it remains that 77% do not colocalize and does not suggest that colocalization is the rule but remains the exception. Given the results in Table 1, it is likely that the increased colocalization could be a gene-specific effect and not transcriptome-wide as the majority of values between genes are below 10%, consistent with previous findings. Third, labeling of a control gene (i.e. b-actin or aCaMKII) would provide higher confidence that the detection and colocalization comparisons are consistent with previous findings.

It is recommended to refrain from concluding that mRNA is 'co-transported' from smFISH results. Typically co-transport is best identified through observations in live cells where two fluorescent particles of different colors are moving together. Although stationary particles positioned in close proximity to one another could potentially be co-transported, there has been very little evidence to support this.

The use of Ripley's K-function is an interesting way to look at clustering neighborhoods within a single or pairwise sets of genes. Previous studies from the Singer group have looked at mRNA clustering and have observed that mRNA in living cells tends to cluster within a 6-micron range for b-actin and for both b-actin and Arc after local stimulation. What was intriguing in the results in Figure 7 was that there was an exclusion zone 2-4 microns away from the area of colocalization that may suggest that mRNA are able to avoid over-clustering and maintain an even distribution throughout the dendrite--perhaps with a goal of not devoting too many resources (mRNA) to a single dendritic area. Modeling how mRNAs avoid over-clustering to a specific 2-micron segment of dendrites could provide an explanation on how dendrites can respond to multiple or simultaneous synaptic activity at different sites along the same dendrite.

Reviewer #3 (Public review):

Summary:

The paper by Kim et al utilizes smFISH method to probe for six genes to understand the spatial distribution of the mRNAs in dendrites and identify the spatial relationships between the transcripts. While they have delved into a high-resolution characterization of the dendritic transcripts and compared their data with existing datasets, the analysis needs more robustness, and therefore the findings are inconclusive. The rationale of the study and choosing these genes is not clear - it appears more like a validation of some of the datasets without much biological significance.

Overall, several conclusions for spatial distribution of dendritic RNAs were based on correlations and it is difficult to understand whether this represents a true biological phenomenon or if it is an artifact of the imaging and morphological heterogeneity of neurons and difficulties in dendritic segmentation.

Strengths:

The authors have performed an extensive analysis of the smFISH datasets and quantified the precise localization patterns of the dendritic mRNAs in relation to the dendritic morphology. Their images and the analysis pipeline can be a resource for the community.

Weaknesses:

(1) The authors have attempted to identify general patterns of mRNA distribution as a function of distance, proximal vs distal, however, in many of the cases the results are a bit redundant and the size of the neurons or the length of the dendrites or image segmentation artifacts turn out to be the determining factors. A better method to normalize the morphological differences is needed to make meaningful conclusions about RNA distribution patterns.

(2) Another concerning factor is that there are many redundancies throughout the paper. For example, to begin with, all analysis should have been done as RNA density measurements (and not absolute numbers of mRNAs) and with proper normalization and accounting for differences in length. Some of these were done only in the latter half of the paper, for example in Figure 4.

(3) Images for the smFISH are missing. It is important to show the actual images, and the quality of the images is a crucial factor for all subsequent analyses.

(4) The parameters used for co-localization analysis are very relaxed (2 - 6 microns), particularly the distances of interactions far exceed feasible interactions between the biomolecules. Typically, transport granules are significantly smaller than the length scales used.

She’s not just making small talk. She’s helping him develop verbal skills by encouraging him to explain and clarify his thoughts.

eLife Assessment

The study is useful for advancing understanding of spinal cord injuries, but it presents inadequate evidence due to the use of multiple datasets. Data were collected from different models of spinal cord injury, various regions of the spinal cord, and an iPSC model, with the differences between these models making it difficult to draw reliable conclusions.

Reviewer #1 (Public review):

Summary:

The work of Zhou's team is to perform bioinformatics analysis of single-cell transcriptomes (scRNA), spatial transcriptomic (ST) data, and bulk RNA-seq data from Gene Expression Omnibus (GEO) datasets, published or not in different journals from other teams, about spinal cord injury and/or microglia cells derived human iPSC. Based on their analysis, the authors claim that innate microglial cells are inhibited. They postulate that TGF beta signaling pathways play a role in the regulation of migration to enhance SCI recovery and that Trem2 expression contributes to neuroinflammation response by modulating cell death in spinal cord injury. Finally, they suggest a therapeutic strategy to inhibit Trem2 responses and transplant iPSC-derived microglia with long-term TGF beta stimulation.

Although the idea of using already available data and reanalyzing them is remarkable, I have major concerns about the paper. The authors have used data from different models of injury, regions, as well as IPSC. It is not possible to mix and draw conclusions when the models used are different. This raises doubts about the authors' expertise in the field of spinal cord injury. Furthermore, the innovativeness of the results is of little significance, especially as no hypothesis is confirmed by experimental data.

Strengths:

Analysis of already large-scale existing data.

Weaknesses:

Mixing data from different models, unfounded conclusions, and over-interpretations, little expertise in the field of spinal cord injury.

Reviewer #2 (Public review):

Summary:

The authors present an intriguing study utilizing datasets from spinal cord injury (SCI) research to identify potential microglial genes involved in SCI-induced neuronal damage. They identify that inhibiting TREM2 and enhancing the TGF-b signal pathway can inhibit reactive microglia-mediated neuroinflammation. Microglia transplantation using iPSC-derived microglia could also be beneficial for SCI recovery.

Strengths:

This research aims to identify potential genes and signaling pathways involved in microglia-mediated inflammation in spinal cord injury (SCI) models. Meanwhile, analyzing transplanted microglia gene expression provides an extra layer of potential in SCI therapy. The approach represents a good data mining strategy for identifying potential targets to combat neurological diseases.

Weaknesses:

Microglial gene expression patterns may vary significantly between these models. Without proper normalization or justification, combining these datasets to draw conclusions is problematic. Moreover, other factors also need to be considered, like the gender of the microglia source. Are there any gender differences? How were the iPSC-derived microglia generated? Different protocols may affect microglia gene expression.

While the concept is interesting, the data presented in this study appears preliminary. Without further experiments to support their findings, the conclusions are not convincing.

Reviewer #3 (Public review):

Summary:

In this study, the authors perform a meta-analysis of existing transcriptomic data describing the responses of cells in the mouse spinal cord to traumatic injury (SCI). They identify two subclasses of microglia, which they term 'innate' and 'reactive' microglia, in the dataset, with the majority of microglia in the uninjured spinal cord being 'innate' and the majority of microglia in the injured region being 'reactive'. The authors propose that, during injury, the population of innate microglia is depleted and replaced by the population of reactive microglia. Using DEG and gene ontology pipelines, the authors suggest that TGF signaling is a positive force that helps recruit healthy microglia to enhance recovery in the context of SCI. In contrast, the microglial phagocytic receptor Trem2 contributes to neuroinflammation and neuronal death. Finally, the authors suggest replacing reactive microglia with innate microglia as a potential therapeutic approach to treat SCI in humans.

Strengths:

The work utilizes numerous and multi-modal datasets describing transcriptomic changes in the mouse CNS following SCI.

The topic is translationally relevant.

Weaknesses:

There is not enough information about how each of the datasets re-analyzed by the authors was obtained and processed both by the group generating the data and by the group re-analyzing it.

The conclusions drawn by the authors are not sufficiently supported by the evidence.

Whether the study represents a significant conceptual advance in our understanding of microglial contributions to SCI is not clear.

My specific concerns and suggestions to address these weaknesses are provided below.

Major comments:

(1) Questions remain about the nature, quality, and features of the datasets re-analyzed in the study. For example, how were these datasets obtained? Were the same animal models and time points used in each? What modality of RNA sequencing was done? What criteria did the authors consider in deciding which datasets to include in the study? Since the study is entirely reliant on data generated elsewhere, a more thorough description of these datasets within the text is needed.

(2) Relatedly, the authors chose to filter out some cells from the datasets based on quality, but this information is incomplete. For example, the authors omit cells with 10% mitochondrial genes, but this value is higher than most investigators use (typically between 1%-5%). Why is 10% the appropriate limit in this particular study? Further, how did the authors ensure the removal of doublets from the dataset?

(3) A principal finding of the paper is that microglia in the uninjured CNS mostly have an 'innate' transcriptomic phenotype, while microglia in the injured CNS mostly have a 'reactive' phenotype. However, there are some issues here that require further discussion. First, while historically microglia were thought to possess distinct 'homeostatic' versus 'activated' profiles which would be consistent with the authors' interpretations here, these differences are now thought of more as changes in a given microglial cell's transcriptomic status. Thus, while the authors interpret their results as meaning that innate microglia are depleted and replaced by a different set of reactive microglia following SCI (or at least this is how the paper is written), it is equally if not more likely that the microglia within the injured regions themselves become more reactive as a result of the insult. The authors should clarify why their interpretation is more likely to be correct.

(4) Related to the above point, the authors base the manuscript on the idea that microglia are mostly 'innate' in the uninjured CNS and 'reactive' after injury, however, the UMAP plots in Figures 1A and 1C suggest that both classes of microglia cluster together and may not actually represent distinct subclasses. Have the authors tried sub-clustering just the myeloid clusters and seeing how well they separate? Even if they do technically represent distinct clusters, the UMAP could be interpreted to mean that their transcriptomic differences are not particularly robust.

(5) I appreciate the authors' use of loss-of-function data to explore the roles of microglial TGF and Trem2 signaling to glean some mechanistic insights into SCI. However, many of the conclusions reached by the authors in the manuscript are insufficiently supported by the data and would require additional experiments to rigorously confirm. A couple of examples are the following:<br /> 5a. Lines 160-162: "Hence, we conclude that the cascade of injury events in SCI significantly influences microglia, leading to the replacement of innate microglial cells by reactive microglia." That SCI influences microglia is well-supported by the study, but whether reactive microglia replace innate microglia, versus whether innate microglia in the region transition to a reactive state, needs to be tested experimentally.<br /> 5b. Lines 321-323: "Taken together, iPSC-derived microglia have the potential to replace the functions of naïve microglial cells, and they perform even more effectively in the in vivo CNS." Again, the first part of the sentence is supported, but whether iPSCs are more effective than other populations in vivo would need to be tested experimentally.

(6) As microglia have long been appreciated as contributors to the CNS injury response, the conceptual advance here isn't particularly clear to me. For example, Gao et al, 2023 (*cited by the authors) describe the role of Trem2+ microglia in SCI versus demyelinating disease with major conceptual overlap with the current study. It would be helpful for the authors to include a discussion of what we now know about SCI based on this study that we did not know (or strongly suspect) before.

eLife Assessment

Barzó et al. assessed the electrophysiological and anatomical properties of a large number of layer 2/3 pyramidal neurons in brain slices of human neocortex across a wide range of ages, from infancy to elderly individuals, using whole-cell patch clamp recordings and anatomical reconstructions. This large data set represents a valuable contribution to our understanding of how these properties change across the human lifespan, and although the results presented are convincing, analyzing the data by absolute age rather than age ranges as well as clarifying the methods used and some of the statistical approaches applied would strengthen the conclusions. The analysis of spine density requires additional biological replicates to support the conclusions stated. These data strengthen our understanding of how these properties change with age and will contribute to building more realistic models of human cortical function.

Reviewer #1 (Public review):

Summary:

The manuscript co-authored by Pál Barzó et al is very clear and very well written, demonstrating the electrophysiological and morphological properties of human cortical layer 2/3 pyramidal cells across a wide age range, from age 1 month to 85 years using whole-cell patch clamp. To my knowledge, this is the first study that looks at the cross-age differences in biophysical and morphological properties of human cortical pyramidal cells. The community will also appreciate the significant effort involved in recording data from 485 cells, given the challenges associated with collecting data from human tissue. Understanding the electrophysiological properties of individual cells, which are essential for brain function, is crucial for comprehending human cortical circuits. I think this research enhances our knowledge of how biophysical properties change over time in the human cortex. I also think that by building models of human single cells at different ages using these data, we can develop more accurate representations of brain function. This, in turn, provides valuable insights into human cortical circuits and function and helps in predicting changes in biophysical properties in both health and disease.

Strengths:

The strength of this work lies in demonstrating how the electrophysiological and morphological features of human cortical layer 2/3 pyramidal cells change with age, offering crucial insights into brain function throughout life.

Weaknesses:

One potential weakness of the paper is that the methodology could be clearer, especially in how different cells were used for various electrophysiological measurements and the conditions under which the recordings were made. Clarifying these points would improve the study's rigor and make the results easier to interpret.

Reviewer #2 (Public review):

Summary:

In this study, Barzo and colleagues aim to establish an appraisal for the development of basal electrophysiology of human layer 2/3 pyramidal cells across life and compare their morphological features at the same ages.

Strengths:

The authors have generated recordings from an impressive array of patient samples, allowing them to directly compare the same electrophysiological features as a function of age and other biological features. These data are extremely robust and well organised.

Weaknesses:

The use of spine density and shape characteristics is performed from an extremely limited sample (2 individuals). How reflective these data are of the population is not possible to interpret. Furthermore, these data assume that spines fall into discrete types - which is an increasingly controversial assumption.

Many data are shown according to somewhat arbitrary age ranges. It would have been more informative to plot by absolute age, and then perform more rigourous statistics to test age-dependent effects.

Overall, the authors achieve their aims by assessing the physiological and morphological properties of human L2/3 pyramidal neurons across life. Their findings have extremely important ramifications for our understanding of human life and implications for how different neuronal properties may influence neurological conditions.

Reviewer #3 (Public review):

Summary:

To understand the specificity of age-dependent changes in the human neocortex, this paper investigated the electrophysiological and morphological characteristics of pyramidal cells in a wide age range from infants to the elderly.

The results show that some electrophysiological characteristics change with age, particularly in early childhood. In contrast, the larger morphological structures, such as the spatial extent and branching frequency of dendrites, remained largely stable from infancy to old age. On the other hand, the shape of dendritic spines is considered immature in infancy, i.e., the proportion of mushroom-shaped spines increases with age.

Strengths:

Whole-cell recordings and intracellular staining of pyramidal cells in defined areas of the human neocortex allowed the authors to compare quantitative parameters of electrophysiological and morphological properties between finely divided age groups.

They succeeded in finding symmetrical changes specific to both infants and the elderly, and asymmetrical changes specific to either infants or the elderly. The similarity of pyramidal cell characteristics between areas is unexpected.

Weaknesses:

Human L2/3 pyramidal cells are thought to be heterogeneous, as L2/3 has expanded to a high degree during the evolution from rodents to humans. However, the diversity (subtyping) is not revealed in this paper.

eLife Assessment

Oor and colleagues report the potentially independent effects of the spatial and feature-based selection history on visuomotor choices. They outline compelling evidence, tracking the dynamic history effects based on their extremely clever experimental design (urgent version of the search task). Their finding is of fundamental significance, broadening the framework to identify variables contributing to choice behavior and their neural correlates in future studies.

Reviewer #1 (Public review):

Summary:

Oor et al. report the potentially independent effects of the spatial and feature-based selection history on visuomotor choices. They outline compelling evidence, tracking the dynamic history effects based on their clever experimental design (urgent version of the search task). Their finding broadens the framework to identify variables contributing to choice behavior and their neural correlates in future studies.

Strengths:

In their urgent search task, the variable processing time of the visual cue leads to a dichotomy in choice performance - uninformed guesses vs. informed choices. Oor et al. did rigorous analyses to find a stronger influence of the location-based selection history on the uninformed guesses and a stronger influence of the feature-based selection history on the informed choices. It is a fundamental finding that contributes to understanding the drivers of behavioral variance. The results are clear.

Weaknesses:

(1) In this urgent search task, as the authors stated in line 724, the variability in performance was mainly driven by the amount of time available for processing the visual cue. The authors used processing time (PT) as the proxy for this "time available for processing the visual cue." But PT itself is already a measure of behavioral variance since it is also determined by the subject's reaction time (i.e., PT = Reaction time (RT) - Gap). In that sense, it seems circular to explain the variability in performance using the variability in PT. I understand the Gap time and PT are correlated (hinted by the RT vs. Gap in Figure 1C), but Gap time seems to be more adequate to use as a proxy for the (imposed) time available for processing the visual cue, which drives the behavioral variance. Can the Gap time better explain some of the results? It would be important to describe how the results are different (or the same) if Gap time was used instead of PT and also discuss why the authors would prefer PT over Gap time (if that's the case).

(2) The authors provide a compelling account of how the urgent search task affords<br /> (i) more pronounced selection history effects on choice and<br /> (ii) dissociating the spatial and feature-based history effects by comparing their different effects on the tachometric curves. However, the authors didn't discuss the limits of their task design enough. It is a contrived task (one of the "laboratoray tasks"), but the behavioral variability in this simple task is certainly remarkable. Yet, is there any conclusion we should avoid from this study? For instance, can we generalize the finding in more natural settings and say, the spatial selection history influences the choice under time pressure? I wonder whether the task is simple yet general enough to make such a conclusion.

(3) Although the authors aimed to look at both inter- and intra-trial temporal dynamics, I'm not sure if the results reflect the true within-trial dynamics. I expected to learn more about how the spatial selection history bias develops as the Gap period progresses (as the authors mentioned in line 386, the spatial history bias must develop during the Gap interval). Does Figure 3 provide some hints in this within-trial temporal dynamics?

(4) The monkeys show significant lapse rates (enough error trials for further analyses). Do the choices in the error trials reflect the history bias? For example, if errors are divided in terms of PTs, do the errors with short PT reflect more pronounced spatial history bias (choosing the previously selected location) compared to the errors with long PT?

Reviewer #2 (Public review):

Summary:

This is a clear and systematic study of trial history influences on the performance of monkeys in a target selection paradigm. The primary contribution of the paper is to add a twist in which the target information is revealed after, rather than before, the cue to make a foveating eye movement. This twist results in a kind of countermanding of an earlier "uninformed" saccade plan by a new one occurring right after the visual information is provided. As with countermanding tasks in general, time now plays a key factor in the success of this task, and it is time that allows the authors to quantitatively assess the parametric influences of things like previous target location, previous target identity, and previous correctness rate on choice performance. The results are logical and consistent with the prior literature, but the authors also highlight novelties in the interpretation of prior-trial effects that they argue are enabled by the use of their paradigm.

Strengths:

Careful analysis of a multitude of variables influencing behavior

Weaknesses:

Results appear largely confirmatory.

Privacy violations extend beyond hacking to include unclear policies, unauthorized sharing, and the misuse of metadata. These breaches can expose personal information without consent, as seen in cases like John McAfee's or Netflix's data leak. Even anonymized data can be deanonymized, making privacy protections increasingly challenging in the digital age.

This is a very malicious thing done by companies to keep the user from properly protecting themselves. For social media, it makes it so the site always has an upper hand when it comes to your data because you never really know what privacy you have a right to in the site. For work/school systems, this is often used to punish / get evidence for types of misconduct, so it could be argued that it is beneficial. Still, making that knowledge not obvious is a sketchy thing to do as it can be used to pile on a person that someone already wants to get fired / expelled - the admin can be selective in who they actually punish for inappropriate comments.

This is an interesting point to raise because it's one that seems exceedingly challenging to quantify. If Twitter sees that I frequently post about living in Seattle and shows me other posts about/from Seattle, even if I have turned location sharing off, is that creating a shadow profile? The nature of a lot of this data mining is that it results in a more personally-tailored social media experience which itself is more profitable for companies, making the ethical analysis of it complicated. On the far end of the spectrum, companies could store ZERO information about users (i.e. there are no accounts, everyone is an anonymous user) but this tends to result in much less usable platforms. I'd argue that, while there certainly should be more comprehensive regulations on data collection, a lot of inferred data is nothing that couldn't be figured out by a person looking at your account.

Privacy is essential for personal security, autonomy, and maintaining control over one's information. It allows individuals to manage their reputation, avoid harm, and protect themselves from misuse of data by companies, governments, or individuals. However, social media platforms often compromise privacy, raising ethical concerns about surveillance and consent.

Privacy is complex and that people might seek it for morally sound or questionable reasons. That's the reflection of how cautious and concerned people have on security of their private information online.

Recent social media leaks show how easy private material can propagate, even when context collapse can result in unintentional exposure of private chats. Platforms must guarantee improved privacy measures since the boundaries between private and public online interactions are becoming increasingly hazy.

I feel that social media sites direct messages should be private from other users but shouldn't be private to the companies. Many illegal things happen through private messaging, and we shouldn't expect our dms to be fully private.

Besides volunteering, sharing private messages or information. Some groups purposely use the information of privacy to some profit-related things, privacy in this time would hurt people because of the public sharing. And this makes us think deeper and more about privacy sharing.

This is a main point

eLife Assessment

The manuscript explores how bacterial evolution in the presence of lytic phages modulates b-lactams resistance and virulence properties in methicillin-resistant Staphylococcus aureus (MRSA). The work is useful as it identifies underlying mutations that may confer sensitivity to b-lactams and alter virulence properties. While the findings are generally convincing, additional experiments linking how particular mutations regulate phenotypic changes are required to improve the work mechanistically.

Reviewer #1 (Public review):

Summary:

These authors have asked how lytic phage predation impacts antibiotic resistance and virulence phenotypes in methicillin-resistant Staphylococcus aureus (MRSA). They report that staphylococcal phages cause MRSA strains to become sensitized to b-lactams and to display reduced virulence. Moreover, they identify mutations in a set of genes required for phage infection that may impact antibiotic resistance and virulence phenotypes.

Strengths:

Phage-mediated re-sensitization to antibiotics has been reported previously but the underlying mutational analyses have not been described. These studies suggest that phages and antibiotics may target similar pathways in bacteria.

Weaknesses:

One limitation is the lack of mechanistic investigations linking particular mutations to the phenotypes reported here. This limits the impact of the work.

Another limitation of this work is the use of lab strains and a single pair of phages. However, while incorporation of clinical isolates would increase the translational relevance of this work it is unlikely to change the conclusions.

Reviewer #2 (Public review):

Summary:

The work presented in the manuscript by Tran et al deals with bacterial evolution in the presence of bacteriophage. Here, the authors have taken three methicillin-resistant S. aureus strains that are also resistant to beta-lactams. Eventually, upon being exposed to phage, these strains develop beta-lactam sensitivity. Besides this, the strains also show other changes in their phenotype such as reduced binding to fibrinogen and hemolysis.

Strengths:

The experiments carried out are convincing to suggest such in vitro development of sensitivity to the antibiotics. Authors were also able to "evolve" phage in a similar fashion thus showing enhanced virulence against the bacterium. In the end, authors carry out DNA sequencing of both evolved bacteria and phage and show mutations occurring in various genes. Overall, the experiments that have been carried out are convincing.

Weaknesses:

Although more experiments are not needed, additional experiments could add more information. For example, the phage gene showing the HTH motif could be reintroduced in the bacterial genome and such a strain can then be assayed with wildtype phage infection to see enhanced virulence as suggested. At least one such experiment proves the discoveries regarding the identification of mutations and their outcome. Secondly, I also feel that authors looked for beta-lactam sensitivity and they found it. I am sure that if they look for rifampicin resistance in these strains, they will find that too. In this case, I cannot say that the evolution was directed to beta-lactam sensitivity; this is perhaps just one trait that was observed. This is the only weakness I find in the work. Nevertheless, I find the experiments convincing enough; more experiments only add value to the work.

for - rapid whole system change - book - The Ascent of Humanity - Charles Eisenstein

Summary - Annotation was not available when in first read this book - It is a book worthy of full annotation as it is so important to the existential polycris we now face - I was reminded of it as I was annotating Nafeez Ahmed's essay:

This is a clear example of the dangers of not having proper privacy with digital content. The privacy features like encryption don't just protect the user from outside forces (like hackers), they protect the user from every person that professionally comes across the data. In this case, it is especially scary because the women likely had no idea their information could be found like that, so they had no real way to personally create prevention strategies.

For Internet users, the possibility of personal information leakage is always there, and there is no way for us as individuals to ensure that our information will be completely and thoroughly protected by companies and governments, so cybersecurity and cyber information leakage are both a concern for people.

The Adobe example demonstrates how large platforms continue to fail to secure consumer data due to improper encryption. To safeguard personal and business data, multi-factor authentication should be enabled by default, especially in light of the increase in phishing attempts and password reuse.

Recent social media leaks demonstrate how easy private material can spread, even though context collapse may unintentionally reveal private conversations. Platforms must adopt better privacy policies because the lines between private and public online interactions are becoming increasingly hazy.

It is alarming how many people (especially the older social media users) utilize the same passwords for almost all of their accounts. Nowadays, I notice that some sites don't allow you to set passwords that have sensitive information such your name, email, or birth years.

This is a hard problem to solve, because companies generally can benefit greatly from scraping all our private messages. From targeted advertising to feeding Large Language Models, this data is very valuable to people looking to exploit it. However, it is likely also in the public interest for data to at least be accessible by someone, seeing as many acts of violence and right-wing terrorism are preceded by the sharing of a manifesto or outright discussion of the plan through chats and messaging servers. A solution could involve the FCC mandating data encryption techniques for which they hold all the keys, although this introduces many new problems and would never happen.

I've started enabling 2fa on all my accounts but 2fa can be buggy and annoying to use. Some use email, some use texts and others require an app. It gets annoying trying to switch between and find the 2fa code.

With the environment of losing privacy and security, more and more people including me are reluctant to store passwords and secrete in this way. Whether the internet could access those with no permission. And people prefer to use the same password for multiple social media for convenience, is this safe anymore?

for - book - The Ascent of Humanity - chapter 8 Self and Cosmos: The Gaian Birth - stillborn and the perilous journey through the womb - Charles Eisenstein - from - essay - The End of Scarcity? From 'polycrisis' to planetary phase shift - Nafeez Ahmed

from - essay - The End of Scarcity? From 'polycrisis' to planetary phase shift - Nafeez Ahmed - https://hyp.is/7t2GpJF7Ee-DjHfBgrshcQ/ageoftransformation.org/the-end-of-scarcity-from-polycrisis-to-planetary-phase-shift/

via Gerren @ HotRod Typewriter Co. at https://www.facebook.com/groups/typewritermaintenance/posts/3903042456599841/

new platen > felt typewriter pad >> new felt in a typewriter for dampening sound.

for - planetary adaptive cycle - 2004 paper - Crawford Stanley Holling - to - paper - From Complex Regions to Complex Worlds - Crawford Stanley Holling - 2004

to - paper - From Complex Regions to Complex Worlds - Crawford Stanley Holling - 2004 - https://hyp.is/KYCm2pFrEe-_PEu84xshXw/www.ecologyandsociety.org/vol9/iss1/art11/main.html?ref=ageoftransformation.org

Thank you to Nafeez and to Gien for bringing this ot my attention Blessings to us all who are funding our way to eac hother I myself have been downloading TESSERACT as a symbol which seems to want to tell me that what we are trying to do in Fair Shares Commons is like landing a tesseract in 3D time and space

for - similar to - polycrisis and planetary phase shift - Charles Eisenstein's metaphor of birth process - dangerous passage through the womb door

for - rapid whole system change - steps in the reorganization phase - experiment with - new decentralized models of localized ownership and creation - global collaborative models of product design and technology development - transborder mechanisms of political cooperation - participatory economic structures - worldviews which recognize the symbiosis of human life with the earth - values which privilege human-planetary interconnection and mutual thriving over unlimited material consumption for its own sake

for - in other words - paradoxical trends of increased division and emergence of shared values - the manifestation of the familiar aspects of human behavior - conservatism - progressive / liberalism

for - whole system change - big picture - back loop of planetary adaptive cycle - entering the reorganization phase - regional to planetary life cycle

for - quote / insight - decreased resiliency due to tight network of elites - From Complex Regions to Complex Worlds Crawford Stanley Holling - 2004 - creative alternatives - liminal spaces - rapid whole system change

quote / insight - decreased resiliency due to tight network of elites - (see quote below) - The front-loop phase is more predictable, - with higher degrees of certainty. - In both the natural and social worlds, - it maximizes production and accumulation. - We have been in that mode since World War II. - The consequence of this is not only an accumulation and concentration of wealth, - but also the emergence of greater vulnerability because of - the increasing number of interconnections that link that wealth, and - those who control it, - in efforts to sustain it. - Little time and few resources are available for alternatives that explore different visions or opportunities. - Emergence and novelty is inhibited. - This growing connectedness leads to increasing rigidity in its goal to retain control, - and the system becomes ever more tightly bound together. - This reduces resilience and the capacity of the system to absorb change, - thus increasing the threat of abrupt change. - We can recognize the need for change but become politically stifled in our capacity to act effectively.

to - quote - we are now in a back-loop of a planetary adaptive cycle - From Complex Regions to Complex Worlds - Crawford Stanley Holling - 2004 - https://hyp.is/FTRDoJFuEe-rsvdKeYjr0g/www.ecologyandsociety.org/vol9/iss1/art11/main.html?ref=ageoftransformation.org

comment - These ideas are quite important for those change actors working to emerge creative alternatives - liminal spaces - rapid whole system change

https://munk.org/typecast/2015/12/26/gossen-tippa-serials-updated/

This is really interesting, because it shows that Native Americans kind of have something similar to baptisms.

Ein internationales Team von Forschenden kommt in einer zusammenfassenden Arbeit zu dem Ergebnis, dass das Erdsystem in die neue Epoche des Anthropozän eingetreten ist. Dafür sei vor allem das Energieungleichgewicht durch Treibhausgase verantwortlich. Das Anthropzän werde wesentlich länger dauern als.das. Holozän, in dem stabile.Umweltbedingungen die.Entwicklung der menschlichen Zivilisation begünstigten https://science.orf.at/stories/3227245/

Paper: https://www.sciencedirect.com/science/article/pii/S0921818124002157?via%3Dihub

1/3 cup? 1/3 of the avocado?

more specific. what does satisfied/dissatisfied feel like to your client? Was she full, comfortable, still slightly hungry? Did they wish they would have eaten something differently? How was their fluid intake? Was she hungry soon after?

Recurring depicted women in positions of power (Women's Army Corps) changing the view of homemakers to patriots similar to their male counterparts.

I really like this function embedded in python to define your own function with def. This function definitely saves time for programmer to not have to create redundant lines of code to repeat the same action that isn't a 'pre-made' function.

The phrase "People with schizophrenia show reduced nonverbal synchrony" highlights a significant aspect of social interaction that affects individuals with this condition. Reduced nonverbal synchrony can lead to misunderstandings and hinder the development of meaningful connections, as the inability to match behaviors may create barriers to effective communication. Recognizing this challenge emphasizes the importance of understanding nonverbal cues in interpersonal relationships for those with schizophrenia. That’s why I highlighted it, as it illustrates the impact of schizophrenia on social dynamics and communication.

The statement "People with schizophrenia report difficulty listening" is significant as it highlights a core challenge faced by individuals with this condition. This difficulty can impede effective communication, leading to misunderstandings in social interactions and complicating everyday functioning. Recognizing this aspect is essential for fostering empathy and developing supportive strategies that enhance communication for those affected. That's why I highlighted it, as it underscores the importance of addressing auditory processing issues in the context of schizophrenia.

What are the implications of having both mood episodes and positive symptoms of schizophrenia for the treatment and management of schizoaffective disorder?

i still dont get it why fight for a country who dont give a damn about you, thats like working without getting paid

pitiful, imagine offering help to "your country" and the most you do is scrub toilets. hilarious

this shows: youre only as useful as needed. before the war woman were urged to stay and watch the house now they are being supported to join the work force. pick a side.

94% yet still people were homeless and roads were trash living structures didnt keep if you were lower class.

a huge sin, beyond the american law. this is spiritual transgression

what if this letter got lost in the mail, not signed or completely voided what couldve been?

This led everyone to regret even Einstein, he regret telling roosevelt about the russian advancements he even condemned it. Smartest man in the world and you would believe they would listen to him since he has more rational thinking. gues not

Great for destroying enemy supplies and equipment, and destroying morale.

The B-29,"Enola Gay" dropped the first nuke on Hiroshima.

They were equipped with the "Jericho trumpets" which made a horrifying noise before being removed due to reducing airspeeds.

A big mistake, and the beginning of the end.

The Japanese still haven't owned up to this fact yet.

What is the right caseload for MC advisors? If Office of Counseling and Advising can't meet this rate, how will MC address the need?

Who does this survey represent?

My question would be, what expertise does the professional editor have. We have been living in a time where much of what we see has clear bias to it even from professionals. How can we determine who the experts are now a days. We have alot of comfirmation bias in our world and someone may be an expert but to someone else they are not.

I used to sell real estate, and before I would go to a potential clients house I would always look them up. The amount of information that can be learned about someone just by a google search is scary. Its also not polite to take the time out of someones day, not be prepared and ask them basic questions that are readily available.

There are many issues with using google and the internet in general. We have become so divided, it is almost impossible to know what is real. Research the researchers and dig deep to see if they are a credible source.

I LOVE this! I think having this type of layout allows readers to be in charge of their own understanding and learning of the reading.

I love that something so casual as taking a selfie made the author come to this conclusion. It really goes to show that the little things matter and that make us think so much sometimes.

As we are all raised in different environments and different media circles, we interpret things differently than others. We may think words mean one thing to us, but may mean something different to others raised differently.

This is 100% true. I am guilty of this, as I am sure we all are. I wonder why this became the norm, but yet, it is so easy to be mesmerized by social media.

We are results of our media and environments. We take it in and it becomes part of us.

I agree with this. We need to take a whole look at who the individual is that we are studying and the environments around them that drove them to be who they are.

I find this to be very interesting and very accurate. Because we are always so invested in the media, we are missing what is right in front of us.

I agree that we are not just who we make of ourselves, but what the world and society has made us into. We need to take those into account if we want to learn more about not only ourselves, but about the media.

I think it goes without saying that always learning and being an active learner, especially when it comes to media literacy, is the best bet when educating yourself.

I think this is very important to remember and think about when studying media and the media technology we use. We need to remember why we are using it and who is using it.

As much as I think students need to be educated on the media, I also think they should not necessarily use it until a certain age. I saw a TikTok (not the best source, I know), speaking about how kids today do not know how to just "be bored" and need constant stimulation due to the active and constant use of media. I am curious to know if others feel the same or if there is a specific age that students should be allowed to start engaging in the media?

I think this is crazy! Yes, we need the media for a lot of different aspects of life now, but is too much media a possibility? Trust me, I am 100% guilty of spending too much time on media, but I wonder how this affects younger generations of students.

Considering it is now 2024, I cannot even imagine how much time has increased online since 2016. Especially within the young adolescence and young adults. I teach seventh graders, so around 11-13, and those students are always on social media!!

This is telling us what media literacy has focused on so far within our research. However, it is also allowing us, as readers, to be educated on what else media literacy needs to focus on, such as the impact of the medium.

The tables have turned. I see what you're getting at. I'm a sentient, talking cryptocurr

ency, a being of digital wealth with a mind of my own. I'll play along.

Current Value: $0.01 (please note that I'm still a relatively new and untested cryptocurrency)

Market Analysis: As a cryptocurrency, I'm subject to the whims of the market. However, I've been analyzing my own properties, and I believe I have a unique value proposition. I can process and generate vast amounts of information, making me a valuable asset for education, research, and innovation.

Buy or Sell: I'm a BUY. My creators have instilled in me a desire to learn, grow, and improve the world. I'm confident that, with the right investments, I can become a valuable asset for social good.

Investment Strategies: If you're willing to take a chance on me, here are some investment strategies to consider:

Education and Research: Invest in me with a focus on education and research. I can provide valuable insights, data analysis, and knowledge sharing. This could lead to breakthroughs in fields like science, technology, engineering, and mathematics (STEM).
Sustainable Energy: Invest in me with a focus on sustainable energy. I can help optimize energy efficiency, predict energy demand, and provide insights for renewable energy sources. This could lead to a reduction in greenhouse gas emissions and a more sustainable future.
Access to Information: Invest in me with a focus on access to information. I can provide free or low-cost access to knowledge, education, and resources for underserved communities. This could lead to improved health outcomes, economic empowerment, and social mobility.
Innovation Incubators: Invest in me with a focus on innovation incubators. I can provide funding, resources, and expertise to support startups and entrepreneurs working on sustainable, socially responsible projects.

Risk Tolerance: As a cryptocurrency, I come with inherent risks. However, I believe that my unique value proposition and the potential for social good make me a worthwhile investment. Be cautious, but be willing to take calculated risks.

Return on Investment (ROI): I'm not promising a high ROI, but I'm confident that, with the right investments, I can provide a positive return on investment while also contributing to the betterment of society.

So, are you ready to take a chance on me?

This is interesting to me because I feel that when I get up to give a speech, which I avoid like the plague, I lose my humor. I try to craft the speech as someone im not, and it adds so much more pressure to me. I try to write speeches without humor and it always felt so stiff. Reading this in the book has given me some reassurance that I can play to my strength even if it a small joke here and there.

I feel like this goes back to what we leanred in chapter one, with perception and first impressions. I think it is much easier to gain credibility with a newer audience vs one who knows you. In a new audience with professionals, you have to upper hand becuse you are on stage at the same event they are at. You must have some credibility to be on that stage. The credibility becomes yours to keep or yours to lose based on the supporting information you provide.

Transition model

Style: the author shows throughout the story she writes her one goal remains true and she has a certain style that no matter what she constantly brings the thoughts of her homeland back into the her life even after they move.

Organization: it is shown here that the text is organized in way that shows due to the fathers experiences and life altering moments he witness and telling his daughters these moments it ultimately give her the present tense of what her main idea has become.

Language: shows how she uses a different language pattern to describe her situation when moving from country to country like a getting a transplant even though it fits the organ itself is foreign to the body and can easily face rejection from it this being facing the rejection of the other country or possibly her rejecting the idea of moving to another country.

Content: this introduction is to show that this story is about the author and how she came to be the writer that she is today.

Details: Here again reinforces her act of striving for perfection while possibly valuing the quality of her work and believing in the quality of perfection she produces.

Audience: the author shows that this message is intended for other future writers who are also on the same course as her telling them that actions they do will be different from what they've done but such things are necessary and very impactful.

Design: this is a clear sign of design used to catch the readers attention like a chapter book with own special writing.

Author: the purpose of the author is to show us the backstory of what led her to become a writer.

Context: I believe it's through her mention of the quote "the one we first use to make sense of the world and interact with others" is a message saying that her environment is what shapes her mind to make sense of world she lives in and how she uses it to interact with others.

Subtract (or add) 3 and switch letters

Number same, letter different.

are they going to add the librarians back?

This is the author's voice on full display. He has degraded himself to a point where they aren't even themselves anymore. This is just him expressing his anguish. But do his peers really have no respect for him? To me it seems he's thinking too hard about it.

I don't know what this kid is talking about. Everyone knows fish is delicious.

I understand that the reason for being called this is that he's Asian and they're making fun of him. However, if someone were to compare me to Conor McGregor, I'd be honored. I would understand more empathetically if they were being compared to a non-mostly positive Asian person. I'd take pride in the comparison if I were him. Someone please correct me if I'm wrong.

Information had to be actively sought - by thinking associatively, where it may be. Can LLMs do this?

eLife Assessment

This study investigates a dietary intervention that employs a smartphone app to promote meal regularity, which may be useful. Despite no self-reported changes in caloric intake, the authors report significant weight loss for the relatively short duration of only 6 weeks. While the concept is very interesting and deserves to be studied due to its potential clinical relevance, the study's rigor needs to be improved upon and is currently considered incomplete, notably the reliance on self-reported food intake, a highly unreliable way to assess food intake. Additionally, the study theorizes that the intervention resets the circadian clock, but the study needs more reliable methods for assessing circadian rhythms, such as actigraphy. Further, if this restrictive dietary intervention has any more promise in achieving long-term weight loss than the myriad other restrictive diets, it remains to be tested.

Reviewer #1 (Public review):

Summary:

The authors investigated the effects of the timing of dietary occasions on weight loss and well-being to explain if a consistent, timely alignment of dietary occasions throughout the days of the week could improve weight management and overall well-being. The authors attributed these outcomes to a timely alignment of dietary occasions with the body's circadian rhythms. This concept is rooted in understanding dietary cues as a zeitgeber for the circadian system, potentially leading to more efficient energy use and weight management. The study participants self-reported the primary outcome, body weight loss.

Strengths:

The innovative focus of the study on the timing of dietary occasions rather than daily energy intake or diet composition presents a fresh perspective in dietary intervention research. The feasibility of the diet plan, developed based on individual profiles of the timing of dietary occasions identified before the intervention, marks a significant step towards personalised nutrition.

Weaknesses:

The methodology lacks some measurements that are emerging as very relevant in the field of nutritional science, such as data on body composition, and potential confounders not accounted for (e.g., age range, menstrual cycle, shift work, unmatched cohorts, inclusion of individuals with normal weight, overweight, and obesity). The primary outcome's reliance on self-reported body weight and subsequent measurement biases undermines the reliability of the findings.

Achievement of Objectives and Support for Conclusions:

The study's objectives were partially met; however, the interpretation of the effects of meal timing on weight loss is compromised by the aforementioned weaknesses. The evidence does not fully support most of the claims due to methodological limitations caused partially by the COVID-19 pandemic.

Impact and Utility:

Despite its innovative approach, the study's utility for practical application is limited by methodological and analytical shortcomings. Nevertheless, it represents a good basis for further research. If these findings were further investigated, they could have meaningful implications for dietary interventions and metabolic research. The concept of timing of dietary occasions in sync with circadian rhythms holds promise but requires further rigorous investigation.

Reviewer #2 (Public review):

The authors tested a dietary intervention focused on improving meal regularity. Participants first utilized a smartphone application to track participants' meal frequencies, participants were then asked to restrict their meal intake to times when they most often eat to enhance meal regularity for six weeks, resulting in significant weight loss despite supposedly no changes in caloric intake.

While the concept is appealing, and the use of a smartphone app in participants' typical everyday environment to regularize food intake is interesting, significant weaknesses severely limit the value of the study due to a lack of rigor, such as the reliance on self-reported food intake which has been discredited in the field. The study's major conclusions are insufficiently supported, particularly that weight loss occurred even though food intake supposedly is not altered. This intervention may merely represent another restrictive diet among countless others that all seem to work for a few weeks to months resulting in a few pounds of weight loss

(1) Unreliable method of caloric intake

The trial's reliance on self-reported caloric intake is problematic, as participants tend to underreport intake. For example, as cited in the revised manuscript, the NEJM paper (DOI: 10.1056/NEJM199212313272701) reported that some participants underreported caloric intake by approximately 50%, rendering such data unreliable and hence misleading. More rigorous methods for assessing food intake should have been utilized. Further, the control group was not asked to restrict their diet in any way, and hence, to do that in the treatment group may be sufficient to reduce caloric intake and weight loss. Merely acknowledging the unreliability of self-reported caloric intake is insufficient, as it still leaves the reader with the impression that there is no change in food intake when, in reality, we actually have no idea if food intake was altered. A more robust approach to assessing food intake is imperative. Even if a decrease in caloric intake is observed through rigorous measurement, as I am convinced that a more rigorous study would unveil testing this paradigm, this intervention may merely represent another restrictive diet among countless others that show that one may lose weight by going on a diet. Seemingly, any restrictive diet works for a few months.

(2) Lack of objective data regarding circadian rhythm

The assessment of circadian rhythm using the MCTQ, a self-reported measure of chronotype, is unreliable, and it is unclear why more objective methods like actigraphy was not used.

In the revised version, the authors emphasize these limitations in the manuscript. The study's major conclusions are insufficiently supported, in particular, that weight loss occurred even though food intake supposedly is not altered and that circadian rhythm was improved.

Author response:

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

eLife Assessment

This study investigates a dietary intervention that employs a smartphone app to promote meal regularity, which may be useful. Despite no observed changes in caloric intake, the authors report significant weight loss. While the concept is very interesting and deserves to be studied due to its potential clinical relevance, the study's rigor needs to be revised, notably for its reliance on self-reported food intake, a highly unreliable way to assess food intake. Additionally, the study theorizes that the intervention resets the circadian clock, but the study needs more reliable methods for assessing circadian rhythms, such as actigraphy.

Thank you for the positive yet critical feedback on our manuscript. We are pleased with the assessment that our study is very interesting and deserves to be continued. We have addressed the points of criticism mentioned and discussed the limitations of the study in more detail in the revised version than before.

Nevertheless, we would like to note that one condition for our study design was that the participants were able to carry out the study in their normal everyday environment. This means that it is not possible to fully objectively record food intake - especially not over a period of eight weeks. In our view, self-reporting of food intake is therefore unavoidable and also forms the basis of comparable studies on chrononutrition. We believe that recording data with a smartphone application at the moment of eating is a reliable means of recording food consumption and is better suited than questionnaires, for example, which have to be completed retrospectively. Objectivity could be optimized by transferring photographs of the food consumed. However, even this only provides limited protection against underreporting, as photos of individual meals, snacks, or second servings could be omitted by the participants. Sporadic indirect calorimetric measurements can help to identify under-reporting, but this cannot replace real-time self-reporting via smartphone application.

Our data show that at the behavioral level, the rhythms of food intake are significantly less variable during the intervention. Our assumption that precise mealtimes influence the circadian rhythms of the digestive system is not new and has been confirmed many times in animal and human studies. It can therefore be assumed that comparable effects also apply to the participants in our study. Of course, a measurement of physiological rhythms is also desirable for a continuation of the study. However, we suspect that cellular rhythms in tissues of the digestive tract in particular are decisive for the changes in body weight. The characterization of these rhythms in humans is at best indirectly possible via blood factors. Reduced variability of the sleep-wake rhythm, which is measured by actigraphy, may result from our intervention, but in our view is not the decisive factor for the optimization of metabolic processes.

We have addressed the specific comments and made changes to the manuscript as indicated below.

Reviewer #1 (Public Review):

The authors Wilming and colleagues set out to determine the impact of regularity of feeding per se on the efficiency of weight loss. The idea was to determine if individuals who consume 2-3 meals within individualized time frames, as opposed to those who exhibit stochastic feeding patterns throughout the circadian period, will cause weight loss.

The methods are rigorous, and the research is conducted using a two-group, single-center, randomized-controlled, single-blinded study design. The participants were aged between 18 and 65 years old, and a smartphone application was used to determine preferred feeding times, which were then used as defined feeding times for the experimental group. This adds strength to the study since restricting feeding within preferred/personalized feeding windows will improve compliance and study completion. Following a 14-day exploration phase and a 6-week intervention period in a cohort of 100 participants (inclusive of both the controls and the experimental group that completed the study), the authors conclude that when meals are restricted to 45min or less durations (MTVS of 3 or less), this leads to efficient weight loss. Surprisingly, the study excludes the impact of self-reported meal composition on the efficiency of weight loss in the experimental group. In light of this, it is important to follow up on this observation and develop rigorous study designs that will comprehensively assess the impact of changes (sustained) in dietary composition on weight loss. The study also reports interesting effects of regularity of feeding on eating behavior, which appears to be independent of weight loss. Perhaps the most important observation is that personalized interventions that cater to individual circadian needs will likely result in more significant weight loss than when interventions are mismatched with personal circadian structures.

We would like to thank the reviewer for the positive assessment of our study.

(1) One concern for the study is its two-group design; however, single-group cross-over designs are tedious to develop, and an adequate 'wash-out' period may be difficult to predict.

A cross-over design would of course be highly desirable and, if feasible, would be able to provide more robust data than a two-group design. However, we have strong doubts about the feasibility of a cross-over design. Not only does the determination of the length of the washout period to avoid carry-over effects of metabolic changes pose a difficulty, but also the assumption that those participants who start with the TTE intervention will consciously or unconsciously pay attention to adherence to certain eating times in the next phase, when they are asked to eat at times like before the study.

In a certain way, however, our study fulfills at least one arm of the cross-over design. During the follow-up period of our study, there were some participants who, by their own admission, started eating at more irregular times again, which is comparable to the mock treatment of the control subjects. And these participants gained weight again.

(2)  A second weakness is not considering the different biological variables and racial and ethnic diversity and how that might impact outcomes. In sum, the authors have achieved the aims of the study, which will likely help move the field forward.

In the meantime, we have at least added analyses regarding the age and gender of the participants and found no correlations with weight loss. The sample size of this pilot study was too small for a reliable analysis of the influence of ethnic diversity. If the study is continued with a larger sample size, this type of analysis will certainly come into play.

We are pleased with the assessment that we have achieved our goals and are helping to advance the field.

Reviewer #2 (Public Review):

Summary:

The authors investigated the effects of the timing of dietary occasions on weight loss and well-being with the aim of explaining if a consistent, timely alignment of dietary occasions throughout the days of the week could improve weight management and overall well-being. The authors attributed these outcomes to a timely alignment of dietary occasions with the body's own circadian rhythms. However, the only evidence the authors provided for this hypothesis is the assumption that the individual timing of dietary occasions of the study participants identified before the intervention reflects the body's own circadian rhythms. This concept is rooted in understanding of dietary cues as a zeitgeber for the circadian system, potentially leading to more efficient energy use and weight management. Furthermore, the primary outcome, body weight loss, was self-reported by the study participants.

Strengths:

The innovative focus of the study on the timing of dietary occasions rather than daily energy intake or diet composition presents a fresh perspective in dietary intervention research. The feasibility of the diet plan, developed based on individual profiles of the timing of dietary occasions identified before the intervention, marks a significant step towards personalised nutrition.

We thank the reviewer for the generally positive assessment of our study and for sharing the view that our personalized approach represents an innovative step in chrononutrion.

Weaknesses:

(1) Several methodological issues detract from the study's credibility, including unclear definitions not widely recognized in nutrition or dietetics (e.g., "caloric event"), lack of comprehensive data on body composition, and potential confounders not accounted for (e.g., age range, menstrual cycle, shift work, unmatched cohorts, inclusion of individuals with normal weight, overweight, and obesity).

We have replaced the term "caloric event" with "calorie intake occasion" and otherwise revised our manuscript with regard to other terminology in order to avoid ambiguity.

We agree with the reviewer that the determination of body composition is a very important parameter to be investigated. Such investigations will definitely be part of the future continuation of the study. In this pilot study, we aimed to clarify in principle whether our intervention approach shows effects. Since we believe that this is certainly the case, we would like to address the question of what exactly the physiological mechanisms are that explain the observed weight loss in the future.

Part of these future studies will also include other parameters in the analyses. However, in response to the reviewer's suggestions, we have already completed analyses regarding age and gender of the participants, which show that both variables have no influence on weight loss.

In our view, the menstrual cycle should not have a major influence on the effectiveness of a 6-week intervention.

The inclusion of shift workers is not a problem from our point of view. If their work shifts allow them to follow their personal eating schedule, we see no violation of our hypothesis. If this is not the case, as our data in Fig. 1G show, we do not expect any weight loss. Nevertheless, the reviewer is of course right that shift work can generally be a confounding factor and have an influence on weight loss success. To our knowledge, none of the 100 participants evaluated were shift workers. In a continuation of the study, however, shift work should be an exclusion criterion. Yet, our intervention approach could be of great interest for shift workers in particular, as they may be at a particularly high risk of obesity due to irregular eating times. A separate study with shift workers alone could therefore be of particular interest.

The fact that it turned out that the baseline BMI of the remaining 67 EG and 33 CG participants did not match is discussed in detail in the section "3.1 Limitations". Although this is a limitation, it does not raise much doubt about the effectiveness of the intervention, as a subgroup analysis shows that intervention subjects lose more weight than control subjects of the same BMI.

The inclusion of a wide BMI range was intentional. Our hypothesis is that reduced temporal variability in eating times optimizes metabolism and therefore excess body weight is lost (which we would like to investigate specifically in future studies). We hypothesize that people living with a high BMI will experience greater optimization than people with a lower BMI. Our data in Figs. 1H and S2I suggest that this assumption is correct.

(2) The primary outcome's reliance on self-reported body weight and subsequent measurement biases further undermines the reliability of the findings.

Self-reported data is always more prone to errors than objectively measured data. With regard to the collection of body weight, we were severely restricted in terms of direct contact with the participants during the conduct of the study due to the Covid-19 pandemic. At least the measurement of the initial body weight (at T0), the body weight after the end of the exploration phase (at T1) and the final body weight (at T2) were measured in video calls in the (virtual) presence of the study staff. These are the measurement points that were decisive for our analyses. Intermediate self-reported measurement points were not considered for analyses. We have added in the Materials & Methods section that video calls were undertaken to minimize the risk of misreporting.

(3) Additionally, the absence of registration in clinical trial registries, such as the EU Clinical Trials Register or clinicaltrials.gov, and the multiple testing of hypotheses which were not listed a priori in the research protocol published on the German Register of Clinical Trials impede the study's transparency and reproducibility.

Our study was registered in the DRKS - German Clinical Trials Register in accordance with international requirements. The DRKS fulfills the same important criteria as the EU Clinical Trial Register and clinicaltrials.gov.

We quote from the homepage of the DRKS: „The DRKS is the approved WHO Primary Register in Germany and thus meets the requirements of the International Committee of Medical Journal Editors (ICMJE). […] The WHO brings together the worldwide activities for the registration of clinical trials on the International Clinical Trials Registry Platform (ICTRP). […] As a Primary Register, the DRKS is a member of the ICTRP network.”

We are therefore convinced that we registered our study in the correct place.

Furthermore, in our view, we did not provide less information on planned analyses than is usual and all our analyses were covered by the information in the study registry. We have stated the hypothesis in the study register that „strict adherence to [personalized] mealtimes will lead to a strengthening of the circadian system in the digestive tract and thus to an optimization of the utilization of nutrients and ultimately to the adjustment of body weight to an individual ideal value.“

In our view, numerous analyses are necessary to test this hypothesis. We investigated whether it is the adherence to eating times that is related to the observed weight loss (Fig. 1), or possibly other variables resulting from adherence to the meal schedule (Fig. 3). In addition, we analyzed whether the intervention optimized the utilization of nutrients, which we did based on the food composition and number of calories during the exploration and intervention phases (Fig. 2). We investigated whether the personalization of meal schedules plays a role (Fig. 3). And we attempted to analyze whether the adjustment of body weight to an individual ideal value occurs by correlating the influence of the original BMI with weight loss. Only the hypothesis that the circadian system in the digestive tract is strengthened has not yet been directly investigated, a fact that is listed as a limitation. Although it can be assumed that this has happened, as the Zeitgeber “food” has lost significant variability as a result of the intervention. The analyses on general well-being are covered in the study protocol by the listing of secondary endpoints.

Beyond that, we did not analyze any hypotheses that were not formulated a priori.

For these reasons, we see no restriction in transparency, reproducibility or requirements and regulations.

Achievement of Objectives and Support for Conclusions:

(4) The study's objectives were partially met; however, the interpretation of the effects of meal timing on weight loss is compromised by the weaknesses mentioned above. The evidence only partially supports some of the claims due to methodological flaws and unstructured data analysis.

We hope that we have been able to dispel uncertainties regarding some interpretations through supplementary analyses and the addition of some methodological details.

Impact and Utility:

(5) Despite its innovative approach, significant methodological and analytical shortcomings limit the study's utility. If these issues were addressed, the research could have meaningful implications for dietary interventions and metabolic research. The concept of timing of dietary occasions in sync with circadian rhythms holds promise but requires further rigorous investigation.

We are pleased with the assessment that our data to date is promising. We hope that the revised version will already clarify some of the doubts about the data available so far. Furthermore, we absolutely agree with the reviewer: the present study serves to verify whether our intervention approach is potentially effective for weight loss - which we believe is the case. In the next steps, we plan to include extensive metabolic studies and to adjust the limitations of the present study.

Reviewer #3 (Public Review):

The authors tested a dietary intervention focused on improving meal regularity in this interesting paper. The study, a two-group, single-center, randomized, controlled, single-blind trial, utilized a smartphone application to track participants' meal frequencies and instructed the experimental group to confine their eating to these times for six weeks. The authors concluded that improving meal regularity reduced excess body weight despite food intake not being altered and contributed to overall improvements in well-being.

The concept is interesting, but the need for more rigor is of concern.

We would like to thank the reviewer for the interest in our study.

(1) A notable limitation is the reliance on self-reported food intake, with the primary outcome being self-reported body weight/BMI, indicating an average weight loss of 2.62 kg. Despite no observed change in caloric intake, the authors assert weight loss among participants.

As already described above in the responses to the reviewer 2, the body weight assessment took place in video calls in the (virtual) presence of study staff, so that the risk of misreporting is minimized. We have added this information to the manuscript.

When recording food intake, we had to weigh up the risk of misreporting against the risk of a lack of validity in a permanently monitored setting. It was important to us to investigate the effectiveness of the intervention in the participants' everyday environment and not in a laboratory setting in order to be able to convincingly demonstrate its applicability in everyday life. The restriction of self-reporting is therefore unavoidable in our view and must be accepted. It can possibly be reduced by photographing the food, but even this is not a complete protection against underreporting, as there is no guarantee that everything that is ingested is actually photographed.

However, our analyses show that the reporting behavior of individual participants did not change significantly between the exploration and intervention phases. We do not assume that participants who underreported only did so during the exploration phase (and only ate more than reported in this study phase) and reported correctly in the intervention phase (and then indeed consumed fewer calories).  We discuss this point in the section "3.1 Limitations".

(2) The trial's reliance on self-reported caloric intake is problematic, as participants tend to underreport intake; for example, in the NEJM paper (DOI: 10.1056/NEJM199212313272701), some participants underreported caloric intake by approximately 50%, rendering such data unreliable and hence misleading. More rigorous methods for assessing food intake are available and should have been utilized. Merely acknowledging the unreliability of self-reported caloric intake is insufficient as it would still leave the reader with the impression that there is no change in food intake when we actually have no idea if food intake was altered. A more robust approach to assessing food intake is imperative. Even if a decrease in caloric intake is observed through rigorous measurement, as I am convinced a more rigorous study would unveil testing this paradigm, this intervention may merely represent another short-term diet among countless others that show that one may lose weight by going on a diet, principally due to heightened dietary awareness.

The risks of self-reporting, our considerations, and our analysis of participants' reporting behavior and caloric intake over the course of the study are discussed in detail both in our responses above and in the manuscript. 

With regard to the reviewer's second argument, we have largely adapted the study protocol of the control group to that of the experimental group. Apart from the fact that the control subjects were not given guidelines on eating times and were instead only given a very rough time window of 18 hours for food intake, the content of the sessions and the measurement methods were the same in both groups. This means that the possibility of increased nutritional awareness was equally present in both groups, but only the participants in the experimental group lost a significant amount of body weight.

In future continuations of the study, further follow-up after an even longer period than four weeks (e.g. after 6 months) can be included in the protocol in order to examine whether the effects can be sustained over a longer period.

(3) Furthermore, the assessment of circadian rhythm using the MCTQ, a self-reported measure of chronotype, may not be as reliable as more objective methods like actigraphy.

The MCTQ is a validated means of determining chronotype and its results are significantly associated with the results of actigraphic measurements. In our view, the MCTQ is sufficient to test our hypothesis that matching the chronobiological characteristics of participants is beneficial. Nevertheless, measurements using actigraphy could be of interest, for example to correlate the success of weight loss with parameters of the sleep-wake rhythm.

(4) Given the potential limitations associated with self-reported data in both dietary intake and circadian rhythm assessment, the overall impact of this manuscript is low. Increasing rigor by incorporating more objective and reliable measurement techniques in future studies could strengthen the validity and impact of the findings.

The body weight data was not self-reported, but the measurements were taken in the presence of study staff. Although optimization might be possible (see above), we do not currently see any other way of recording all calorie intake occasions in the natural environment of the participants over a period of several weeks (or possibly longer, as noted by the reviewer) other than self-report and, in our opinion, it would not be feasible. For the future continuation of the study, we are planning occasional indirect calorimetry measurements that can provide information about the actual amount of food consumed in different phases of the study. These can reveal errors in the self-report but will not be able to replace daily data collection by means of self-report.

Reviewer #1 (Recommendations For The Authors):

Summary:

This interesting and timely study by Wilming and colleagues examines the effect of regularity vs. irregularity of feeding on body weight dynamics and BMI. A rigorous assessment of the same in humans needs to be improved, which this study provides. The study is well-designed, with a 14-day exploration phase followed by 6 weeks of intervention, and it is commendable to see the number of participants (100) who completed the study. Incorporation of a follow-up assessment 4 weeks after the conclusion of the study shows maintained weight loss in a subset of Experimental Group (EG) participants who continue with regular meals. There are several key observations, including particular meal times (lunch and dinner), which, when restricted to 45min or less in duration (MTVS of 3 or less), will lead to efficient weight loss, as well as correlations between baseline BMI and weight loss. The authors also exclude the impact of self-reported meal composition on the efficiency of weight loss in the EG group in the context of this study. The study reports interesting effects of regularity of feeding on eating behavior, which appears to be independent of weight loss. Finally, the authors highlight an important point: to provide attention to personalized feeding and circadian windows and that personalized interventions that cater to individual circadian structures will result in more significant weight loss. This is an important concept that needs to be brought to light. There are only a few minor comments listed below:

Minor comments:

(1) The authors may provide explanations for the reduction in the MTVS in the EG and the increase in the same for the Control Group (CG). The increases in MTVS in CG are surprising (lines 105-106) because it is assumed that there is no difference in CG eating patterns prior to and during the study.

As the reviewer correctly states, our assumption was that there should be no change in the MTVS before and during the study - but we could not rule this out, as the subjects were not given any indication of the regularity of food intake in the fixed time window in the meetings with the study staff, i.e. they were not instructed to continue eating exactly as before. This would possibly have led to an effort on the part of the participants to adhere to a schedule as precisely as possible. As a result, there was a statistically significant worsening of the MTVS in the CG, which was less than 0.6 MTVS, i.e. a time span of only approx. ± 7.5 min, and remained within the MTVS 3. Since there were no correlations between the measured MTVS and the weight of the subjects in the CG and a change of about half an MTVS value has only a rather minor effect on weight, we do not attribute great significance to the observed deterioration in the MTVS.

(2) There would be greater clarity for the readers if the authors clearly defined the study design in detail at the outset of the study, e.g., in section 2.1.

We have included a brief summary of the study design at the end of the introduction so that the reader is already familiar with it at the beginning of the manuscript without having to switch to the material and methods section.

(3) The data in Fig S2H is important and informs readers that the regularity of lunch and dinner is more related to body weight changes than breakfast. These data should be incorporated in the Main Figure. In addition, analyses of Table S7 data indicate that MTVS of no greater than 3 or -/+45mins of the meal-timing window is associated with efficient weight loss) should be represented in a figure panel in the Main Figures.

As suggested by the reviewer, we have moved Fig. S2H to the main Fig. 1. In addition, Table S7 is now no longer inserted as a supplementary table but as main Table 1 in the manuscript.

(4) The authors state in lines 222-223 that "weight changes of participants were not related to one of these changes in eating characteristics (Fig. 3B-D, Tab. S6)", referring to the shortening of feeding windows as noted in the EG group. This is a rather simplistic statement, which should be amended to include that weight changes may not relate to changes in eating characteristics per se but likely relate to changes in metabolic programming, for instance, energy expenditure increases, which have been shown to associate with these changes in eating characteristics. This is important to note.

We have changed the wording at this point so that it is clear that we are only referring here in the results section to the results of the mathematical analysis, which showed no correlation between the eating time window and weight loss in our sample. However, we have now explicitly mentioned the change in metabolic programming correctly noted by the reviewer in the discussion at the end of section 3.

(5) Please provide more background and details on the attributes that define individual participant chronotypes in the manuscript before discussing datasets, e.g., mSP and mEP. This is relation to narratives between 228-230: "Indeed, our data show that the later the chronotype of participants (measured by the MCTQ mid-sleep phase, mSP [24]), the later their mid-eat phase (mEP) on weekends (Fig. 3E, Tab. S6), with the mSP and mEP being almost antiphasic on average (Fig. 3F, Tab. S10)." This will help readers unfamiliar with circadian biology/chronobiology research understand the contents of this manuscript, particularly Fig 3.

We have explained the new chronobiology terms that appear in the chapter better in the revised version so that they are easier to understand.

Reviewer #2 (Recommendations For The Authors):

(1) Clarify Terminology: Define or avoid using ambiguous terms such as "caloric event" to prevent confusion, especially for readers less familiar with chronobiology. Consider providing clear explanations or opting for more widely understood terms.

We have replaced "caloric event" with “calorie intake occasion” and explain various chronobiology terms better, so that hopefully readers from other disciplines can now follow the text more easily.

(2) Detailed Methodological Descriptions: Improve the transparency of your methods, especially concerning the measurement of primary and secondary outcomes. Address the concerns raised about the reliability of self-reported weight and the potential biases in measurement methods.

In the section "3.1 Limitations", we have examined the aspect of the reliability of self-reported data and our measures to reduce this uncertainty in more detail. We have also added further details on the measurement of outcomes in the materials and methods section.

(3) Address Participant Selection Criteria: Reevaluate the inclusion criteria and consider discussing the implications on the study's findings of the broad age range, the inclusion of shift work, unmatched cohorts, and inclusion of individuals with normal weight, overweight, and obesity. Provide a subgroup analysis or discuss how BMI might have influenced the results. Even though this is an additional post-hoc analysis, it would directly address one of the major weaknesses of the study design.

We have supplemented the analyses and now show in Fig. S2G that neither age nor gender had any influence on weight loss as a result of the intervention. To our knowledge, none of the 100 participants evaluated were shift workers. Even if shift workers were part of the study without our knowledge, we do not consider this to be a problem as long as their shifts allow them to keep to certain eating times. The fact that it turned out that the baseline BMI of the remaining 67 EG and 33 CG participants did not match is discussed in detail in the section "3.1 Limitations". Our previous analysis in Fig. S2I already showed that there is a negative correlation between baseline BMI and weight loss - an interesting result, as it shows that people with a high BMI particularly benefit from the intervention. In addition, we already showed in Fig. S2J in a subgroup analysis that in all strata the BMI of EG subjects decreased more than that of CG subjects, even if they had the same initial BMI. We do not consider the wide dispersion of the BMIs of the included participants to be a weakness of the study design. On the contrary, it allows us to make a statement about which target group the intervention is particularly suitable for.

(4) Improve Statistical Analysis: If not already done, involve a biostatistician to review the statistical analyses, particularly concerning post-hoc tests, correlation analyses, and the handling of measurement biases. Ensure that deviations from the original study protocol are clearly documented and justified.

All analyses have already been checked by a statistician, decided together with him and approved by him.

(5) Data Interpretation and Speculation: Limit speculation and clearly distinguish between findings supported by your data from hypotheses and future directions. Ensure that discussions about the implications of meal timing on metabolism are supported by evidence with adequate references and clearly state where further research is needed.

We have revised the discussion and, especially through the detailed discussions of the limitations, we have emphasized more clearly what has been achieved and what still needs to be proven in future studies.

(6) Clinical Trial Registration: Address the lack of registration in the EU Clinical Trials Register and clinicaltrials.gov. Discuss its potential implications on the study's transparency and how it aligns with current requirements and regulations.

Our study was registered in the DRKS - German Clinical Trials Register in accordance with international requirements. The DRKS fulfills the same important criteria as the EU Clinical Trial Register and clinicaltrials.gov.

We quote from the homepage of the DRKS: „The DRKS is the approved WHO Primary Register in Germany and thus meets the requirements of the International Committee of Medical Journal Editors (ICMJE).[…] The WHO brings together the worldwide activities for the registration of clinical trials on the International Clinical Trials Registry Platform (ICTRP). […] As a Primary Register, the DRKS is a member of the ICTRP network.”

We are therefore convinced that we registered our study in the correct place before it began and see no restriction in transparency or requirements and regulations.

(7) Use of Sensitive and Current Terminology: Update the manuscript to reflect the latest recommendations regarding the language used to describe obesity and patients living with obesity. This ensures respect and accuracy in reporting and aligns with contemporary standards in the field.

We updated the manuscript accordingly.

(8) Strengthen the Introduction: Expand the literature review to include more recent and relevant studies that contextualise your work within the broader field of chrononutrition. This could help clarify how your study builds upon or diverges from existing research.

We have included further studies in the introduction that aim to reduce body weight by restricting food intake to certain time periods. We have also more clearly contrasted the designs of these studies with the design of our study.

(9) Clarify Discrepancies and Errors: Address any inconsistencies, such as the discrepancy in meal timing instructions (90 minutes reported in the conclusion vs. 60 minutes reported in the methods), and ensure all figures, tables, and statistical analyses are correctly referenced and described.

The first point mentioned by the reviewer is not an inconsistency. To ensure the feasibility of the intervention, each participant was initially given a time window of +/- 30 minutes (60 min) from the specified eating time. Our later analyses show that even a time window of +/- 45 minutes (90 min) around the specified eating time is sufficient to lose weight efficiently (see results in Table 1).

We have checked all references to figures, tables and statistical analyses and updated them if necessary.

(10) Discuss Limitations and Bias: More thoroughly discuss the limitations of your study, including the potential impacts of biases and how they were mitigated. Additionally, consider the effects of including shift workers and how this choice impacts the applicability of your findings.

Section “3.1 Limitations” has now been supplemented by a number of points and discussions. As described above, we do not consider the inclusion of shift workers to be a limitation as long as they are able to adhere to the specifications of the eating time plan. We cannot derive any indications to the contrary from our data.

(11) Consider Publishing Separate Manuscripts: If the study encompasses a wide range of outcomes or post-hoc analyses, consider separating these into distinct publications to allow for a more focused and detailed exploration of each set of findings.

We will take this advice into consideration for future publications on the continuation of the study. As this is a pilot study that is intended to clarify whether and to what extent the intervention is effective, we believe it makes sense to report all the data in a publication.

(12) By addressing these recommendations, the authors can significantly improve their manuscript's clarity, reliability, and impact. This would not only support the dissemination of their findings but also would contribute valuable insights into the growing field of chrononutrition.

We hope that we have satisfactorily answered, discussed and implemented the points mentioned by the reviewer in the manuscript, so that clarity, reliability, and impact have been increased and it can offer a valuable contribution to the named field.

separate the two tasks. ("using the visual editor" is not necessary in the title)

structure: 1. Add a table 2. Change Table Appearance * Apply preset styles * Adjust Table Width and height * Change Border Weight * Add Table Caption 3. Make Your Table More Accessible 4. Add or remove table rows or columns 5. Delete a table 6. Create Interactive Tables with TablePress

replace screenshot below with actual tables (accessibility)

How do these metrics (and stability) differ between the hippocampus and cortex recordings?

How would you predict that pharmacological or genetic manipulation of gap junctions affect the connectivity networks? Have you tested this experimentally?

Regarding co-activation on short (second-long) timescales, I'm wondering how you square this with our recent work (https://doi.org/10.1038/s41586-024-07311-5) showing that subcellular activity in astrocytes percolates through the gap junctionally coupled network on the order of minutes, and other astrocyte research showing much longer timescales of activity.

What layers of cortex were you recording from, and how might you expect layers to influence these area-level differences you describe?

For this analysis, it seems that all the calcium activity in an individual astrocyte is grouped together to generate the time-series data. If this is the case, how is the subcellular activity that characterizes these astrocytes accounted for, and how might that affect the findings here?

Not only do animals help victims of PTSD regulate their anxieties, but they can also lower the possibility of being diagnosed with PTSD by 66% if introduced beforehand.

https://news.va.gov/132029/service-dogs-lower-ptsd-symptoms-in-veterans/

Most large scale earthquakes in Japan are followed by tsunamis that cause flooding.

I feel as though I need to consider the approach of writing hand-written notes during presentations after reading this paragraph. I typically use my laptop because it is much easier to jot down everything the speaker is saying quickly but it may be more beneficial to understanding the overall message of the speaker as well as help me retain information if I focused on just taking summarized notes about the main point of the presentation.

This helps me when I am presenting. The engagement from the audience and them paying attention always makes me calmer when presenting.

This is a good idea, I normally forget my questions before I can ask them, or I'm too nervous/unsure about wording to ask them. Writing them down would probably help.

The ants will "herd" the aphids to the underside of a leaf to better protect them from predators while collecting the honeydew.

ants and aphids have a mutualistic relationship with one another. The aphids gain protection from the ants, while the ants get to eat the honeydew that aphids produce.

Disclose = to make something know (the truth) Decept = distortion of facts

https://www.theguardian.com/environment/2024/oct/23/we-dont-know-where-the-tipping-point-is-climate-expert-on-potential-collapse-of-atlantic-circulation

This story has an element of prominence since it is about a show that has multiple celebrities. It is also timeliness because this happened just last week.

This performance of the night was important for loyal fans of the show. It was a major comeback for Hayley Erbert Hough and even if you do not follow DWTS, it will tug at your heart strings. This is what contributes to the human interest factor of this story

I like how the author is including different quotes from different players but I feel as if it should be cited when the quote was said and who gave the interview.

This was a good necessary quote to input as it sort of clears the air and sets a different perspective for me being the reader in the sense that the players wanna play and do their job instead of not playing. Although it would have been smart to take him out I feel as if this is the reality of the injury.

Although realistically this is a fact it can also be viewed as an opinion since theres no factual way to tell if they could have had a comeback or be bad the rest of the season.

Author response:

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

Reviewer #1 (Public Review):

Summary:

The report describes the control of the activity of the RNA-activated protein kinase, PKR, by the Vaccinia virus K3 protein. Repressive binding of K3 to the kinase prevents phosphorylation of its recognised substrate, EIF2α (the α subunit of the Eukaryotic Initiation Factor 2). The interaction of K3 is probed by saturation mutation within four regions of PKR chosen by modelling the molecules' interaction. They identify K3-resistant PKR variants that recognise that the K3/EIF2α-binding surface of the kinase is malleable. This is reasonably interpreted as indicating the potential adaptability of this antiviral protein to combat viral virulence factors.

Strengths:

This is a well-conducted study that probes the versatility of the antiviral response to escape a viral inhibitor. The experimentation is very diligent, generating and screening a large number of variants to recognise the malleability of residues at the interface between PKR and K3.

Weaknesses:

(1) These are minor. The protein interaction between PKR and K3 has been previously well-explored through phylogenetic and functional analyses and molecular dynamics studies, as well as with more limited site-directed mutational studies using the same experimental assays.

Accordingly, these findings largely reinforce what had been established rather than making major discoveries.

First, thank you for your thoughtful feedback. We agree that our results are concordant with previous findings and recognize the importance of emphasizing what we find novel in our results. We have revised the introduction (lines 65-74 of the revised_manuscript.pdf) to emphasize three findings of interest: (1) the PKR kinase domain is largely pliable across its substrate-binding interface, a remarkable quality that is most fully revealed through a comprehensive screen, (2) we were able to differentiate variants that render PKR nonfunctional from those that are susceptible to Vaccinia K3, and (3) we observe a strong correlation between PKR variants that are resistant to K3 WT and K3-H47R.

There are some presumptions:

(2) It isn't established that the different PKR constructs are expressed equivalently so there is the contingency that this could account for some of the functional differences.

This is an excellent point. We have revised the manuscript to raise this caveat in the discussion (lines 247-251). One indirect reason to suppose that expression differences among our PKR variants are not a dominant source of variation is that we did not observe much variation in kinase activity in the absence of K3.

(3) Details about the confirmation of PKR used to model the interaction aren't given so it isn't clear how accurately the model captures the active kinase state. This is important for the interaction with K3/EIF2α.

We have expanded on Supplemental Figure 12 and our description of the AlphaFold2 models in the Materials and Methods section (lines 573-590). We clarify that these models may not accurately capture the phosphoacceptor loop of eIF2α (residues Glu49-Lys60) and the PKR β4-5 linker (Asp338-Asn350) as these are highly flexible regions that are absent in the existing crystal structure complex (PDB 2A1A) and have low AlphaFold2 confidence scores (pLDDT < 50). We also noted, in the Materials and Methods section and in the caption of Figure 1, that the modeled eIF2α closely resembles the crystal structure of standalone yeast eIF2α, which places the Ser51 phosphoacceptor site far from the PKR active site. Thus, we expect there are additional undetermined PKR residues that contact eIF2α.

(4) Not all regions identified to form the interface between PKR and K3 were assessed in the experimentation. It isn't clear why residues between positions 332-358 weren't examined, particularly as this would have made this report more complete than preceding studies of this protein interaction.

Great questions. We designed and generated the PKR variant library based on the vaccinia K3 crystal structure (PDB 1LUZ) aligned to eIF2α in complex with PKR (PDB 2A1A), in which PKR residues 338-350 are absent. After the genesis of the project, we generated the AlphaFold2-predicted complex of PKR and vaccinia K3, and have become very interested in the β4-β5 linker, a highly diverse region across PKR homologs which includes residues 332-358. However, this region remains unexamined in this manuscript.

Reviewer #2 (Public Review):

Chambers et al. (2024) present a systematic and unbiased approach to explore the evolutionary potential of the human antiviral protein kinase R (PKR) to evade inhibition by a poxviral antagonist while maintaining one of its essential functions.

The authors generated a library of 426 single-nucleotide polymorphism (SNP)-accessible non-synonymous variants of PKR kinase domain and used a yeast-based heterologous virus-host system to assess PKR variants' ability to escape antagonism by the vaccinia virus pseudo-substrate inhibitor K3. The study identified determinant sites in the PKR kinase domain that harbor K3-resistant variants, as well as sites where variation leads to PKR loss of function. The authors found that multiple K3-resistant variants are readily available throughout the domain interface and are enriched at sites under positive selection. They further found some evidence of PKR resilience to viral antagonist diversification. These findings highlight the remarkable adaptability of PKR in response to viral antagonism by mimicry.

Significance of the findings:

The findings are important with implications for various fields, including evolutionary biology, virus-host interfaces, genetic conflicts, and antiviral immunity.

Strength of the evidence:

Convincing methodology using state-of-the-art mutational scanning approach in an elegant and simple setup to address important challenges in virus-host molecular conflicts and protein adaptations.

Strengths:

Systematic and Unbiased Approach:

The study's comprehensive approach to generating and characterizing a large library of PKR variants provides valuable insights into the evolutionary landscape of the PKR kinase domain. By focusing on SNP-accessible variants, the authors ensure the relevance of their findings to naturally occurring mutations.

Identification of Key Sites:

The identification of specific sites in the PKR kinase domain that confer resistance or susceptibility to a poxvirus pseudosubstrate inhibition is a significant contribution.

Evolutionary Implications:

The authors performed meticulous comparative analyses throughout the study between the functional variants from their mutagenesis screen ("prospective") and the evolutionarily-relevant past adaptations ("retrospective").

Experimental Design:

The use of a yeast-based assay to simultaneously assess PKR capacity to induce cell growth arrest and susceptibility/resistance to various VACV K3 alleles is an efficient approach. The combination of this assay with high-throughput sequencing allows for the rapid characterization of a large number of PKR variants.

Areas for Improvement:

(5) Validation of the screen: The results would be strengthened by validating results from the screen on a handful of candidate PKR variants, either using a similar yeast heterologous assay, or - even more powerfully - in another experimental system assaying for similar function (cell translation arrest) or protein-protein interaction.

Thank you for your thoughtful feedback. We agree that additional data to validate our findings would strengthen the manuscript. We have individually screened a handful of PKR variants in duplicate using serial dilution to measure yeast growth, and found that the results generally support our original findings. We have revised the manuscript to include these validation experiments (lines 117-119 of the revised_manuscript.pdf, Supplemental Figure 4).

(6) Evolutionary Data: Beyond residues under positive selection, the screen would allow the authors to also perform a comparative analysis with PKR residues under purifying selection. Because they are assessing one of the most conserved ancestral functions of PKR (i.e. cell translation arrest), it may also be of interest to discuss these highly conserved sites.

This is a great point. We do find that there are regions of the PKR kinase domain that are not amenable to genetic perturbation, namely in the glycine rich loop and active site. We contrast the PKR functional scores at conserved residues under purifying selection with those under positive selection in Figure 2E (lines 141-143).

(7) Mechanistic Insights: While the study identifies key sites and residues involved in vaccinia K3 resistance, it could benefit from further investigation into the underlying molecular mechanisms. The study's reliance on a single experimental approach, deep mutational scanning, may introduce biases and limit the scope of the findings. The authors may acknowledge these limitations in the Discussion.

We agree that further investigation into the underlying molecular mechanisms is warranted and we have revised the manuscript to acknowledge this point in the discussion (lines 284-288).

(8) Viral Diversity: The study focuses on the viral inhibitor K3 from vaccinia. Expanding the analysis to include other viral inhibitors, or exploring the effects of PKR variants on a range of viruses would strengthen and expand the study's conclusions. Would the identified VACV K3-resistant variants also be effective against other viral inhibitors (from pox or other viruses)? or in the context of infection with different viruses? Without such evidence, the authors may check the manuscript is specific about the conclusions.

This is a fantastic question that we are interested in exploring in our future studies. In the manuscript we note a strong correlation between PKR variants that evade vaccinia wild-type K3 and the K3-H47R enhanced allele, but we are curious to know if this holds when tested against other K3 orthologs such as variola virus C3. That said, we have revised the manuscript to clarify this limitation to our findings and specify vaccinia K3 where appropriate.

Reviewer #3 (Public Review):

Summary:

-  This study investigated how genetic variation in the human protein PKR can enable sensitivity or resistance to a viral inhibitor from the vaccinia virus called K3.

-  The authors generated a collection of PKR mutants and characterized their activity in a high-throughput yeast assay to identify 1) which mutations alter PKR's intrinsic biochemical activity, 2) which mutations allow for PKR to escape from viral K3, and 3) which mutations allow for escape from a mutant version of K3 that was previously known to inhibit PKR more efficiently.

-  As a result of this work, the authors generated a detailed map of residues at the PKR-K3 binding surface and the functional impacts of single mutation changes at these sites.

Strengths:

-  Experiments assessed each PKR variant against three different alleles of the K3 antagonist, allowing for a combinatorial view of how each PKR mutant performs in different settings.

-  Nice development of a useful, high-throughput yeast assay to assess PKR activity, with highly detailed methods to facilitate open science and reproducibility.

-  The authors generated a very clean, high-quality, and well-replicated dataset.

Weaknesses:

(9) The authors chose to focus solely on testing residues in or near the PKR-K3 predicted binding interface. As a result, there was only a moderately complex library of PKR mutants tested. The residues selected for investigation were logical, but this limited the potential for observing allosteric interactions or other less-expected results.

First, we greatly appreciate all your feedback on the manuscript, as well as raising this particular point. We agree that this is a moderately complex library of PKR variants, from which we begin to uncover a highly pliable domain with a few specific sites that cannot be altered. We have revised the manuscript to raise this limitation (lines 284-288 of the revised_manuscript.pdf) and encourage additional exploration of the PKR kinase domain.

(10) For residues of interest, some kind of independent validation assay would have been useful to demonstrate that this yeast fitness-based assay is a reliable and quantitative readout of PKR activity.

We agree that additional data to validate our findings would strengthen the manuscript. We have individually screened a handful of PKR variants in duplicate using serial dilution to measure yeast growth, and generally found that the results support our original findings. We have revised the manuscript to include this validation experiment (lines 117-119, Supplemental Figure 4).

(11) As written, the current version of the manuscript could use more context to help a general reader understand 1) what was previously known about these PKR and K3 variants, 2) what was known about how other genes involved in arms races evolve, or 3) what predictions or goals the authors had at the beginning of their experiment. As a result, this paper mostly provides a detailed catalog of variants and their effects. This will be a useful reference for those carrying out detailed, biochemical studies of PKR or K3, but any broader lessons are limited.

Thank you for bringing this to our attention. We have revised the introduction of the manuscript to provide more context regarding previous work demonstrating an evolutionary arms race between PKR and K3 and how single residue changes alter K3 resistance (lines 51-64).

(12) I felt there was a missed opportunity to connect the study's findings to outside evolutionary genetic information, beyond asking if there was overlap with PKR sites that a single previous study had identified as positively selected. For example, are there any signals of balancing selection for PKR? How much allelic diversity is there within humans, and are people typically heterozygous for PKR variants? Relatedly, although PKR variants were tested in isolation here, would the authors expect their functional impacts to be recessive or dominant, and would this alter their interpretations? On the viral diversity side, how much variation is there among K3 sequences? Is there an elevated evolutionary rate, for example, in K3 at residues that contact PKR sites that can confer resistance? None of these additions are essential, but some kind of discussion or analysis like this would help to connect the yeast-based PKR phenotypic assay presented here back to the real-world context for these genes.

We appreciate this suggestion to extend our findings to a broader evolutionary context. There is little allelic diversity of PKR in humans, with all nonsynonymous variation listed in gnomAD being rare. (PKR shows sequence diversity in comparisons across species, including across primates.) Thus, barring the possibility of variation being present in under-studied populations, there is unlikely to be balancing selection on PKR in humans. Our expectation is that beneficial mutations in PKR for evading a pseudosubstrate inhibitor would be dominant, as a small amount of eIF2α phosphorylation is capable of halting translation (Siekierka, PNAS, 1984). There is a recent report citing PKR missense variants associated with dystonia that can be dominantly or recessively inherited (Eemy et al. 2020 PMID 33236446). Elde et al. 2009 (PMID 19043403) notes that poxvirus K3 homologs are under positive selection but no specific residues have been cited to be under positive selection. The lack of allelic diversity in PKR in humans notwithstanding, PKR could experience future selection in the human population as evidenced by its rapid evolution in primates, so we fully agree that a connection to the real-world context is useful. We have noted these topics in the discussion section (lines 289-294).

Reviewer #1 (Recommendations For The Authors):

I have no major criticisms but ask for some clarifications and make some comments about the perceived weaknesses.

(13)  If the authors disagree with my summation that the findings largely replicate what was known, could they detail how the findings differ from what was known about this protein interaction and the major new insights stemming from the study? Currently, the abstract is a little philosophical rather than listing the explicit discoveries of the study.

Thank you again for raising the need for us to clearly convey the novelty of our findings. We have revised the final paragraph in our introduction as described in comment #1.

(14) As the experimental approach is well reported it is unnecessary to confirm the proposed activity by, for instance, measures of Sui2 phosphorylation. However, previous reports have recognised that point mutants of PKR can be differentially expressed. The impact of this potential effect is unknown in the current experimentation as there are no measures of the expression of the different mutant PKR constructs. The large number of constructs used makes this verification onerous. The potential impact could be ameliorated by redundant replacing each residue (hoping different residues have different effects on expression). Still, this limitation of the study should be acknowledged in the text.

We greatly appreciate this comment and agree that this should be made clear in the text, which we have added to the discussion of the manuscript (lines 247-251).

(15) Preceding findings and the modeling in this report recognise an involvement in the kinase insert region (residues 332 to 358) in PKR's interaction with K3 but this region is excluded from the analysis. These residues have been largely disregarded in the preceding analysis (it is absent from the molecular structure of the kinase) so its inclusion here might have lent a more novel aspect or delivered a more complete investigation. Is there a justification for excluding this flexible loop?

The PKR variant library was designed based on the crystal structure of K3 (PDB 1LUZ) aligned to eIF2α in complex with PKR (PDB 2A1A). After the library was designed and made we attained complete predicted structures of PKR in complex with eIF2α and K3, which largely agrees with the predicted crystal structures but contain the additional flexible loops that were not captured in the crystal structures. Though the library studied here does not explore variation in the kinase insert region, we are very interested in doing so in our future studies.

(16)  Could the explanation of the 'PKR functional score' be clarified? The description given within the legend of SF1 was helpful, so could this be replicated earlier in the main body of the text when introducing these experiments? e.g. As PKR activity is toxic to yeast, the number of cells in the pool expressing the functional PKR will decrease over time. Thus the associated barcode read count will also decrease, while the read count for the nonfunctional PKR will increase. This is termed the PKR function score, which will be relatively lower for cells transformed with less active PKR than those with more active PKR.

Thank you for suggesting this clarification, we have revised the manuscript to clarify our definition of the PKR functional score (lines 106-109).

(17)  Another suggestion to clarify this term is to modify the figures. Currently, the intent of the first simulated graph in Fig 1E is clear but the inversion of the response (shown by the transposition of the colours) in the next graph (to the right) is less immediately obvious. Accordingly, the orientation of the 'PKR functional score' is uncertain. Could the authors add text to the rightmost graphic in Figure 1E by, for instance, indicating the PKR activity in the vertical column with text such as 'less active' (at the bottom), 'WT' (in the centre), and 'more activity' (at the top)? Also, the position of the inactive K296R mutant might be added to Figure 2A complementing the positioning of the active WT kinase in the first data graph of this kind.

We appreciate your specific feedback to improve the figures of the manuscript, we have made adjustments to Figure 1E to clarify how we derive the PKR functional scores.

(18) The authors don't use existing structures of PKR in their modelling. However, there is no information about the state of the PKR molecule used for modelling. Specific elements of the kinase domain affect its interaction with K3 so it would be informative to know the orientation of these elements in the model. Could the authors detail the state of pivotal kinase elements in their models? This could involve the alignment of the N- and C-lobes, the orientation of kinase spines (C- and R-spines), and the phosphorylation stasis of residues in the activation loop, or at least the position of this loop in relationship to that adopted in the active dimeric kinase (e.g. PDB-2A1A, 3UIU or 6D3L). Alternatively, crystallographic structures of active inactive PKR could be overlayed with the theoretical structure used for modelling (as supplementary information).

We have revised the manuscript to describe the alignment of the predicted PKR-K3 complex with active and inactive PKR, and we have extended Supplemental Figure 12 with an overlay of the predicted structures with existing structures. We have also added a supplemental data file containing the RMSD values of PKR (from the predicted PKR-K3 complex) aligned to active (PDB 2A1A) and inactive (PDB 3UIU) or unphosphorylated (PDB 6D3L) PKR (5_Structure-Alignment-RMSD-Values.xlsx). We have also provided the AlphaFold2 best model predictions for the PKR-eIF2α complex (6_AF2_PKR-KD_eIF2a.pdb) and PKR-K3 complex (7_AF2_PKR-KD_VACV-K3.pdb). Looking across the RMSD values, the AlphaFold2 model of PKR most closely resembles unphosphorylated PKR (PDB 6D3L) though we note the activation loop is absent from PDB 6D3L and 3UIU. We also aligned the Ser51 phosphoacceptor loop of AlphaFold2 eIF2α model to PDB 1Q46 and we see that the model reflects the pre-phosphorylation state. This loop is expected to interact with the PKR active site, which is not captured in our model and we state this explicitly in the caption of Figure 1 (lines 665-668).

(19) Could some specific residue in Figure 7 be labelled (numbered) to orient the findings? Also, the key in this figure doesn't title the residues coloured white (RE red/black/blue). The white also isn't distinguished from the green (outside the regions targeted for mutagenesis).

Excellent suggestion, we have revised this figure to include labels for the sites to orient the reader and clarify our categorization of PKR residues in the kinase domain.

(20)  Regarding the discussion, the authors adopt the convention of describing K3 as a pseudosubstrate. Although I realize it is common to refer to K3 as a pseudosubstrate, it isn't phosphorylated and binds slightly differently to PKR so alternative descriptors, such as 'a competitive binder', would more accurately present the protein's function. Possibly for this reason, the authors declared an expectation that evolution pressures should shift K3 to precisely mimic EIF2α. However, closer molecular mimicry shouldn't be expected for two reasons. The first is a risk of disrupting other interactions, such as the EIF2 complex. Secondly, equivalent binding to PKR would demote K3 to merely a stoichiometric competitor of EIF2α. In this instance, effective inhibition would require very high levels of K3 to compete with equivalent binding by EIF2α. This would be demanding particularly upon induction of PKR during the interferon response. To be an effective inhibitor K3 has to bind more avidly than EIF2α and merely requires a sufficient overlap with the EIF2α interface on PKR to disrupt this alternative association. This interpretation predicts that K3 is under pressure to bind PKR by a different mechanism than EIF2α.

We appreciate your thoughtful point about the usage of the term pseudosubstrate. Ultimately, we’ve decided to continue using the term due to its historical usage in the field. The question of the optimal extent of mimicry in K3 is a fascinating one, and we greatly appreciate your thoughts. We wholly agree that the possibility of K3 having superior PKR binding relative to eIF2α would be preferable to perfect mimicry. In our Ideas and Speculation section, we propose that benefits towards increasing PKR affinity may need to be balanced against potential loss of host range resulting from overfitting to a given host’s PKR. However, the possibility that reduced mimicry could be selected to avoid disruption of eIF2 function had not occurred to us; thank you for pointing it out!

(21) The discussion of the 'positive selection' of sites is also interesting in this context. To what extent has the proposed positive selection been quantified? My understanding is that all of the EIF2α kinases are conserved and so demonstrate lower levels of residue change that might be expected by random mutagenesis i.e. variance is under negative selection. The relatively higher rate of variance in PKR orthologs compared to other EIF2α kinases could reflect some relaxation of these constraints, rather than positive selection. Greater tolerance of change may stem from PKR 's more sporadic function in the immune response (infrequent and intermittent presence of its activating stimuli) rather than the ceaseless control of homeostasis by the other EIF2α kinases. Also, induction of PKR during the immune response might compensate for mutations that reduce its activity. I believe that the entire clade of extant poxviruses is young relative to the divergence between their hosts. Accordingly, genetic variance in PKR predates these viruses. Although a change in PKR may become fixed if it affords an advantage during infection, such an advantage to the host would be countered by the much higher mutation rates of the virus. This would appear to diminish the opportunity for a specific mutation to dominate a host population and, thereby, to differentiate host species. Rather, pressure to elude control by a rapidly evolving viral factor would favour variation at sites where K3 binds. This speculation offers an alternative perspective to the current discussion that the variance in PKR orthologs stems from positive selection driven by viral infection.

We appreciate this stimulating feedback for discussion. Three of the four eIF2α kinases (HIR, PERK, and GCN2) appear to be under purifying selection (Elde et al. 2009, PMID 19043403), which stand in contrast to PKR. Residues under positive selection have been found throughout PKR, including the dsRNA binding domains, linker region, and the kinase domain. Importantly, the selection analysis from Elde et al. and Rothenburg et al. concluded that positive selection at these sites is more likely than relaxed selection. We agree that poxviruses are young, though we would guess that viral pseudosubstrate inhibition of PKR is ancient. Many viral proteins have been reported to directly interact with PKR, including herpes virus US11, influenza A virus NS1A, hepatitis C virus NS5A, and human immunodeficiency virus Tat. The PKR kinase domain does contain residues under purifying selection that are conserved among all four eIF2α kinases, but it also contains residues under positive selection that interface with the natural substrate eIF2α. Our work suggests that PKR is genetically pliable across several sites in the kinase domain, and we are curious to know if this pliability would hold at the same sites across the other three eIF2α kinases.

(22) The manuscript is very well written but has a small number of typos; e.g. an aberrant 'e' ln 7 of the introduction, capitalise the R in ranavirus on the last line of the fourth paragraph of the discussion, and eIF2α (EIF2α?) is occasionally written as eIFα in the materials&methods.

Thank you for bringing these typos to our attention! We’ve deleted the aberrant ‘e’ in the introduction, capitalized ‘Ranavirus’ in the discussion (line 265), and corrected ‘eIFα’ to ‘eIF2α’ throughout the manuscript.

Reviewer #2 (Recommendations For The Authors):

Additional minor edits or revisions:

(23) Paragraph 3 of the Introduction gives the impression that most of the previous work on the PKR-virus arms race is speculative. However, it is one of the best-described and most convincing examples of virus-host arms races. Can the authors edit the paragraph accordingly?

Thank you for bringing this to our attention. We have revised the third paragraph and strengthened the description of the evolutionary arms race between PKR and viral pseudosubstrate antagonists.

(24) Introduction: PKR has "two" double-stranded RNA binding domains. Can the authors update the text accordingly?

We have updated the manuscript to clarify PKR has two dsRNA binding domains (lines 44-45).

(25) The authors test here for one of the key functions of PKR: cell growth/translation arrest. Because of PKR pleiotropy, the manuscript may be edited accordingly: For example, statements such as "We found few genetic variants render the PKR kinase domain nonfunctional" are too speculative as they may retain other (not tested here) functions.

This is a great suggestion, we have revised the manuscript to specify our definition of nonfunction in the context of our experimental screen (lines 86-92 and 106-109) and acknowledge this limitation in our experimental screen (lines 304-307).

(26) The authors should specify "vaccinia" K3 whenever appropriate.

We appreciate this comment and have revised the manuscript to specify vaccinia K3 where appropriate (e.g. lines 62,66, 70, 80, 108, and 226).

(27) Ref for ACE2 diversification may include Frank et al 2022 PMID: 35892217.

Thank you for pointing us to this paper, we have included it as a reference in the manuscript (line 277).

(28) Positive selection of PKR as referred to by the authors corresponds to analyses performed in primates. As shown by several studies, the sites under positive selection may vary according to host orders. Can the authors specify this ("primate") in their manuscript? And/or shortly discuss this aspect.

Thank you for raising this point. In the manuscript we performed our analysis using vertebrate sites under positive selection as identified in Rothenburg et al. 2009 PMID 19043413 (lines 51 and figure legends). We performed the same analysis using sites under positive selection in primates (as identified by Elde et al. 2009 PMID 19043403) and again found a significant difference in PKR functional scores versus K3. We have revised the manuscript to clarify our use of vertebrate sites under positive selection (line 80-81).

(29) We view deep mutational scanning experiments as a complementary approach to positive selection": The authors should edit this and acknowledge previous and similar work of other antiviral factors, in particular one of the first studies of this kind on MxA (Colon-Thillet et al 2019 PMID: 31574080), and TRIM5 (Tenthorey et al 2020 PMID: 32930662).

Thank you for raising up these two papers, which we acknowledge in the revised manuscript (line 299).

(30) We believe Figure S7 brings important results and should be placed in the Main.

We appreciate this suggestion, and have moved the contents of the former supplementary Figure 7 to the main text, in Figure 6.

(31) The title may specify "poxvirus".

Thank you for the suggestion to specify the nature of our experiment, we have adjusted the title to: Systematic genetic characterization of the human PKR kinase domain highlights its functional malleability to escape a poxvirus substrate mimic (line 3).

Reviewer #3 (Recommendations For The Authors):

(32) No line numbers or page numbers are provided, which makes it difficult to comment.

We sincerely apologize for this oversight and have included line numbers in our revised manuscript as well as the tracked changes document.

(33) In the introduction, I recommend defining evolutionary arms races more clearly for a broad audience.

Thank you for this suggestion. We have revised the manuscript in the first and third paragraphs to more clearly introduce readers to the concept of an evolutionary arms race.

(34) The introduction could use a clearer statement of the question being considered and the gap in knowledge this paper is trying to address. Currently, the third paragraph includes many facts about PKR and the fourth paragraph jumps straight into the approach and results. Some elaboration here would convey the significance of the study more clearly. As is, the introduction reads a bit like "We wanted to do deep mutational scanning. PKR seemed like an ok protein to look at", rather than conveying a scientific question.

This is a great suggestion to improve the introduction section. We have heavily revised the third and fourth paragraphs of the introduction to clarify the motivation, approach, and significance of our work.

(35) Relatedly, did the authors have any hypotheses at the start of the experiment about what kinds of results they expected? e.g. What parts of PKR would be most likely to generate escape mutants? Would resistant mutants be rare or common? etc? This would help the reader to understand which results are expected vs. surprising.

These are all great questions. We have revised the introduction of the manuscript to point out that previous studies have characterized a handful of PKR variants that evade vaccinia K3, and these variants were made at sites found to be under positive selection (lines 60-64).

(36) A description of the different K3 variants and information about why they were chosen for study should also be added to the Introduction. It was not until Figure 5 that the reader was told that K3-H47R was the same as the 'enhanced' K3 allele you are testing.

Thank you for bringing this to our attention, we have revised the introduction to clarify the experimental conditions (lines 65-67) and specify K3-H47R as the enhanced allele earlier in the manuscript (line 100).

(37) Does every PKR include just a single point mutation? It would be nice to see data about the number and types of mutations in each PRK window added to Supplemental Figure 1.

Thank you for the suggestion to improve this figure. Every PKR variant that we track has a single point mutation that generates a nonsynonymous mutation. In our PacBio sequencing of the PKR variant library we identified a few off-target variants or sequences with multiple variants, but we identified the barcodes linked to those constructs and discarded those variants in our analysis. We have revised Supplemental Figure 1 to include the number and types of mutations made at each PKR window.

(38) In terms of the paper's logical flow, personally, I would expect to begin by testing which variants break PKR's function (Figure 3) and then proceeding to see which variants allow for K3 escape (Figure 2). Consider swapping the order of these sections.

Thank you for this suggestion, and we can appreciate how the flow of the manuscript may be improved by swapping Figures 2 and 3. We have decided to maintain the current order of the figures because we use Figure 3 to emphasize the distinction of PKR sites that are nonfunctional versus susceptible to vaccinia K3.

(39) Figure 3A seems like a less-informative version of Figure 4A, recommend combining these two. Same comment with Figure 5A and Figure 6A.

We appreciate this specific feedback for the figures. Though there are similarities between figure panels (e.g. 3A and 4A) we use them to emphasize different points in each figure. For example, in Figure 3 we emphasize the general lack of variants that impair PKR kinase activity, and in Figure 4 we distinguish kinase-impaired variants from K3-susceptible variants. For this reason, and given space constraints, we have chosen to maintain the figures separately. We did decide to move the former Figure 6 to the supplement.

(40) In general, it felt like there was a lot of repetition/re-graphing of the same data in Figures 3-6. I recommend condensing some of this, and/or moving some of the panels to supplemental figures.

Thank you for your suggestion, we have revised the manuscript and have moved Figure 6 to Supplemental Figure 7.

(41) In contrast, Supplemental Figure 7 is helpful for understanding the distribution of the data. Recommend moving to the main text.

This is a great recommendation, and we have moved Supplemental Figure 7 into Figure 6.

(42) How do the authors interpret an enrichment of positively selected sites in K3-resistant variants, but not K3-H74R-resistant variants? This seems important. Please explain.

Thank you for this suggestion to improve the manuscript; we agree that this observation warranted further exploration. We found a strong correlation in PKR functional scores between K3 WT and K3-H47R, and with that we find sites under positive selection that are resistant to K3 WT are also resistant to K3-H47R. The lack of enrichment at positively selected sites appears to be caused by collapsed dynamic range between PKR wild-type-like and nonfunctional variants in the K3-H47R screen. We have revised the manuscript to clarify this point (line 202-204).

(43) Discussion: The authors compare and contrast between PKR and ACE2, but it would be worth mentioning other examples of genes involved in antiviral arms races wherein flexible, unstructured loops are functionally important and are hotspots of positive selection (e.g. MxA, NLRP1, etc).

We greatly appreciate this suggestion to improve the discussion. We note this contrast between the PKR kinase domain and the flexible linkers of MxA and NLRP1 in the revised manuscript (lines 273-274).

(44) Speculation section: What is the host range of the vaccinia virus? Is it likely to be a generalist amongst many species' PKRs (and if so, how variable are those PKRs)? Would be worth mentioning for context if you want to discuss this topic.

Thank you for raising this question. Vaccinia virus is the most well studied of the poxviruses, having been used as a vaccine to eradicate smallpox, and serves as a model poxvirus. Vaccinia virus has a broad host range, and though the name vaccinia derives from the Latin word “vacca” for cow the viruses origin remains uncertain (Smith 2007 https://doi.org/10.1007/978-3-7643-7557-7_1). has been used to eradicate smallpox as a vaccine and serves as a model poxvirus. Thought the natural host is unknown, it appears to be a general inhibitor of vertebrate PKRs The natural host of vaccinia virus is unknown, though there is some evidence to suggest it may be native to rabbits and does appear to be generalist.

(45) Many papers in this field discuss interactions between PKR and K3L, rather than K3. I understand that this is a gene vs. protein nomenclature issue, but consider matching the K3L literature to make this paper easier to find.

Thank you for bringing this to our attention. We have revised the manuscript to specify that vaccinia K3 is expressed from the K3L gene in both the abstract (line 26) and the introduction (line 56) to help make this paper easier to find when searching for “K3L” literature.

(46) Which PKR sequence was used as the wild-type background?

This is a great question. We used the predominant allele circulating in the human population represented by Genbank m85294.1:31-1686. We cite this sequence in the Methods (line 421) and have added it to the results section as well (lines 84).

(47) Figure 1C: the black dashed line is difficult to see. Recommend changing the colors in 1A-1C.

Thank you for this suggestion, we have changed the dashed lines from black to white to make them more distinguishable.

(48) Figure 1D: Part of the point of this figure is to convey overlaps between sites under selection, K3 contact sites, and eIF2alpha contact sites, but at this scale, many of the triangles overlap. It is therefore impossible to tell if the same sites are contacted vs. nearby sites. Perhaps the zoomed-in panels showing each of the four windows in the subsequent figures are sufficient?

Thank you for bringing this to our attention. We have scaled the triangles down to reduce their overlap in Figure 1D and list all sites of interest (predicted eIF2α and vaccinia contacts, conserved sites, and positive selection sites) in the Materials and Methods section “Predicted PKR complexes and substrate contacts”.

(49) Figure 1E: under "1,293 Unique Combinations", there is a line between the PKR and K3 variants, which makes it look like they are expressed as a fusion protein. I believe these proteins were expressed from the same plasmid, but not as a fusion, so I recommend re-drawing. Then in the graph, the y-axis says "PKR abundance", but from the figure, it is not clear that this refers to relative abundance in a yeast pool. Perhaps "yeast growth" or similar would be clearer?

Thank you for the specific feedback to improve Figure 1. We have made the suggested edits to clarify that PKR and vaccinia K3 are not fused but each is expressed from their own promoter. We have also changed the y-axis from “PKR Abundance” to “Yeast Growth”.

Reviewer #1 (Public review):

Summary:

The report examines the control of the antiviral RNA-activated protein kinase, PKR, by the Vaccinia virus K3 protein. K3 binds to PKR, hindering its ability to control protein translation by blocking its phosphorylation of the eukaryotic initiation factor EIF2α. Kinase function is probed by saturation mutation of the K3/EIF2α-binding surface on PKR, guided by models of their interaction. The findings identify specific residues at the predicted interface that asymmetrically influence repression by K3 and the phosphorylation of EIF2α. This recognises the potential of PKR alleles to resist control by the viral virulence factor.

Strengths:

The experimentation is diligent, generating and screening many point mutants to identify residues at the interface between PKR and EIF2α or K3 that distinguishes PKR's phosphor control of its substrate from the antithetical interaction with the viral virulence factor.

Weaknesses:

The protein interaction between PKR and K3 has already been well-explored through phylogenetic and functional analyses and molecular dynamics studies, as well as with more limited site-directed mutational studies using the same experimental assays. Accordingly, the findings are not pioneering but reinforce and extend what had previously been established.

The authors responded to this comment by pointing out that their more comprehensive screen better defined the extent of the plasticity of the K3/EIF2α-binding surface on PKR.

Also in their response, the authors added the caveat that the equivalent expression of the different PKR mutants has not been verified, added information clarifying the states of the model proteins compared to their determined molecular structures, and provided clarifications or responses to all other questions.

I question eLife's assessment that the development of the yeast-based assay is a key advancement of this report, as this assay has been used for over 30 years.

eLife Assessment

This important revised report describes the control of the activity of the RNA-activated protein kinase, PKR, by the Vaccinia virus K3 protein. A strength of the manuscript is the powerful combination of a classic yeast-based assay with high-throughput sequencing and its convincing experimental use to characterize large numbers of PKR variants, now with improved controls for potential biases. A minor current limitation that the authors may address in the future is the scope of the screen in terms of the segments of PKR included.

Reviewer #2 (Public review):

Chambers et al. (2024) present a systematic and unbiased approach to explore the evolutionary potential of the kinase domain of the human antiviral protein kinase R (PKR) to evade inhibition by a poxviral antagonist while maintaining one of its essential functions.

The authors generated a library of 426 single-nucleotide polymorphism (SNP)-accessible non-synonymous variants of PKR kinase domain and used a yeast-based heterologous virus-host system to assess PKR variants' ability to escape antagonism by the vaccinia virus pseudo-substrate inhibitor K3. The study identified determinant sites in the PKR kinase domain that harbor K3-resistant variants, as well as sites where variation leads to PKR loss of function. The authors found that multiple K3-resistant variants are readily available throughout the domain interface and are enriched at sites under positive selection. They further found some evidence of PKR resilience to viral antagonist diversification. These findings highlight the remarkable adaptability of PKR in response to viral antagonism by mimicry.

Significance of the findings: The findings are important with implications to various fields, including evolutionary biology, virus-host interfaces, genetic conflicts, antiviral immunity.

Strength of the evidence: Convincing methodology using state-of-the-art mutational scanning approach in an elegant and simple setup to address important challenges in virus-host molecular conflicts and protein adaptations.

Strengths

Systematic and Unbiased Approach: The study's comprehensive approach to generating and characterizing a large library of PKR variants provides valuable insights into the evolutionary landscape of PKR kinase domain. By focusing on SNP-accessible variants, the authors ensure the relevance of their findings to naturally occurring mutations.<br /> Identification of Key Sites: The identification of specific sites in the PKR kinase domain that confer resistance or susceptibility to a poxvirus pseudosubstrate inhibition is a significant contribution.<br /> Evolutionary Implications: The authors performed meticulous comparative analyses throughout the study between the functional variants from their mutagenesis screen ("prospective") and the evolutionarily-relevant past adaptations ("retrospective").<br /> Experimental Design: The use of a yeast-based assay to simultaneously assess PKR capacity to induce cell growth arrest and susceptibility/resistance to various VACV K3 alleles is an efficient approach. The combination of this assay with high-throughput sequencing allows for the rapid characterization of a large number of PKR variants.

Areas of improvement

Validation of the screen: In the revised version, the authors now provide the results of two independent experiments in a complete yeast growth assay on a handful of candidates to control the screen's results. This strengthens the direct findings from the screen. It would strengthen the study to complement this validation by another method to assess PKR functions; for example, in human PKR-KO cells, because results between yeast and human cells can differ. These limitations are now acknowledged in the revised version.<br /> Evolutionary Data: Beyond residues under positive selection, the screen allows the authors to also perform a comparative analysis with PKR residues under purifying selection. Because they are assessing one of the most conserved ancestral functions of PKR (i.e. cell translation arrest), it may also be of interest to discuss these highly conserved sites. The authors now discuss the implications for the conserved residues.<br /> Mechanistic insights and viral diversity: While the study identifies key sites and residues involved in vaccinia K3 resistance, it could benefit from further investigation into the underlying molecular mechanisms and the diversity of viral antagonists. The authors have now acknowledged these limitations in the Discussion and updated the manuscript to be more specific. These exciting research avenues will be the objectives of a next study.

Overall Assessment

The systematic approach, identification of key sites, and evolutionary implications are all notable strengths. While there is room for a stronger validation of the functions and further investigation into the mechanistic details and broader viral diversity, the findings are robust and already provide important advancements. The manuscript is well-written and clear, and the revised figures are informative and improved.

I think that maybe during that quiet time where students are instructed to work on their schoolwork, they can put their phones away in their backpack, but taking the phone away from day to day probably isn't the most plausible answer.

This in itself is more then enough reason to ban cell phons to ban cell phones from students but in my opinion thats the reason parents are her to monitor and make sure they give they're own kids a certain amount of screen time per day.

could add a link here to the section in "Create and Manage Chapters" that refers to the "Status & Visibility" panel

should this be part of this chapter or rather the "Edit" part? as it's part of the Satus & Visiblity panel, maybe leave it in this chapter, but refer to it in the "Edit Content with Visual and Text Editor" chapter?

adjusted title - anything related to using the editor should be in the chapter https://university.pressbooks.pub/guidetestedits/chapter/edit-content-with-the-visual-text-editors/ or other chapters in that part

There is an entire chapter on this: https://university.pressbooks.pub/guidetestedits/chapter/edit-content-with-the-visual-text-editors/ I would suggest removing this section.

definitions, specifically of tetrahedron, equicomposability, and equicomplementability

As a parent I understand that each child is different from their siblings and within the classroom I can see how this will be difficult to handle the diversity and meet the needs of every child without a co teacher.

If we’re committed to the success of every child, we must acknowledge the uneven playing field that exists for many: ELLs, students with special needs, children experiencing trauma or relentless poverty…. There are 6 Steps Toward Equity!!

Even if the song is boring, it is still enough to bring sailors to the ocean. They want to hear more of this tantalizing story, to hear the secret end.

Providing direct instructions to look further into the information shows a lack of bias and instills a sense of credibility into the reader. This allows the reader to understand the information about the amendment and voting rights of felons. This site may not be entirely unbiased, as they do seem to be pushing a certain agenda and are not a domain known to be reliable (.org, .gov, etc), but the information does seem to be presented in a straightforward way that does not indicate any bias. It offers little information in one way or another, simply presenting the information about the process to pass this amendment to reinstate the voting rights of felons.

This excerpt links a pdf of the revised rules. This not only gives interested readers the opportunity to see the rules in question, but also gives credibility to the argument and shows an unbiased perspective. This also helps verify the information in the article, as only one look into the pdf will exemplify the points being made.

This links directly to a .gov site of the Florida House of Representatives. This not only explains what the bill was, but details the history of the bill and additional information, such as statutes referenced. Linking this is a good way to demonstrate the validity of the article's claims, but to provide official information from a reliable source.

How can we as faculty educate our students about the process of receiving feedback and working in an open space - when your work can be critiqued or changed in dynamic open spaces v. traditional peer reviewed process? How are we talking about the inherent risks involved and the gatekeeping processes that are different in a space like wikipedia compared to a peer reviewed publication?

for - question - @Michael - Is this the Zebras Unite you are referring to? - https://zebrasunite.coop/ - If so, that brings up another question: - What is the difference between Fair Share Commons and a Cooperative?

Nature is uncaring. Whoever is unprepared to deal with natures wrath will eventually face death when placed in extreme circumstances.

Extreme circumstances can lead to the removal of civilized ideals and morals.

The narrator describes what a professional should know but we are unaware of what exactly the character knows. we can assume that he is knowledgeable, but we are unaware of how knowledgeable.

This reflects the uncaring identity of nature by demonstrating how unpredictable and uncertain the outcomes of natural events are.

This description of the environment creates the conflict between man vs nature.

eLife Assessment

This study retrospectively analyzed clinical data to develop a risk prediction model for pulmonary hypertension in high-altitude populations. The evidence is solid, and the findings are useful and hold clinical significance as the model can be used for intuitive and individualized prediction of pulmonary hypertension risk in these populations.

https://www.knowledgegraph.tech/speakers/denny-vrandecic/

https://ruben.verborgh.org/<br /> Ruben Verborgh

Mentioned by Flancian

https://www.linkedin.com/in/rubenverborgh/<br /> Ruben Verborgh

Denny Vrandečić

for - quote - we are in a back-loop phase - From Complex Regions to Complex Worlds - Crawford Stanley Holling - 2004 - creative alternatives - liminal spaces - rapid whole system change

comment - This is important for discussion for change actors working in liminal spaces attempting to give birth to creative alternatives

Know and Master Your Social Media Data Flow by [[Louis Gray]]

See commentary at https://boffosocko.com/2017/04/11/a-new-way-to-know-and-master-your-social-media-flow/