Reviewer #1 (Public Review):

The core conclusions in this manuscript are well supported. First, the genomic data clear support a recent origin of the Baltic and Arctic ecotypes from an Atlantic-like ancestor, without major bottlenecks and with gene exchange at least on the one sampled sympatric location but probably also more widely. The genome scans strongly suggest the involvement of multiple loci in divergence. This is supported by the quantitative trait locus analysis but this support is relatively weak because the number of markers used was small and markers were rather unevenly distributed on the genetic map, leaving little power to detect QTL in some areas. An analysis of the power of the QTL analysis is lacking and there might also be an issue about whether the measured trait truly captures rhythmicity.

The presence of segregating variants across all sampled populations for the loci that appear to underlie local adaptation, plus the absence of strong sweep signatures, is good evidence that adaptation to the Baltic environment was based on standing genetic variation. This is supported by the known presence of local adaptation to tidal regimes within the Atlantic ecotype, providing a mechanism for the maintenance of standing variation. Clustering of putatively adaptive loci in one region of chromosome 1 seems clear, although it is not formally compared to a random distribution.

The authors make an interesting point that it is only when many genes are involved that Gene Ontology enrichment is expected to be informative. Here, they also had clear a priori expectations which are neatly fulfilled, further suggesting that differentiated loci are good candidates for a role in adaptation.

The broader context provided for the analysis of the fascinating marine midge system is brief and could usefully be expanded. It should make clear that, despite some focus on major gene effects that can be assigned to individual loci, there is widespread evidence for polygenic adaptation. Even where structural variants are known to have major effects, many other regions of the genome typically contribute to local adaptation. It would also be helpful to refer to theoretical expectations regarding distributions of effect sizes and clustering of locally-adaptive loci, with or without gene flow.

Reviewer #2 (Public Review):

Fuhrman et al. explore a fascinating system to study the evolution and genetic architecture of ecological adaptation in marine midges. They use a number of approaches including analyses of whole genome sequences and QTL mapping to explore population structure and the loci associated with the timing and mode of reproduction. I have some concerns about the analyses and interpretations which I outline below.

1) My primary concern is in the design and interpretation of the QTL analysis. The QTL approach used here has low power, both due to the sample size and the number of markers used (it looks like ~8 per chromosome). The authors use an analysis of the sex determining locus as a "control" but because of the complete heritability of this trait in most systems it is more of a straw man to me. The authors conclude that the architecture of the trait is polygenic based on this, but we are missing key information to evaluate this.

2) There are some issues with the presentation and interpretation of the population genetic analyses. Many assumptions are made about whether introgression or ILS occurred and there are statements that are not accurate about it being "impossible" to distinguish between these scenarios.

3) Some of the analyses associated with ecological adaptation that follow on the QTL results struck me as ad hoc and with the potential to lead to spurious results. I am not familiar with the BayPass approach but since it is the approach that explicitly accounts for population structure it seems the one that would be most appropriate for the authors to focus on in a revised manuscript. The use of phylogenetic windows that associate with ecotype is concerning to me as given the level of ILS and gene flow that appears to be present in this system is would be very challenging to distinguish signal from noise.

4) There were issues with the GO analysis that should be addressed. Because the gene universe used for GO enrichment is a subset of the full gene set, GO enrichment results will be biased. This will mostly lead to false positives (i.e. overrepresentation of a GO category due to evaluating a subset of genes that fall in that category).

Reviewer #1 (Public Review):

The authors define regulatory networks across 77 tissue contexts using software they have previously published (PECA2, Duren et al. 2020). Each regulatory network is a set of nodes (transcription factors (TF), target genes (TG), and regulatory elements (RE)) and edges (regulatory scores connecting the nodes). For each context, the authors define context-specific REs, as those that do not overlap REs from any of the other 76 contexts, and context-specific regulatory networks as the collection of TFs, TGs, and REs connected to at least one context-specific RE. This approach essentially creates annotations that are aggregated across genes, elements, and specific contexts. For each tissue, the authors use linkage disequilibrium score regression (LDSC) to calculate enrichment for complex trait heritability within the set of all REs from the corresponding context-specific regulatory network. Heritability enrichments in context-specific regulatory network REs are compared with heritability enrichments in regions defined using other approaches.

Reviewer #2 (Public Review):

In this manuscript the authors develop a method, SpecVar, to perform heritability estimation from regulatory networks derived from gene expression and chromatin accessibility data. They apply this approach to public datasets available in ENCODE and Roadmap Epigenomics consortia as well as GWAS phenotype associations in UK Biobank. It promises to be a powerful method to interpret mechanisms from genetic associations. Below are some strengths and weaknesses of the paper.

Strengths

- The method performs heritability enrichment on two major genomic data types: gene expression and chromatin accessibility.<br /> - This method leverages gene regulatory networks to perform the heritability estimation, which may better capture complex disease architecture.<br /> - The authors perform an extensive comparison to other LDSC-based approaches using different tissue datasets.

Weaknesses<br /> - This approach may represent a modest advance over existing LDSC methods when looking at other complex traits.<br /> - The authors only compare with LDSC using different functional annotations as input, which may not be appropriate. A more broad comparison with other heritability methods would be helpful.<br /> - The method seems to be applied to "paired" data, but this is still bulk profiles not paired single-cell RNA/ATAC data.

The authors successfully applied a regulatory network approach to improving the heritability estimation of complex traits by using both gene expression and chromatin accessibility data. While the results could be further strengthened by comparing them to other network and non-network-based methods, it provides important insight into a few traits beyond the standard LDSC model with different functional annotations.

Given that this method is based on the widely used LDSC approach it should be broadly applied in the field. However, the authors should consider adapting this to single-cell data as well as admixed human population genetic data.

Reviewer #3 (Public Review):

Identifying the critical tissues and cell types in which genetic variants exert their effects on complex traits is an important question that has attracted increasing attention. Feng et al propose a new method, SpecVar, to first construct context-specific regulatory networks by integrating tissue-specific chromatin states and gene expression data, and then run stratified LD score regression (LDSC) to test if the constructed regulatory network in tissue is significantly associated with the trait, measured by a statistic called trait relevance score in this study. They apply their method to 6 traits for which there exists prior evidence on the most relevant tissues in the literature, and then further apply to 206 traits in the UK Biobank. They find that compared to LDSC using other sources of information to define context-specific annotations, their method can "improve heritability enrichment", "accurately detect relevant tissues", helps to "interpret SNPs" identified from GWAS, and "better reveals shared heritability and regulations of phenotypes" between traits. However, I think it requires more work to understand where exactly the benefits come from and the statistical properties of their proposed test statistic (e.g., how to perform hypothesis tests with their relevance score and whether the false positive rate is under control). In addition, it's not clear to me what they can conclude about the shared heritability (which means genetic correlation) by comparing their relevance score correlation across tissues to the phenotypic correlation between traits.

They show that SpecVar gives much higher heritability enrichment than the other methods in the trait-relevant tissues (Fig. 2). The fold enrichment from SpecVar is extremely high, e.g., more than 600x in the right lobe of the liver for LDL. First, I think a standard error should be given so that the significance of the differences can be assessed. Second, it is very rare (hence suspicious) to observe such a huge enrichment. Since SpecVar is based on LDSC, the same methodology that other methods in comparison depend on, the differences to the other methods must come from the set of SNPs annotated for each tissue. I think it is important to understand the difference between the SpecVar annotated SNPs and those from other methods. For example, is the extra heritability enrichment mainly from the SpecVar-specific annotation or from the intersection narrowed down by SpecVar?

They propose to use the relevance score (R score) to prioritise trait-relevant tissues. In Fig. 3, they show tissue-trait pairs with the highest R scores, and from there they prioritise several tissues for each trait (Table 1). I can see that some tissue has an outstanding R score, however, it is not clear to me where they draw the line to declare a positive result. The threshold doesn't seem to be even consistent across traits. For example, for LDL, only the right lobe of the liver is identified although other tissues have R scores greater than 100, whereas, for EA, Ammor's horn and adrenal gland are identified although their R scores are apparently smaller than 100. It seems to me they use some subjective criteria to pick the results. It leads to a serious question on how to apply their R score in a hypothesis test: how to measure the uncertainty of their R score? What significance threshold should be used? Whether the false positive rate is under control? Without knowing these statistical properties, readers won't be able to use this method with confidence in their own research.

Another related comment to the above is to investigate false positive associations, they should show the results for all tissues tested to see if SpecVar tends to give higher R scores even in tissues that are not relevant to the trait. It would also be useful to include some negative control traits, such as height for brain tissues.

Fig. 3 shows that tissues prioritised by LDSC-SAP and LDSC-SEG seem to make less sense than those from SpecVar. However, some of the results are not consistent with the LDSC-SEG paper (Finucane et al 2018). For example, LDL was significantly associated with the liver in Finucane et al (Fig. 2), but not in this study. How to explain the difference?

The authors highlight an example where SpecVar facilitates the interpretation of GWAS signals near FOXC2. They find GWAS-significant SNPs located in a CNCC-specific RE downstream of FOXC2 and reason these SNPs affect brain shape by regulating the expression of FOXC2. I think more work can be done to consolidate the conclusion. For example, if the GWAS signals are colocalised with the eQTL for FOXC2 in the brain. Also, note that the top GWAS signal is actually on the left of the CNCC-specific RE (Fig. 4b). A deeper investigation should be warranted.

They show that SpecVar's relevance score correlation across tissues can better approximate phenotypic correlation between traits. However, the estimation of the phenotypic correlation between traits is neither very interesting nor a thing difficult to do (it can be directly estimated from GWAS summary statistics). A more interesting question is to which extent the observed phenotypic correlation is due to common genetic factors acting in the shared tissues/cell types/pathways/regulatory networks between traits. Note that in their Abstract, they use words "depict shared heritability and regulations" but I don't seem to see results supporting that.

Line 396-402: "For example, ... heritability could select most relevant tissues ... but failed to get correct tissues for other phenotypes ... P-value could obtain correct tissues for CP ... but failed to get correct tissues for ... SpecVar could prioritize correct relevant tissues for all the six phenotypes." Honestly, I find hard to judge which tissues are "correct" or "incorrect" for a trait in real life. It would be more straightforward to compare methods using simulation where we know which tissues are causal.

Reviewer #1 (Public Review):

Yuan et al. propose that the magnesium transporter unextended (uex) controls Drosophila sleep via Mg2+ efflux, Ca2+-dependent CREB signaling, and a CNK-ERK pathway. UEX protein levels display daily oscillations in fly heads, whereas UEX-depleted flies show long sleep with low levels of Ca2+ and synaptic plasticity. Transgenic expression of wild-type UEX or the mammalian uex homolog CNNM1 possibly rescues the uex mutant sleep, supporting their evolutionary conservation. UEX forms a protein complex with the ERK signaling suppressor CNK, and UEX depletion appears to de-repress ERK activation. As expected, pan-neuronal CNK depletion phenocopies uex mutant sleep. Taken together, the authors suggest a novel mechanism whereby magnesium shapes animal sleep through the specific MAPK pathway. Overall, the authors reported quite a few exciting phenotypes in UEX-depleted flies using a range of analyses (e.g., behaviors, gene expression, Mg2+/neural imaging, and biochemistry). However, evidence for their causal link to sleep regulation is missing, and some key conclusions remain justified by more rigorous analyses. It is noteworthy that Wu et al. have previously demonstrated the Mg2+ efflux transporter activity of UEX and mapped its memory-enhancing function to a specific group of adult neurons in the fly brain (https://pubmed.ncbi.nlm.nih.gov/33242000/). Given the intimate interactions between sleep and memory, these findings may have broad implications in sleep-relevant physiology and disorders. However, a number of important control studies and statistical assessments are not included, but are necessary to conclusively interpret the data.

Reviewer #2 (Public Review):

The authors described the analysis of a magnesium transporter UEX as a sleep-regulating gene in Drosophila melanogaster. They also proposed the UEX regulates sleep through its downstream Ca2+-dependent CREB signaling and a CNK-dependent ERK pathway. The involvement of UEX in sleep regulation is novel and potentially interesting, but the data presented in the manuscript does not fully support the conclusions the authors proposed. Most of the data are derived from elav-GAL4, which is a non-specific pan-neuronal GAL4 driver. Since as the authors described, UEX functions to alter sleep in various brain regions, the relationship between UEX and other molecules in Ca2+-dependent CREB signaling and a CNK-dependent ERK pathway may be indirect in the sleep-regulating pathway, which means it may involve multiple regions of the brain using different pathways, and the sleep phenotype is the summation of different functions of UEX.

Reviewer #3 (Public Review):

In this manuscript, Yuan et al. examined the relationship between a magnesium transporter and sleep behavior. They find that the knockdown of a magnesium efflux transporter (uex) in neurons increases bout length of inactivity and recovery activity of the flies with neuronal knockdown of uex with a human homolog CNNM1. The authors suggest a model in which Mg2+ promotes sleep through the inhibition of Ca2+ levels that are wake-promoting in the mushroom body and PDF+ neurons. Overall, the idea explored here that ion homeostasis in the neurons contributes to behavior is an area that is timely and interesting to the neuroscience community. The transgenic lines of human CNNMs could be a useful tool for scientists studying metal transport and ion homeostasis in flies. Unfortunately, the results of the experiments do not entirely support the authors' conclusions.

The authors fall short of showing that the increased inactivity is sleep behavior as Mg2+ changes in neurons could be affecting the mobility of the fly. To validate that the increased inactivity is sleep, the authors should have used a combination of negative geotaxis, arousal threshold, or multibeam/video monitoring. Another characteristic of sleep is the presence of compensatory rebound following sleep deprivation. Here, when the authors sleep deprive the flies with uex knockdown, the flies do not have increased rebound sleep over control flies. Together the current data suggest that the increased inactivity may not be sleep and more evidence to the contrary should be shown.

In Fig 1, the authors show that there is a huge developmental effect on rest:activity rhythms when using the elav-gal4>uex RNAi compared to the inducible elav-geneswitch > uex RNAi, but in Fig 2, the authors use gal4 drivers rather than an inducible system. Use of an inducible system such as geneswitch, AGES, or TARGET is important to rule out developmental effects. Again, in Fig 4, the authors use the gal4 rather than the geneswitch for knocking down the other magnesium channels/transporters so it is unclear whether any sleep increase may be due to the role magnesium plays in development. In Fig 6 elav-gal4 was also used instead of GS. According to previously published work on UEX in fly neurons (Wu et al. eLife 2020 PMID: 33242000), UEX is primarily in the mushroom body and much lower expression in the PI or PDF+ neurons of the adult brains, further suggesting that sleep increases in the PDF+ and PI gal4s driving uex RNAi may be developmental.

From this work, the authors suggest that Mg2+ is sleep-promoting, and in the absence of uex efflux transporter to remove the Mg2+, Mg2+ increases to the point of inhibiting Ca2+, a wake-promoting signal; however, not all the Mg2+ transporters assayed efflux out Mg2+, but rather regulate the influx of Mg2+ into the cells. If a channel regulating Mg2+ influx is inhibited, the prediction would be that Mg2+ would be decreased and thus the flies should sleep less. But in Fig 4H that was not the case. All the Mg2+ transports/channel RNAi lines increased sleep. The authors do not reconcile this data with their proposed model. It is possible the Mg2+ transporter RNAi lines result in increased Mg2+ in the relevant neuronal subgroup in which case Mg2+ levels should be measured in the RNAi lines.

Reviewer #1 (Public Review):

Modified rabies viral vectors allow high throughput mapping of neuronal circuits with cell type specificity. However, lack of standardization in this field limits extrapolation of useful information, beyond the identity of anatomically connected regions, such as differential input densities and connectivity motifs. in this manuscript, Tran-Van-Minh et al., attempt to develop a statistical approach which will allow consolidation of new, as well as previously-acquired datasets, to yield biologically significant insights into the logic underlying rabies vectors' expansion from single starter cells.

This question the authors address is of high importance and the presentation of this manuscript is timely, as rabies tracing experiments increase at an exponential rate. The authors provide a largely complete description of the main pitfalls and caveats in current analysis approaches and common misconceptions in the interpretation of results. In addition, they correctly diagnose the potential of this methodology to extract pertinent information from such experiments and provide the reader with useful tips on how to better design, analyze and interpret them.

While such a paper is undoubtedly called for, and has the potential to substantially improve circuit mapping experiments, with little cost to the experimenter, there are a few critical flaws in their premises, which will limit a widespread adoption of their analysis approach. While the authors discuss caveats in design and interpretation of results, they do not implement these suggestions into their own experiments, such as the need for accurate estimation of starter cells and automated cell counting which is not biased by strong signal coming from dendritic arbors/axons in densely-labeled regions. While the authors claim that the reduction in residuals and relative conformity across experiments following log-transformation of n(i) and n(s) shows that their approach is robust, the large degree of variability across experiments, the datasets of some are biologically implausible, showing a decrease in n(i) as a function of n(s), suggests that this transformation disposes of useful information, which might help detect anomalies in data acquisition. The fact that the most rigorously-acquired dataset which was presented by the Allen Institute (BRAIN Initiative Cell Census Network, Nature, 2021) is the only one which, does not comply with the transformation, attests further to this caveat. This is probably the reason why the estimated number of individual neurons retrogradely labeled by a single starter cell (~1400) is more than an order of magnitude higher than any previously-published estimation, including those which include tracing from single-cell, and is highly unrealistic.

In addition, the model selected by the authors to fit the various datasets does not take into proper account saturation of n(i), as all proposed functions are growing functions. Here, the most suitable model which should be used to describe the nature of RV expansion is a cumulative distribution function, while the rest are either private cases (e.g. linear fit given low n(s) or zero divergence) or are biologically unrealistic (e.g. exponential fit). Again here, the only dataset which appears to fit this function the best comes from the BRAIN Initiative Cell Census Network (Nature, 2021).

In conclusion, while such work is called for and has the potential of becoming a staple in the connectivity toolbox, many of the premises presented here will need to be significantly adjusted, before the approach could be put into widespread use.

Reviewer #2 (Public Review):

Appropriate brains functions require the precise wiring of vast numbers of synaptic connections in broadly distributed neural circuits. Monosynaptic retrograde rabies virus tracing has become a common approach in neuroscience to assay presynaptic inputs into a given postsynaptic region. However, quantification and interpretation of rabies tracing data is confounded by the lack of uniform and appropriate measuring approaches across different studies and laboratories, as well as the lack of knowledge of the trans-synaptic transfer properties of different rabies viruses in various brain regions.

The current study comprehensively applies mathematical approaches to an example rabies tracing dataset in layer 5 of mouse visual cortex, as well as previously published datasets, to propose more standardized methodologies for rabies data analysis and interpretation. The major strength of the study is the rigorous and unbiased mathematical approaches applied to their data and a range of previously published studies in the field. Inclusion of representative image data would be helpful for readers and would further strengthen the study. Given the ubiquitous use of rabies virus tracing in the field, yet lack of insight into this crucial aspect of its use, this will provide a useful resource for the neuroscience community.

Reviewer #1 (Public Review):

The manuscript by Chen and colleagues contains a number of important findings. First, they showed with careful imaging, cell cycle characterization, and models that replisomes of fast-growing E. coli form factories and super factories (factories involving cousin replisomes), they estimated that factories split after the replication of 1/3 of the chromosome. Second, they used a replication fork block to monitor the interdependence of replisomes of factories. Third, they show that orphaned replisomes replicate slowly and frequently require a repair process to complete the replication cycle. This is the first characterization of an advantage of replicating bacterial genomes in a factory rather than as independent replisomes. This is an important discovery that simultaneously confirms the existence of DNA factories, a much-debated topic, and reveals one of their functions. The existence and characterization of factories were mostly addressed through imaging methods; here the combination of imaging with molecular genomics assays is a real asset for the manuscript.

Reviewer #2 (Public Review):

In most organisms, DNA replication is restricted to a relatively few cytologic structures termed replication factories. Studies indicate that such factories contain multiple replication forks. Although these observations suggest that replication fork colocalization has functional significance, the biological rationale for replication factories has remained elusive. To address this issue, the current study utilizes E. coli, a bacterium with a circular chromosome that replicates its DNA bidirectionally from a single origin of replication. During the first half of an E. coli DNA replication cycle, these two forks spatially co-localize into a single "factory." The experimental plan of this study is to block one of the two replication forks at various informative genomic locations and see if such blocks affect the progression and efficiency of the non-blocked fork. Using this approach, the authors find that blocking the progression of one fork at an early point in replication slows the progression of the corresponding unblocked fork and considerably increases its probability of replication fork collapse. This study considerably advances the field by demonstrating for the first time a possible biological purpose behind the replication factory - that factory formation in some yet unknown manner helps coordinate and stabilize bidirectionally oriented replication forks.

Although others have tried to study replication factories using similar experimental logic, this well-written study by Chen et. al. examines the problem with higher sensitivity and resolution using a very elegant and synergistic approach that combines 3-dimensional microscopy, deep DNA sequencing, and old-fashion cell biology with a series of carefully engineered E. coli strains containing a conditional replication fork block in different informative genomic locations. These approaches in combination allow one to make a direct experimental correlation between cytologically defined replication factories (3D fluorescent imaging of labelled replication factors with image deconvolution), and fork progression via an analysis of copy number (genomics). Their experimental approach and accompanying analysis pipeline will be of general interest to the research community.

In addition to a very careful analysis of factory formation that helps resolve several previous discrepancies on this subject, the authors used this approach to show that blocking one replication fork early in DNA replication coordinately decreases both the rate of fork progression and the level of fork stability in the unblocked sister fork. This conclusion is supported by their genomic analysis that shows the velocity of the unblocked fork slows when the other fork is blocked. To further elucidate this observation, the authors examined the likelihood that elevated replication fork collapse contributed to the decreased fork rate. As the restart of a collapsed replication fork depends upon genetic recombination, the role of recombination in fork progression in this situation was examined. Two questions were asked in this system: 1) Is the progression of the unblocked fork specifically reduced in the absence of genetic recombination (with a mutation in RecB)? and 2) Using chromatin IP, does this slow fork specifically recruit binding of a catalytically-dead Holliday-junction resolvase (RuvC)? The results from both experiments strongly support the conclusion that replication factories in some yet unknown manner are needed to stabilize the bidirectionally orientated replication forks. Although this strong conclusion indicates that the unblocked fork specifically creates DNA lesions, this approach does not unambiguously distinguish between damage resulting directly from fork collapse and damage caused by other aspects of defective DNA replication.

Reviewer #1 (Public Review):

In this study, the authors investigate multi-attribute economic decisions in humans. More specifically, they investigate the impact of adding a decoy option to a binary choice, the effect of which has been debated in the previous literature with recent studies finding effects in different directions. By re-analyzing large datasets from some of these studies and by applying computational modeling to the data, the authors propose that a subjective encoding rule comparing attributes separately, and not computed as one multiplicative value, explains participants' behavior on different levels of granularity. Context effects were well captured by a selective integration model. The work has many positive features, including praising open science, the analysis of the potential confounds of previously used designs, and both quantitative and qualitative comparisons of several well-justified models.

Reviewer #2 (Public Review):

Summary

This manuscript re-examines a distractor effect of decoy options on risky choice reported in previous research by re-analyzing data from previously published experiments that reported these effects. The previous studies reported that adding an unavailable decoy option to a choice set consisting of two available risky choices increased the discriminability between the two available risky choices, especially when the expected value difference between the two available risky options was small, by increasing the expected value of the unavailable distractor. The authors argue convincingly that the distractor effect is an artifact of two other confounding factors: one is that there is a covariance between the distractor's expected value and the subjective utility difference between the two targets; the second is that the expected value of the distractor alternative could covary with its relative position in the reward-probability space, and its relative position in the multi-attribute space could induce a well-known context effect. The first alternative explanation was established by comparing binary choice with and without the distractor present and finding the same effect in binary choice without any distractor present. The second was established by showing that the distractor effect was most pronounced when it was close to the higher-value target in the multi-attribute space, inadvertently producing a previously well-known attraction effect. These results clarify the role that an unavailable distractor plays in decisions between two risk alternatives.

Evaluation

This is a very comprehensive and somewhat complex manuscript. It does a good job of detective work to get at the bottom of the distractor effect reported in previous articles (including this journal). It essentially contains two main sections. The first section is designed to establish the conclusion that the distractor effect is an artifact of a confounding variable, the additive utility difference between the two available choices, and generalized linear model analyses were used to make this point. The second section is designed to show that the distractor effect also covaries with a well-known context effect called the attraction effect, and they use mathematical modeling of choice and response time to understand this part. Different hypotheses about how the risk information was integrated tested by varying how the drift rate was calculated in a racing drift diffusion model for choice and response time. In particular, they contrasted a divisive expected value type of integration hypothesis with a selective attention type of additive utility hypothesis. They concluded from these mathematical modeling analyses that an additive utility model for integrating the risk information was used in these experiments to evaluate the risky gambles.

Strengths the manuscript makes a very compelling case for the conclusion that the distractor effect was confounded with the additive utility difference between the available alternatives. This was achieved comparing the binary choice results, with and without the distractor, and finding little or no difference between these two conditions. The manuscript is also commendable for its rigorous mathematical modeling of the context effect of the distractor on the binary choices when the distractor was present.

One weakness is that the contribution is somewhat narrowly focused with respect to the phenomenon that it addresses - the distractor effect in risky choice. However, I do think it is important for understanding this particular phenomenon. The other main weakness is the complexity of the manuscript. The manuscript is very long with numerous detailed statistical analyses and computational modeling analyses. Generally speaking, the authors did a good job describing and summarizing all these analyses, and they made effective use of figures to illustrate the ideas and conclusions. However, there are several spots that are somewhat difficult to follow (see specific comments), and the reader is pressed to think pretty hard and fairly long and with a lot of effort to absorb all the points.

One other major concern I have regards the conclusion that the participants in these studies use an additive rather than a multiplicative rule to integrate the risk information. The additive rule is problematic in general because it fails to predict the reversal in the effect of probability on payoffs when the payoffs change sign. More specifically, increasing the probability of winning increases the probability of choosing an option when the payoff is positive, but the effect reverses when the payoff is negative. One needs to impose some pretty ad hoc assumptions to make the additive model account for this fundamental interaction between probability and payoff. Of course, the experiments reported here did not include negative payoffs, and so didn't run into this problem. In fact, when the payoffs are positive, it is possible to transform the multiplicative model to an additive model by a log transform. This transformation is only possible for the simple type of gamble investigated in this manuscript - a single amount to win with some probability of winning, otherwise win or lose nothing. If the gambles involved more than one outcome, then the theorist needs to deal with a sum of products and the log transform is no longer possible. For these reasons I am very skeptical about the general application of a summation rule for probability and value in risk choice. The authors do address this issue to some extent. They point out the abundance of other research supporting a multiplicative rule, and they speculate that the additive rule may have occurred within the restrictions of this special situation. The latter discussion is a good start, but I suggest that the authors discuss this fundamental issue in more depth.

Reviewer #3 (Public Review):

The authors re-analyze published datasets of value-based decision making with and without unavailable distractors, i.e., with ternary and binary choices. By setting the accuracy of binary choices as baseline, they show that a phantom distractor effect appears even without the presence of distractor. This result suggests that distractor effects could be partially explained by target-related covariation. They test how reward and probability are integrated under their datasets. The additive model wins over the multiplicative model in predicting both true and phantom distractor effects in binary choices. Then they test how multiple alternatives interfere with each other in ternary choices. They find that the model with the assumption of rank dominance wins over normalization models. They also replicate the correlation between individual-level decision noise and distractor-related parameters, which implies distractor effects can be emergent properties from a normative decision policy.

I see three strengths of this work.

First, the highlight of this work is that they explore the integration of the multi-attribute and multi-alternative information by bridging distinct distractor effects and providing a unified explanation. The result has a potential impact on a neuroscience topic that attracts a lot of attention in recent years-how the brain represents multiple features and items (e.g. Rigotti, Nature, 2013; Flesch et al., Neuron, 2022; Fusi et al., Curr. Opin. Neurobiol., 2016).

Second, the results of the trial-by-trial baseline approach warn that, due to the complexity of multi-attribute and multi-alternative problem, the studies of the effect should be designed and analyzed with care to prevent possible confounding factors from high dimensionality.

Third, besides static models that can only account for accuracy, the authors implement a dynamic accumulator frame to test all hypotheses. The dynamic accumulator models take into account both accuracy and reaction time. This approach strengthens their model comparison.

Overall, I think this paper is an impressive piece of work that clarifies the true effect of distractors by well-designed analysis and provides a model that bridges distinct distractor effects. Their analysis supports their claims.

Reviewer #1 (Public Review):

This manuscript provides the first cellular analysis of how neuronal activity in axons (in this case the optic nerve) regulates the diameter of nearby blood vessels and hence the energy supply to neuronal axons and their associated cells. This is an important subject because, in a variety of neurological disorders, there is damage to the white matter that may result from a lack of sufficient energy supply, and this paper will stimulate work on this important subject.

Axonal spiking is suggested to release glutamate which activates NMDA receptors on myelin-making oligodendrocytes wrapped around the axons: the oligodendrocytes - either directly or indirectly via astrocytes - then generate prostaglandin E2 which relaxes pericytes on capillaries, thus decreasing the resistance of the vascular bed and (presumably) increasing blood flow in the nerve.

Strengths of the paper

The paper identifies some important characteristics of axon-vascular coupling, notably its slow temporal development and long-lasting nature, the involvement of PgE2 in an oxygen-dependent manner, and a role for NMDARs. Rigorous criteria (constriction and dilation of capillaries by pharmacological agents) are used to select functioning pericytes for analysis.

Weaknesses of the paper

The study focuses exclusively on pericytes. It would have been interesting to assess whether arteriolar SMCs also contribute to regulating blood flow.

The slow (~10 minutes) time scale of the responses seen is remarkable when compared to grey matter neurovascular coupling which occurs in seconds. The authors suggest that this reflects movement of messengers along the nerve, but the action potential moves so rapidly down the nerve that it will activate the release of vasodilators essentially at the same time everywhere along the nerve, and there will be no concentration (or voltage) gradient to drive such a movement of messengers (unless there is a spatially localized unique site in the vascular bed where propagating responses are generated). Thus it remains unclear why the responses are so slow.

The activity-evoked dilation is thought to reflect PgE2 release at what is probably a physiological O2 concentration, but not at the hyperoxic 95% O2 used in most of the experiments. The involvement of NMDA receptors is apparently only shown (using OL-specific receptor subunit deletion) at the unphysiological high O2 level. This raises two questions: are NMDA receptors also involved in the response at low O2 (as schematized in Fig 6), and what is the messenger downstream of NMDARs at high O2 (NO would be an obvious candidate, previously shown to contribute to NVC in the grey matter).

Reviewer #2 (Public Review):

This paper describes a new concept of "axe-vascular coupling" whereby action potential traffic along white matter axons induces vasodilation in the mouse optic nerve. This is an initial report dissecting some of the mechanisms that are undoubtedly complex as in gray matter NVC. I like the novel AVC concept.

Some minor corrections and suggestions:

1) p3: "The cerebral white matter (WM) in the adult brain is particularly vulnerable to cerebrovascular diseases such as ischemia":this may be misleading since WM is actually far less vulnerable to ischemia than gray matter

2) p4-5: "The ON exhibited a median of 175.8 pc/mm2 {plus minus} 35.7 pc/mm2, more than twice the number of pericytes observed in the corpus callosum [...] and lower than cortex ": this seems incorrect, the density in cortex is not significantly different than ON

3) p5: what is the unit 'pc'? (A cellI I presume but please define at first use)

4) p7 : "To evaluate if pericytes have and retain their contractile properties, we applied the vasoconstrictor U46619 (100 nM) for 15 min followed by acetylcholine (ACh - 100 μM) as a vasodilator": if they saw an effect, how would the authors know these were mediated my pericytes and not smooth muscle cells?

5) in Fig. 3i there is a sharp step after U466... application: is this an artifact or evidence of a delayed constriction? Could a clearer trace be shown that does not confuse?

6) Fig. 4I: what does "20% CAP (norm)" mean? Why not just mV for the y-axis? Also what pulse width was used for stimulation?

7) Fig.5: it would be good to show both the CAPs (at various frequencies) and the vasorespones at 95% vs 20% O2. In particular, are the ONs able to sustain conduction at the higher frequencies (showing overlays as in 4I), and if not, could this at least partially account for the different responses at the two O2 levels?

8) Fig. 6G,H is somewhat misleading as it implies no change in AVC, at odds with 6E. Suggest some clearer labeling to reduce confusion surrounding this very important point.

9) P18 authors state radius of a MON is 150um but on p4 they say "150 μm - 200 μm thickness", pls clarify.

10) p19-20: as part of their second messenger speculation authors may also want to include NO that has been shown to induce important effects in WM. Indeed, testing the tat uncoupling peptides could be interesting to see of oligodendroglial NMDARs have a similar singling arrangement with NOS as do neurons. This may have important implications for WM neuroprotective strategies in stroke that have typically focused on gray matter mechanisms.

Reviewer #3 (Public Review):

This study used the ex vivo optic nerve preparation from adult mice to examine the organization of blood vessels and the mechanisms or neurovascular coupling (NVC). Strengths of the study include the benefits of the isolated preparation, which allows visualization of vessels and pericytes with high resolution and control over axonal activity and the extracellular environment, and the elegant analyses performed. Imaging at high resolution is critical, because vessel diameter changes can be small and slow to develop. The authors leverage this preparation to define the organization of blood vessels and pericytes in the nerve. They then examine the extent of NVC, showing that some aspects appear to be distinct. In particular, dilation does not present rapidly (over minutes) during axon stimulation, but rather emerges after the stimulation, increasing progressively over tens of minutes. It is similarly dependent on oligodendrocyte NMDARs and prostaglandin E4 receptors, but the latter only appears to be engaged during low oxygen conditions. There are several notable limitations of these studies. Less is known about NVC in the intact optic nerve, so it is unclear how well this preparation mimics the in vivo environment. All studies of NVC were performed in the presence of U46619 (an agonist of prostaglandin H2 receptors) to pre-constrict the vessels, which may interfere with NVC. The degree of vessel change was small and slow to develop, and the magnitude and timecourse of the dilation were not closely linked to the stimulation frequency, raising concerns about tissue stability and cell viability. Finally, the studies examined the role of oligodendrocyte NMDARs in NVC using a conditional gene knockout strategy to inactive the NR1 subunit in these cells. To control for possible developmental effects, additional studies could be performed using acute application of NMDAR antagonists, as this preparation contains only neuronal axons, and a further analysis of vessel structure and pericyte organization should be performed using the methodologies developed to characterize their properties in control nerves. Importantly, extracellular stimulation of the nerve, which triggers near simultaneous activation of axons may not mimic activity patterns in these nerves that occur during vision.

Reviewer #1 (Public Review):

The goal of this work is to study whether sleep and wake regulate cerebellar structural plasticity. Scanning electron microscopy and 3D reconstruction were used to characterize structural changes in spines and synapses in the mouse cerebellar cortex. The net number and size of synapses did not change between sleep and wake. However, the number of small portion of spines ("naked" spines without synapses) increased during sleep, and the number of branched spines (contains more than one synapse) increased during wake.<br /> The methodological approach (scanning electron microscopy) is laborious but adequate. However, it cannot follow the same synapses longitudinally, and live imaging, even only in superficial regions, is an ideal approach to achieve the goals.

The results did not find changes in the total number of spines or synapses during sleep and wake. Structural changes were found in small number of spines lacking a synapse. Whether these changes are functionally important is unclear. The results support circuit-specific, rather than global, effect of sleep on synaptic plasticity.

The role of sleep range from cellular maintenance to memory consolidation. Multiple evidence suggests that sleep regulates synaptic plasticity. Although this work did not attempt to understand the functional role of sleep in regulating learning and memory, it discovered that sleep promotes synaptic pruning in specific circuits of the cerebellum. Future similar anatomical studies in additional brain regions, including excitatory and inhibitory circuits, combine with physiological and behavioral assays are expected to provide insights to the role of sleep in regulating synaptic plasticity, learning and memory.

Reviewer #2 (Public Review):

This study used serial block face scanning electron in the mouse posterior vermis. The analysis showed that Purkinje cell "naked" spines, are ~5% of all spines after wakefulness but grow to ~10% of all spines after sleep. Additional analysis revealed that the observed sleep-wake difference is best explained by a change in the number of "branched" synapses, Branched spines are proposed to convert to single spines during sleep. It is speculated that sleep promotes the pruning of branched synapses. This is a beautiful study that must have taken considerable effort in addition to expertise. No such data exist in the literature and the observations are interesting in light of the prior data from cortex published by the same group.

Major critique:

• The abstract and in particular the second half is very difficult to follow and should be rewritten. It might be easier to follow if the authors compare to previous work in cortex<br /> • The figures are very well done. However, I am missing a model diagram explaining the model proposed for changes in naked spine during the sleep-wake cycle and the proposed functional consequences.<br /> • The authors have previously studied the effect of sleep on the ultrastructure of glial cells, astrocytes and oligoes. This might be a separate study, but it would be of interest to discuss the role of Bergmann glial cells in synaptic plasticity. One major difference is that Bergmann glia express AMPA receptors, unlike cortical astrocytes and these are important for the proximity of astrocytic processes to synapses.

Reviewer #1 (Public Review):

Zebrafish have become a well-established model organism for studies of damage and repair in hair cell sensory organs due to organ accessibility and robust mechanisms for repair and regeneration. While lateral line organs in zebrafish are widely used to study hair cell physiology, zebrafish also contain hair cell organs in their inner ears that have the potential to be more comparable to mammalian counterparts. Using single-cell RNA seq in embryonic through adult stages, this study found that hair cells and supporting cells of the zebrafish inner ear are distinct from those of the lateral line. Additionally, they identified distinct cellular subtypes within the maculae and cristae of the ear.

This work provides some novel findings, including identifying distinct markers in the macula and crista hair cells and supporting cells as well as detecting a domain in the zebrafish utricle that coincides with features of the striolar region in mouse utricle. However, while much of the focus of in situ expression analysis was the spatial separation of calcium-binding proteins capb1b and capb2b in the maculae, it was not clear how Capb1 & 2 expression corresponds to striolar and extrastriolar regions in the mammalian utricle, where Capb2 is expressed throughout. In addition, the authors assumed the zebrafish utricle and saccule perform similar functions (i.e. hearing) and contain hair cells with similar frequency tuning. It would improve this study to consider the unique functions and frequency sensitivities of the utricle and the saccule, given that different expression of voltage-gated channels may give insight into the specific physiology of these two sensory organs.

Reviewer #2 (Public Review):

This is an interesting study with a primary value in generating new transcriptional data sets for zebrafish hair cells and non-sensory cells in the inner ear. The data will, no doubt, be useful for future studies of hair cell function, development, and regeneration. The data also reveal transcriptional differences between similar cell types in different structures and transcriptional similarities between fish and mammalian cell types within analogous structures. Overall the strength of evidence in support of the results is strong.

Reviewer #3 (Public Review):

The authors describe the use of single-cell RNA sequencing (scRNA-seq) of the zebrafish inner ear at various stages ranging from embryos to adults and they characterize 3 major cell types: supporting cells, progenitor cells, and hair cells. While scRNA-seq experiments have been performed on adult inner ear tissues and the lateral line previously, a detailed characterization of the cellular subtypes in the inner ear at the embryonic through adult stages has not been accomplished before at the transcriptomic and spatial levels and is an important contribution to the field.

In the manuscript, the authors describe the transcriptomic profiles of the inner ear at single-cell resolution followed by spatial validation. In agreement with previously published research, they identify 3 major cell types in the inner ear and use advanced bioinformatic analysis to identify distinct support and hair cell subtypes that reside in the hearing vs balance organs. They elucidate the transcriptomic differences between support and hair cell types that reside in the larval lateral line vs inner ear and demonstrate that these systems are different. Finally, they provide the groundwork for comparisons between zebrafish and mouse transcriptomic profiles and show conservation in the hair cell population. Most importantly, the authors validate their transcriptomic sequencing findings at the single-cell level with spatial information in the inner ear tissues using in situ hybridization assays.

The work performed takes several stages of inner ear development as well as sub-organ dissections coupled to scRNA-seq to carefully identify key cell types and map them to their matching mouse counterparts (when they exist). This work represents the groundwork for many comparative studies across species at the molecular level.

Reviewer #1 (Public Review):

Here, Sreejith et al. studied the mutant phenotypes of Drosophila c19orf12 homologs, CG3740 and Nazo (CG11671). They successfully discovered the tissue-autonomous role of Nazo in maintaining gut lipid droplets, although the underlying molecular mechanism remains elusive.

1. The phenotypic characterization of Nazo mutants is quite solid. The authors show the gut lipid droplet phenotype in mutants under various conditions (normal, starved, tissue specific rescued, HFD, HFD and trace, etc). It is convincing that Nazo functions autonomous in enterocytes to maintain gut lipid droplet pool.<br /> 2. The finding that Nazo mutant are sensitive to oxidative stress is also consistent with the previous role of c19orf12.<br /> 3. The underlying mechanism of how Nazo mutant/knockdown causes gut lipid droplet depletion is very preliminary. The authors proposed that Nazo affects the level of lipid droplet protein Plin2, which subsequently affects the Brummer lipase-mediated lipolysis, determining lipid droplet abundance. What is the mechanistic link between Nazo and Plin2, if the proposed mechanism is correct? It is well-known that the protein level of Plin2 is associated with the abundance of lipid droplets. Therefore, it is not surprising that Plin2 level is reduced in gut lysate of Nazo mutant (Figure 7C). Along the same line, it is not surprising that RNAi of Bmm rescues the depleted lipid droplet phenotype of Nazo mutants.<br /> 4. In figure 7C, the reduction of Plin2 is marginal. Whether Plin2 partial loss-of-function has the same phenotype?<br /> 5. The ER-LD contact site localization of Nazo need to be strengthened. Is the HA-tagged Nazo functional? It the localization Drosophila protein specific?<br /> 6. Does Nazo interact with Plin2?

Reviewer #2 (Public Review):

The authors present a genetic, physiological and cellular analysis of Drosophila mutants lacking one or both of the Drosophila homologs of the functionally uncharacterized c19orf12 gene. This gene is mutated in patients suffering from a familial neurodegenerative disorder called Neurodegeneration with Brain Iron Accumulation (NBIA).

To this aim the authors extend a previous study based on in vivo RNAi in Drosophila by generating deletion mutants for nazo and CG3740, which encode the two fly c19orf12 homologs. Nazo single and nazo CG3740 double mutant flies are viable but show lifespan reduction and compromised climbing ability.<br /> While these results are generally credible they are methodologically not sound.<br /> The authors compare to "WT" controls but the method section refers to no wild type but rather to a w[1118] mutant, which is widely used as control. However, there is no record that the nazo and/or CG3740 mutants have been backcrossed in this control background. Statistics is missing on lifespan experiment and the documented climbing distances are not credible (26cm compared to 4cm in 4 sec for controls in Ref. 46).

Lifespan reduction and climbing deficits at mid-age can be indicative of neurodegeneration or of developmental metabolic defects alike. The observed early-age mortality argues for a developmental rather than a degenerative effect. Comparing climbing capability at young age can sort this out and should at least be discussed.

Surprising for genes, implicated in neurodegeneration, fly c19orf12 homologs are (predominantly) expressed in the gut. Accordingly, the authors performed comparative gut transcriptome analysis to find a number of lipid metabolism genes regulated. Microscopic inspection revealed lower gut lipid droplet (LD) abundance in nazo single and nazo CG3740 double mutants compared to controls. How they might be related to the regulated lipid metabolism genes remains unaddressed.

The authors exclude an implication of the (gut) microbiome / the immune system, as well as abnormal food intake and of fatty acid absorption on this phenotype.<br /> Notably, the assay for fatty acid absorption is neither described in the methods section nor referenced or validated.

Next the authors convincingly demonstrate that the nazo function on LDs is cell-autonomous in the gut by enterocyte-targeted expression of (i) a new (unvalidated) RNAi transgene and (ii) a nazo transgene in the nazo mutant background. To what extent the rescue of nazo function in the enterocytes reverts representative mutant phenotypes such as lifespan reduction, climbing ability or adipose tissue lipid storage remains unaddressed.

The authors demonstrate that adipose tissue LDs and- correspondingly - organismal storage fat content is reduced in nazo deletion and global knockdown mutants, which causes starvation sensitivity. The central question, however, remains open: is this due to nazo function in the adipose tissue (where human c19orf12 is highly expressed) or an indirect effect of compromised nazo in the gut?

The authors report some additional nazo/CG3740 mutant phenotypes such as oxidative stress sensitivity, elevated body H2O2 and an inflated crop in response to high fat diet (HFD). While these are interesting observations, they altogether do not advance the understanding of the gene(s) function(s). On the contrary, it appears counterintuitive, that flies with inflated crop on HFD due to a gut peristaltic defect (data not shown), manage to dynamical accumulated LDs in the gut as witnessed by Fig. 6B.

Entry point to the mechanistic understanding of Nazo is the subcellular localization of the protein. The authors use overexpression of a (functionally unvalidated) tagged-Nazo in Drosophila tissue culture cells supplemented with fatty acid to trigger LD formation, to show that Nazo locates to LD contact sites of the ER. While this tissue culture system is widely used, in vivo data are required to substantiate this central finding. The myc-tagged, functionally validated rescue transgene offers an obvious tool for such an analysis.

Next the authors demonstrate that the gut LD storage capacity is not generally limited in nazo mutants but can be reversibly expanded by dietary intervention. Based on this result the authors favour lipid catabolism over anabolism dysregulation to explain the LD depletion phenotype of nazo mutants and turn to genetic experiments to substantiate this hypothesis.

Ubiquitous knockdown of Brummer/DmATGL is the only one out of three lipases tested (data of one not shown), which suppresses the nazo/CG3740 LD phenotype. The informational value of these experiments is compromised by the lack of essential controls; most importantly the consequences on gut LDs of knocking down these lipases in flies with normal nazo/CG3740 function. Also, ubiquitous lipase knockdown (instead of gat-targeted) limits the interpretation as indirect effects on gut LDs cannot be excluded.

Overexpression of Perilipin-2, which shields LDs, is conceptually similar to the down-regulation of lipases. Accordingly, the authors show that ubiquitous overexpression of Plin-2 (but not of a different LD protein) partially suppresses the nazo LD phenotype. Again, the study design suffers from not showing the consequences of overexpression of these proteins on gut LDs of flies with normal nazo/CG3740 function. Without these data the specificity of the genetic interactions of nazo cannot be assessed.

Central to the model of the authors is the lack of Nazo causing a decrease of Plin-2 protein abundance. This claim is poorly supported by the Western blot data presented Fig. 7C. Moreover, at least in adipose tissue Plin-2 has a LD-associated and a cytoplasmic pool. Accordingly, global abundance is a weak predictor for LD-associated Plin-2, which calls for alternative and complementary experimental approaches (e.g. in vivo imaging of tagged proteins, immunohistochemistry, co-IPs) to substantiate the working model of the authors.

Reviewer #3 (Public Review):

This is an interesting study by Sreejith and colleagues exploring the function of the Nazo protein which is a Drosophila homolog of c19orf12. This study presents an interesting combination of genetics, biochemistry, and imaging to define the role of the Nazo protein in gut triglyceride homeostasis. Major findings include that (1) nazo mutants and double mutants nazo/CG3740 shows depletion of lipid droplet, (2) These mutants are also sensitive to starvation and oxidative stress. (3)Later authors also showed that Nazo localizes to the ER-lipid droplet contact sites. (4) Also, nazo mutants are rescued by overexpression of Perilipin2 and knockdown of lipase Brummer. The authors conclude that Nazo is a membrane contact site protein that regulates the activity of lipase Brummer.

The paper is well written, and I found the data convincing and supportive of the final conclusions. I did not find any significant weaknesses, and I think this will be of great interest to those in the field of lipid droplets and membrane contact sites.

Reviewer #1 (Public Review):

The authors Rem et al., examine the mechanism of action of APP, a protein implicated in Alzheimer's disease pathology, on GABAB receptor function. It has been reported earlier that soluble APP (sAPP) binds to the Sushi domain 1 of the GABAB1a subunit. In the current manuscript, authors examine this issue in detail and report that sAPP or APP17 interacts with GABABR with nano Molar affinity. However, binding of APP to GABAB receptor does not influence any of the canonical effects such as receptor function, K+ channel currents, spontaneous release of glutamate, or EPSC in vivo. The experimental evidence provided to support the conclusions is thorough and statistically sound. The range of techniques used to address each of the aims has been carefully curated to draw meaningful conclusions.

The authors use HEK293T heterologous cell line to confirm the affinity of APP17 for the receptor, ligand displacement, and receptor activation. They also use this method to study PKA activation downstream of the GPCR. They use slice electrophysiology to measure changes in glutamatergic transmission EPSC and then in vivo 2-photon microscopy to measure functional changes in vivo.

The work is significant for the field of Alzheimer's and also GABAB receptor biology, as it has been assumed for sAPP acts via GABAB receptors to influence neurotransmission in the brain. The results presented here open up the question yet again, what is the physiological function of sAPP in the brain?

The manuscript is clearly written and easy to follow. The main criticism would be that the manuscript fails to identify the mechanism downstream of APP17 interaction with GB1a SD1.

Reviewer #2 (Public Review):

Amyloid-β precursor protein (APP) regulates synaptic activity in part through the release of secreted APP (sAPP) acting at cell-surface receptors. In 2019 two articles (Dinamarca et al, 2019; Rice et al, 2019) were published showing that sAPP binds with high affinity with GABAB receptors. These receptors regulate neuronal excitability and synaptic release. In the Rice et al. paper, it was concluded that sAPP plays a physiological role by regulating GABAB receptors by modulating synaptic transmission, consistent with the direct activation of these receptors by sAPP. This article has received major attention in the field of Alzheimer's disease and synaptic biology.

The present work was designed to fully explore the functional consequences of sAPP binding to GABAB receptors, in particular, because it was unclear how a conformational change in SD1 - the region of GABAB receptors that binds sAPP - potentially induced by sAPP could increase GBR activity.<br /> The work does confirm that the peptide APP17 which derives from sAPP binds with nanomolar affinity with GABAB receptors. The authors use a diverse range of techniques, ranging from biophysical assays in recombinantly expressed receptors to electrophysiology and live imaging in cultured neurons, slices, and in vivo neuronal activity. In none of these assays, could the authors demonstrate any functional effect of sAPP mediated by an action on GABAB receptors.

This work from a team that has exquisite knowledge of the different aspects of GABAB receptors represents an important and very convincing clarification for the field, and it would therefore be very useful if this information is rapidly available.

Reviewer #1 (Public Review):

This paper by Hubmann et al investigates the role of a large ECM protein in lymphangiogenesis using the zebrafish model to test genetic interactions and with state-of-the-art reporter readouts to evaluate expression patterns and vascular behaviors. The experiments examine the effects of svep1 deletion on facial lymphatics and produce the surprising result that svep1 and vegfc appear to be required in non-overlapping areas of this lymphatic vascular bed. They then use these tools and mutants for tie1 or tie2 to examine genetic interactions in this area and in parachordal lymphangioblast migration in the trunk, showing that tie1 but not tie2 shows epistasis with svep1. The data are overall rigorous and quantified to show the statistical significance of the stated results. The work provides important insights into a complex signaling pathway that is widely utilized in vascular development. The evidence is convincing in supporting the findings and is predicted to be of interest to vascular biologists and others interested in Tie/Tek signaling such as cancer biologists.

Reviewer #2 (Public Review):

In their current manuscript Hussmann et al., present a very detailed phenotypic analysis of the role of svep1 in lymphatic development in zebrafish. They show that svep1 is essential for the development of particular aspects of facial lymphatics (the FCLV and BLECs) in a fashion complementary to VEGF-C. Furthermore, they show that the loss of tie1 phenocopies svep1 mutants not only with respect to lymphatic defects but also in blood vessels (DLAV).

Overall, the manuscript is clearly written, the experiments are carefully executed, and the quality of data is very high and support the author's main conclusions: 1) that Svep1 and Tie1 genetically interact during lymphatic and blood vessel development and 2) that this function is independent and complementary to VEGF-C. 3) The authors confirm and extend on a previous study (Jiang et al. 2020) showing that tie2 (tek) has no overt role in vascular development in zebrafish in blood as well as in lymphatic vessels.

The strength of the paper lies in the careful combination and comparison of different mutant alleles and the use of state-of-the-art imaging. These analyses show that Svep1 and Tie1 interact at the genetic level. In vivo cell tracking experiments show that Tie1 and Svep1 regulate particular aspects of lymphatic cell migration.

An obvious remaining question concerns the epistatic relationship and the molecular mechanism of Tie1/Svep1 interaction. The authors suggest a non-autonomous requirement of Svep1 in the ECM regulating the availability of Tie1 ligands (Ang-1/-2?) in LECs. Since bona fide ligands for Tie1 have not yet been identified in zebrafish further studies will be needed to test this model.

Reviewer #3 (Public Review):

This manuscript describes a Vegfc-independent mechanism of lymphatic vessel formation that is controlled by Svep1 and an orphan endothelial receptor tyrosine kinase Tie1. Based on similarities in the phenotype of svep1 and tie1 mutant zebrafish in the head and trunk vasculature, as well as genetic interaction between the two during parachordal lymphangioblast migration, the authors propose that svep1 is a component of the tie1 signaling pathway. Specifically, svep1 and tie1 mutants show a unique phenotype with the absence of facial collecting lymphatic vessel (that forms in Vegfc mutants) while other facial vessels (that are dependent on Vegfc/Vegfr3 signaling) were only partially affected. Svep1 and tie1 mutants also show similar defects in the formation of brain LECs, the number and migration of parachordal lymphangioblasts from the horizontal myoseptum, and DLAV formation. In contrast, tie2, which is the major angiopoietin receptor in mammals, was found to be dispensable for vascular development in zebrafish.

The presented experiments are performed well and the data are conclusive. The novel findings are the identification of a role of tie1 in zebrafish lymphatic development, and svep1 as a component of the tie1 signaling pathway. The latter raises the possibility that svep1 regulates the activity of Angiopoietin or even acts as a ligand for tie1. However, the conclusion on Svep1 and Tie1 being in the same pathway is based solely on the comparison of mutant phenotypes and genetic interaction studies. Any biochemical data on how svep1 regulates tie1 signaling would greatly strengthen this conclusion.

Reviewer #1 (Public Review):

Gormley et al. conduct a comprehensive Mendelian randomisation (MR) analysis to study the causal effect of obesity and metabolic disorder on oral and oropharyngeal cancer. This work follows on from observational studies that found evidence of associations between these variables and continued on from a previous MR study that focused on the effect of circulating lipid traits on these cancer outcomes. In this study, the authors found limited evidence for an effect of obesity-related traits on either cancer type, contradicting the previous observational studies and thus implying that the latter may have been affected by confounding. Sensitivity analyses that adjusted for potential pleiotropy and outlying instrumental variants agreed with the main study findings. Risk factors including smoking, risk-taking (a proxy for sexual behavior), and alcohol consumption were also stratified for, and only evidence was found for smoking as a mediator of the observed effect.

Strengths:<br /> Strengths of this study include the thorough MR analysis conducted, with all required sensitivity analyses and checks conducted and summarised appropriately, including the use of recent MR methods such as SIMEX where required. For the disease outcomes, oral and oropharyngeal cancer, the authors used summary statistics from the single largest trans-ethnic published meta-analysis GWAS available. Known risk factors were stratified for in sensitivity analyses to follow up any results of interest. Where measures of risk factors were not directly available (e.g. sexual behaviour), genetically-correlated proxy measures were used.

Weaknesses:<br /> While this study has several strengths, there are also a number of limitations present, many of which the authors outline in the paper. In particular, the sample size of the outcome GWAS used was rather small which may hinder the power. Also, some of the cancer subtypes of interest that had previously been investigated in observational studies were not available as GWAS, and so could not be included in this study. The authors only used European or trans-ancestry GWAS, as these are the only ancestries presently available, but a future investigation into other ethnicities is warranted. They also used risk-taking (self-defined via questionnaire) as a proxy for sexual behaviour, which despite having a strong genetic correlation, may not be the best substitute.

Reviewer #2 (Public Review):

The authors used Mendelian randomisation to study the relationships between metabolic traits and oral/oropharyngeal/head and neck cancers. This study was conducted as the relationships between these traits and cancers are unclear based on observational data. Evidence for relationships between these traits and cancers is inconclusive, which is a relevant finding in the context of previous observational data.

Strengths include using large studies to develop the instrumental variables used in MR and examining multiple metabolic traits. Weaknesses include relatively low power to detect associations and a lack of discussion around any possible pleiotropy of SNPs associated with any of the metabolic traits. Based on these strengths and weaknesses, it is unclear whether the authors achieved their goal and whether the results support their conclusions.

This work is relevant to researchers interested in oral cancers and their etiology. Several issues would need to be addressed to make the evidence more reliable.

Reviewer #1 (Public Review):

This is an elegant article where the authors define valuable criteria to identify and classify high-confidence miRNA in fungi. The data supporting the conclusions are solid, and the results are essential to unify the annotation criteria in these organisms. Interestingly, the paper shows that miRNAs in fungi look and position within the genome more similarly to their animal counterpart but may act like plant miRNAs.

The conclusions of this paper are well supported by data, but some aspects need to be clarified and extended.

Reviewer #2 (Public Review):

The work systematically reassesses fungal mi/miRNA-like characteristics and annotation confidence and identifies that many of the loci fail to meet the key points of the methods developed for animal or plant miRNAs. Therefore the authors establish a set of criteria suitable for the annotation of fungal miRNAs and provide a centralized annotation of identified mi/milRNA hairpin RNAs in fungi based on their established rules.

Here are some comments and suggestions for the manuscript to be improved:<br /> 1. The title mentions "ancestral links", however, the main context of this paper does not include the evolution of fungal mi/milRNAs or show the origins of conserved mi/milRNAs in fungi. The authors are suggested to consider a more appropriate title for this work.<br /> 2. The work proposes a fungal mi/milRNAs hairpin precursor recovery pipeline with three minimal criteria to annotate fungal mi/milRNA loci, which allows nearly half of the loci to pass these rules. To highlight the innovation of this annotation, it is strongly suggested that the authors compare their established pipeline and criteria for fungi with those used in animal or plant miRNAs in detail, and emphasize the advantages of the established pipeline. A figure showing the established pipeline and detailed parameters is needed.<br /> 3. The established "standard rules" for fungal mi/milRNA annotation still require more evaluation. It would be better if there is experimental validation to improve confidence.

Reviewer #3 (Public Review):

The authors provide a centralized annotation of miRNA and miRNA-like hairpins in fungi. They aim to develop a standardized pipeline and criteria for miRNA annotation in fungi focusing only on sRNAs derived from hairpin structures, seeking to identify essential characteristics of fungal miRNA and miRNA-like.

Overall this paper will be of interest to readers trying to understand the characteristics and functions of miRNA and miRNA-like hairpins in fungi. The conclusions of this paper are mostly well supported by data, but some aspects of the methodology need to be clarified and extended. The absence of follow-up experiments somewhat limits the impact of this paper. Subsequent work should focus on searching and validating targets of miRNA in fungi. In particular, the strong mi/milRNAs candidates detected in their work.

Reviewer #1 (Public Review):

In this manuscript, the author characterizes the lattice of kinesin-decorated microtubule reconstituted from porcine tubulins in vitro and Xenopus egg extract using cryo-electron tomography and subtomogram averaging. Using the SSTA, they looked at the transition in the lattice of individual microtubules. The authors found that the lattice is not always uniform but contains transitions of different types of lattices. The finding is quite interesting and probably will lead to more investigation of the microtubule lattice inside the cells later on for this kind of lattice transition.

The manuscript is easy to read and well-organized. The supporting data is very well prepared.

Overall, it seems the conclusion of the author is justified. However, the manuscript appears to show a lack of data. Only 4 tomograms are done for the porcine microtubules. Increasing the data number would make the manuscript statistically convincing. In addition, having the same transition with the missing wedge orientation randomly from different subtomograms will allow a better average of transition without the missing wedge artifact.

Another thing that I found lacking is the mapping of the transition region/alignment in the raw data. However, it is not easy for me or the reader to understand how each segment is oriented relative to each other apart from the simplified seam diagrams in the figures, and also the orientation of the seam corresponding to the missing wedge in the average. With these improvements, I think the conclusion of the manuscript will be better justified.

Reviewer #2 (Public Review):

Differences in protofilament and subunit helical-start numbers for in vitro polymerized and cellular microtubules have previously been well characterized. In this work, Guyomar et al. analyze the fine organization of tubulin dimers within the microtubule lattice using cryo-electron tomography and subtomogram averaging. Microtubules were assembled in vitro or within Xenopus egg cytoplasmic extracts and plunge frozen after addition of a kinesin motor domain to mark the position of tubulin dimers. By generating subtomogram averages of consecutive sections of each microtubule and manually annotating their lattice geometry, the authors quantified changes in lattice arrangement in individual microtubules. They found in vitro polymerized microtubules often contained multiple seams and lattice-type changes. In contrast, microtubules polymerized in the cytoplasmic extract more frequently contained a single seam and fewer lattice-type transitions.

Overall, their segmented subtomogram averaging approach is appropriately used to identify regions of lattice-type transition and quantify their abundance. This study provides new data on how often small holes in the lattice occur and suggests that regulators of microtubule growth in cells also control lateral tubulin interactions. However, not all of the claims are well supported by their data and the presentation of their main conclusions could be improved.

Reviewer #3 (Public Review):

Protofilament number changes have been observed in in vitro assembled microtubules. This study by Guyomar and colleagues uses cryo-ET and subtomogram averaging to investigate the structural plasticity of microtubules assembled in vitro from purified porcine brain tubulin at high concentrations and from Xenopus egg extracts in which polymerization was initiated either by addition of DMSO or by adding a constitutively active Ran. They show that the microtubule lattice is plastic with frequent protofilament changes and contains multiple seams. A model is proposed for microtubule polymerization whereby these lattice discontinuities/defects are introduced due to the addition of tubulin dimers through lateral contacts between alpha and beta tubulin, thus creating gaps in the lattice and shifting the seam. The study clearly shows quantitatively the lattice changes in two separate conditions of assembling microtubules. The high frequency of defects they observe under their microtubule assembly conditions is much higher than what has been observed in vivo in intact cells. Their observations are clear and supported by the data, but it is not at all clear how generalizable they are and whether the defect frequencies they see are not a result of the assembly conditions, dilutions used and presence of kinesin with which the lattice is decorated. The study definitely has implications for mechanistic studies of microtubules in vitro and raises the question of how these defects vary for protocols from different labs and between different tubulin preparations.

Reviewer #1 (Public Review):

In this manuscript, Garratt and collaborators describe exposure to male vs. female olfactory cues as an intervention modulating mouse lifespan. They use urine exposure (early life) and soiled bedding exposure (after weaning) to determine the impact of sex-specific olfactory cues on lifespan. They use wild-type and Gnao1 neural-specific mutants to determine potential dependency on vomeronasal function as well. They also recorded information about body temperature, body weight, glucose levels, grip strength, and balance. Overall, they identify female-olfactory cues as increasing the lifespan of female but not male mice, regardless of genotype.

This study reports an intriguing new intervention with an impact on mouse lifespan (with a female-specific effect). However, some of the data with negative results are not plotted/shown, and although all experiments were performed in wild-type and mutant background, all the shown data is pooled and not split by genotype. Overall, this study will be valuable to the field provided that a few analytical points are addressed for clarity and reproducibility and that all methodological details are included.

Reviewer #2 (Public Review):

In this study, the authors were attempting to determine if early life exposure to specific olfactory cues leads to changes in lifespan. They exposed young mice to urine from male or female adult mice, or no urine for the control groups. They were also interested in determining if the Gao gene was responsible for any effect they found due to its impact on olfaction. They found that females exposed to female urine lived longer than control or male-exposed female mice, and there was no effect of exposure on male lifespan. These effects were found to be completely independent of the Gao gene.

I felt the overall methods were good, and they had sufficient power to look at the lifespan effects. However, the authors used spent bedding from male and female mice as the source of the smell exposure, and I would worry that spent bedding would have traces of fecal matter in it. This could suggest that any effect they see would be due to microbiome differences from the bedding exposure, not the smell of urine.

While the results are interesting, I'm not sure they will have a huge impact on the field. Early life exposures have previously been shown to affect aging and lifespan, and there were overall very minor effects seen of these olfactory exposures in female mice.

Reviewer #3 (Public Review):

Garratt et al. investigated that transient exposure of young mice during their first two months of life with olfactory cues from con-specific adults would have long-lasting effects on their late-life health and lifespan. They find that the olfactory cues have sex-specific effects on lifespan, which only the lifespan of young females can be extended by odours from adult females but no other combinations, neither young females with adult males nor young males with either sex. Interestingly, their data also suggested that depletion of G protein Gαo in the olfactory system played no role in the lifespan extension, indicating it might be another unknown factor(s) mediating this sex-specific effect on longevity in mice. While the conclusions of this study are well supported by the data, there are some issues with parts of the data analysis and presentation that would need to be clarified and extended.

1) The authors suggested that the G protein Gαo played no role in lifespan extension in the case that transient exposure of young females with olfactory cues from female adults, as they showed in Figure 1. However, it is not clear if the depletion of G Gαo (Gαo mutant) itself has effects on lifespan, compared to its wild type. It would be important to show the lifespan curves from wild type and Gαo mutant individually alongside the pooled lifespan curves, as well as regarding data in a table, followed with a proper discussion.

2) Regarding the functional tests, the authors showed that there was only a small fraction of experiments showed differences between treatments, which were all in figure 2. However, it is necessary to also show the data with no differences, particularly since the conclusion of the study suggested the underlying mechanisms are not clear yet. In my opinion, body weight, plasma glucose, and body temperature all deserve to have their figures individually with all data points.

3) As the authors mentioned in the Introduction, the age at sexual maturity correlates positively with the median lifespan across mice strains (Yuan et al. 2012, Wang et al. 2018). Also, young female mice that were exposed to male odours during their developmental stage accelerated sexual maturity (Drickamer 1983), and the same happened to young males that were exposed to the odours from the opposite sex (Vandenbergh 1971). It is, therefore, surprising to see in this study, the exposure of young females or young males to the olfactory information from their opposite sex had no effects on lifespan. One of the solutions to solve this disparity is to measure the sexual maturity of the mice in this study. The authors should seek the possibility to check the record of when the first litter of pups was born between treatments (Shindyapina et al. 2022) or examine preputial separation and vaginal opening (Hoffmann 2018), for instance.

In sum, this is a great piece of work suggesting the importance of sex differences on olfactory cues mediated lifespan and pointing out some directions for future works.

Joint Public Review:

In this work Malis et al introduce a novel spin-labeling MRI sequence to measure cerebrospinal fluid (CSF) outflow. The glymphatic system is of growing interest in a range of diseases, but few studies have been conducted in humans due to the requirement for and invasiveness of contrast injections. By labeling one hemisphere of the brain the authors attempt to assess outflow through the superior sagittal sinus (SSS), one of the major drainage pathways for CSF, signal changes across time were assessed to extract commonly used metrics. Additionally, correlations with age are explored in their cohort of healthy volunteers. The authors report the movement of labeled CSF from the subarachnoid space to the dura mater, parasagittal dura, and ultimately SSS, evidence of leakage from the subarachnoid space to the SSS, and decreases in CSF outflow metrics with older age.

1. I don't think that the description of Parasagittal dura in figure 1 is correct. There is no anatomical structure at the top of SSS that is known as PSD. The location of the lymphatic structures is also incorrect. Please review "Anatomic details of intradural channels in the parasagittal dura: a possible pathway for flow of cerebrospinal fluid" Neurosurgery 1996 Fox at al. There is usually no obvious tissue between the upper wall of the SSS and the calvarium, which can also be seen in the authors' fig 2A and 2B. All of the tissues located lateral to the SSS are known as PSD. Also, the SSS wall is not as thick as the authors stated and is known as PSD in this region. For this reason, the authors need to revise Fig 1 and it should be changed to PSD in the areas referred to as the SSS wall in the article.

2. The authors described tagged CSF in two pathways: from dura mater to PSD and SAS into the SSS and directly from SAS to SSS. Flow from dura mater to PSD and SAS in the main and supplement cannot be seen. Only a flow from PSD to SSS can be seen. Also, regular dura cannot carry flow-collagen-rich fibrous tissue, except parasagittal dura. There is no flow from dura to the CSF in the figures.

3. The authors have conducted many tests to prevent venous contamination. However, measurements were made based on SSS flow rates in all tests. Small parenchymal venous structures, and small cortical-SAS veins might be tagged due to different flow patterns and T2- Relaxation times.

4. The rate of CSF formation in humans is 0.3 - 0.4 ml min-1. ( Brinker et al 2014. Fluids Barriers CNS). We can assume that the absorption rate is also similar to the CSF formation for the entire system brain and Spine. Therefore, the absorption rate of this very small amount of CSF by SSS is very low in seconds. It is hard to detect by MR and especially CSF flow from the PSD to SSS. The authors concluded that using this technique the rate averaged less than a couple of seconds, rather than on the order of hours or days as previously reported with the use of intrathecal administration of GBCA (Ringstad et al., 2020).

5. Overall, I think that the CSF flow from the PSD to the CNS described by the authors - the CSF flow, might be the venous flow that drains into the SSS slowly, predominantly in the rich venous channels, venous lacunae, and previously described channels in the PSD. Additional explanations are needed.

6. The study is generally well described and to the best of my knowledge an innovative approach. The findings are broadly consistent with what might be expected from the literature and the authors make a good argument in support of their findings. However, the lack of validation is a major limitation of the presented work. In introducing a novel technique a comparison with an existing approach, such as Gd enhanced contrast techniques, or phase contrast would have been expected. Several considerations could have been mentioned/addressed in more detail e.g. what effect labeling efficiency, tortuosity of vessels, lack of gating, the effectiveness of the intensity thresholding to remove the signal from blood, etc may have on the quantification, etc. Without a more thorough validation, it is difficult to evaluate the findings. While scans were conducted on two volunteers to assess reproducibility this is a very small sample and it is notable that scans were conducted consecutively, which might be expected to reduce variance relative to scans further apart e.g. on different dates, scanned by a different operator and no information is provided on how the two scans were positioned (i.e. separately vs copied from the first to the second scan), some metrics showed large percentage differences, which were more pronounced in one subject than the other. Without further data, it is difficult to interpret the reproducibility results. No assessment of the effect of physiological parameters e.g. breathing, cardiac pulsations, or factors affecting glymphatic clearance e.g. amount of sleep the evening before was given.

7. Given these limitations it is hard to adequately assess the likely impact or utility. In recent years several groups have published work e.g. doi.org/10.1038/s41467-020-16002-4 , doi.org/10.1016/j.neuroimage.2021.118755 assessing the blood-CSF barrier. However, previous work has generally focused on larger structures, and by labeling in the oblique-sagittal plane it is unclear how drainage and blood flow rates may affect the presented values here.

8. Some validation data would greatly increase the value of the reported work. I would therefore encourage the authors to consider acquiring some additional datasets to compare measures of CSF draining against another method e.g. 2-D or 4-D phase contrast, or Gd-based contrast-enhanced techniques. Some additional points to consider are noted below.

8. Abstract

CSF outflow may also be imaged with phase contrast MRI (albeit in a limited way).<br /> Demographics would fit better in Results, breakdown could be given for the young and old groups i.e. n, ages, sex.<br /> Conclusion - unless further validation can be provided I think some of the claims should be toned down.

9. Introduction

The authors emphasise the role of Nedergaard, however, there was some relevant earlier work (e.g. Rennels et al, PMID: 2396537).

10. Methods

It would be more conventional to summarise the volunteer characteristics in the Results.<br /> Given the age difference between the two groups, and the fact that for conventional ASL we know of differences in labelling efficiency and the need for a different post-labelling duration in more elderly patients how did the authors account for this?<br /> More broadly what would the effect of differences in labeling efficiency be, given the labeling plane is unlikely to be perpendicular to the draining vessels?<br /> While the authors mention circadian effects there is no mention of controlling for other factors before the scan e.g. caffeine consumption, smoking, etc.<br /> Various mechanisms have been hypothesised to drive glymphatic pulsations. Assessing how physiological signals correlated with the flow may have been a useful proof of concept. Why was it not considered necessary to use a gated acquisition? Did the authors consider the potential impact of respiratory and cardiac pulsations on their measurements?<br /> ROI segmentation - manually selected by two raters, was this done independently and blinded? How were consensus ROIs agreed?<br /> Intensity values outwith MEAN +/- 2 SD were excluded from further analyses. This is justified as removing pulsatile blood. However, was this done independently for tag-on and tag-off? Does this mean slight differences were present in the number of voxels between the two?<br /> The starting points and parameter ranges are given in Eq'n 3, how were the ranges defined? Was there a reason for constraining the fit to positive values only, is there a risk of bias from this?<br /> While the main results appear to have a reasonable sample size n=2 for the reproducibility analysis is very limited. Additional datasets would be useful in properly interpreting the results.

11. Results<br /> While the authors have taken some measures to reduce potential contamination from blood I would be concerned about the risk of surface vessels affecting the signal, and there does not seem to be an evaluation of how effective their measures are.<br /> The labeling pulse is applied in the oblique sagittal orientation, but in tandem with differing rates of blood flow and CSF drainage from the labeling plane does that not risk circulating flow from other slices potentially affecting the values?<br /> Figure 4. The authors focus on the parasagittal dura, but in both the subtraction image and panel C showing different slices at TI=1250 ms some movement appears visible in the opposing hemisphere. Similarly in S2 If the signal does represent CSF movement then this seems counterintuitive and should be explained.<br /> In Figures 4 and 5 the angulation of the TIME-SLIP tag pulse seems quite different. What procedure was used to standardise this, and what effect may this have on the results?

12. Discussion<br /> Phrasing error 'which will be assessed in future studies'.<br /> I would suggest that some of the claims of novelty be moderated e.g. 'may facilitate establishment of normative values for CSF outflow' seems a stretch given multiple pathways exist and this is only considered one.<br /> More consideration should be given to some of the points mentioned in the results. The lack of validation should be properly discussed.

Reviewer #1 (Public Review):

The authors use what is potentially a novel method for bootstrapping sequence data to evaluate the extent to which SARS-CoV-2 transmissions occurred between regions of the world, between France and other European countries, and between some distinct regions within France. Data from the first two waves of SARS-CoV-2 in Europe were considered, from 2020 into January 2021. The paper provides more detail about the specific spread of the virus around Europe, specifically within France, than other work in this area of which I am aware.

An interesting facet of the methodology used is the downsampling of sequence data, generating multiple bootstraps each of around 500-1000 sequences and conducting analysis on each one. This has the strength of sampling, in total, a large number of sequences, while reducing the overall computational cost of analysis on a database that contains in total several hundred thousand sequences. A question I had about the results concerns the extent of downsampling versus the rate of viral migration: If between-country movements are rapid, a reduced sample could be misleading, for example characterising a transmission path from A to B to C as being from A to C by virtue of missing data. I acknowledge that this would be a problem with any phylogeographic analysis relying on limited data. However, in this case, how does the rate of migration between locations compare to the length of time between samples in the reduced trees? Along these lines, I was unclear to what extent the reported proportions of intra- versus inter-regional transmissions (e.g. line 223) would be vulnerable to sampling effects.

A further question around the methodology was the use of an artificially high fixed clock rate in the phylogenetic analysis so as to date the tree in an unbiased way. Although I understood that the stated action led to the required results, given the time available for review I was unable to figure out why this should be so. Is this an artefact of under-sampling, or of approximations made in the phylogenetic inference? Is this a well-known phenomenon in phylogenetic inference?

The value of this kind of research is highlighted in the paper, in that genomic data can be used to assess and guide public health measures (line 64). This work elucidates several facts about the geographical spread of SARS-CoV-2 within France and between European countries. The more clearly these facts can be translated into improved or more considered public health action, through the evaluation of previous policy actions, or through the explication of how future actions could lead to improved outcomes, the more this work will have a profound and ongoing impact.

Reviewer #2 (Public Review):

This study represents an important contribution to our understanding of SARS-CoV-2 transmission dynamics in France, Europe and globally during the early pandemic in 2020 and the authors should be congratulated for tackling this important question. Through evaluation of the contributions of intra- and inter-regional transmission at global, continental, and domestic levels, the authors provided compelling, although as of yet correlative and incomplete, evidence towards how international travel restrictions reduced inter-regional transmission while permitting increased transmission intra-regionally. Unfortunately, however this work suffers from a number of serious analytical shortcomings, all of which can be overcome in a major revision and re-analysis.

With this genomic epidemiology analysis, the authors disentangled the relative contributions of different geographic levels to transmission events in France and in Europe in the first two COVID-19 waves of 2020. By partitioning the analysis into three complementary, but distinct, geographic levels, the migration flows in and out of continents, countries in Europe, and regions in France were inferred using maximum likelihood ancestral state reconstruction. The major strengths of this paper were the inclusion of multiple geographic levels, the comparison of different rate symmetries in the ancestral character estimation, and the comprehensive qualitative descriptions of comparisons over time and geographies. However, there were also major weaknesses that need to be addressed and are described in more detail below. They include summing across replicates that were drawn with replacement and were not independent; inadequate justification for excluding underrepresented geographies; the assertion that positive correlation between intra-regional transmission and deaths validates the accuracy of the analysis; considering the framework the authors have chosen for this analysis the analysis would accommodate and benefit strongly from increasing the size of the sequence sets selected for analysis in each replicate; and the sparsity of quantitative (over qualitative or exploratory) comparisons and statistics in the reporting of results. In particular, it would greatly strengthen the paper if the authors could better evaluate the effect of travel restrictions on importations and exportations by testing hypotheses, quantifying changes in the presence of restrictions, or estimating inflection points in importation rates.

General comments on the Background: Need to elaborate on how this study fits into the big picture in the first paragraph. Should discuss how phylodynamics contributes to understanding of viral outbreaks, SARS-CoV-2 epidemiology and viral evolution.

The authors should consider a hypothesis driven framework for their analyses, for example considering the geographically central position of France what hypotheses stem from this considering sources of viral importations and destinations of exportations from/to Europe vs other international? Or other a priori expectations.

To address the computational limits of phylogenetic reconstruction, 100 replicates of fewer than 1000 sequences each were sampled for each epidemic wave at each level. The inter- and intra-regional transmissions were averaged and then summed across replicates in order to compare the relative roles played by each geography towards transmission. While we see the logic in using the sum across replicates, this is highly likely to bias results, especially since in the methods, this is described as sampling with replacement between replicates (LX). The validity of summing replicates needs to be discussed and are likely most appropriately presented as mean or median. Also, these samples are quite small considering the computational capacity of the maximum likelihood tools being used. We recommend repeating the analysis with a substantially larger number of sequences per sample.

Reviewer #1 (Public Review):

Ge et. al., examined sodium-glucose cotransporter-2 inhibitors (SGLT2i) in Alport syndrome (AS), and demonstrate that it was beneficial in AS through reduced lipotoxicity in podocytes as a key mechanism of action. The SGLT2i empagliflozin has been previously shown to have positive effects on hyperglycemia control, as well as on cardiovascular and renal outcomes of type II diabetes mellitus through tubuloglomerular feedback, but its effect on glomerular diseases such as AS are unknown to date. The authors have previously identified that cholesterol efflux in podocytes plays a critical pathogenic role in a diabetic kidney disease setting. The evidence that authors provide in favor of their hypothesis in a disease of non-metabolic origin such as AS, was supported as the SGLT2i was effective in reducing the deleterious effects of lipotoxicity in podocytes, ameliorated glomerular injury and proteinuria, and extending the life span of Col4a3 knockout mice. They further show that empagliflozin treatment mitigated AS podocytes from cell death through apoptosis, but did not impact the cell's cytotoxicity. These results support the notion that empagliflozin affects the regulation of important metabolic switch in mouse kidneys, perhaps through decreasing lipid accumulation in podocytes.

However, the authors solely rely on one IHC staining image of a human biopsy to demonstrate SGLT2 expression in podocytes in vivo. Although the authors have done several experiments which greatly increase the confidence in their findings that empagliflozin is beneficial in AS and would have clinical significance, their data does not rule out the possibility that empagliflozin has beneficial effects through the other glomerular cells in AS, or limited to impacting lipids in podocytes in AS.

Reviewer #2 (Public Review):

The manuscript by Ge et al investigated the therapeutic benefits of the SGLT2 inhibitor empagliflozin in Alport syndrome (AS). They established the immortalized tubular cells and podocytes using wildtype (WT) mice and mice with AS. They showed that cultured human and mouse podocytes express similar levels of SGLT2 protein as compared to tubular cells. In vitro, they demonstrated that AS podocytes accumulate more lipid droplets and show increased levels of apoptosis in comparison to WT podocytes. Empagliflozin significantly reduces lipid droplets and apoptosis in AS podocytes. Furthermore, empagliflozin inhibits glucose/pyruvate-driven respiration in AS podocytes. In vivo, empagliflozin prolongs the lifespan of AS mice. Compared to untreated AS mice, empagliflozin improves kidney function and reduces the content of triglycerides and cholesterol esters in the kidney cortices of AS mice. Overall, the manuscript is nicely written, well-arranged, and easy to read. The experimental methods are reliable, and the conclusions are supported by the results.

Reviewer #3 (Public Review):

Using cultured human podocytes the expression of SGLT2 is established using immunostaining and western blotting. An analysis of podocyte RNA wasn't performed, but the expression in cultured podocytes was comparable to that seen in human cultured proximal tubular cells. This work then paved the way for treatment of immortalized cells obtained from an Alport syndrome mouse model (Col4A3-/-), representing an autosomal recessive form of Alport syndrome. Podocytes from Alport syndrome mice showed a lipid droplet accumulation which was reduced to some extent by SGLT2 inhibition. In a series of metabolic experiments, it was shown that SGLT2 inhibition reduced the formation of pyruvate as a metabolic substrate in Alport podocytes. In vivo experiments showed an improvement in survival of Col4a3-/- mice treated with SGLT2 inhibition. When compared to ace inhibitor, SGLT2 inhibition has a similar effect on renal function and no additive effect was seen with SGLT2 inhibitor plus ace inhibitor. Like the cell assays, the in vivo treatment seemed to prevent the podocyte lipid accumulation in Alport syndrome mice.

This data in cells and animals generally supports the findings in SGLT2 inhibitor human studies, where Alport syndrome patients with proteinuria and progressive CKD seem to benefit. The work paves the way for a dedicated trial of SGLT2i in Alport patients and a reassessment of the human podocyte disease phenotype in this condition, before and after treatment. There are patients with mutations in SGLT2 with familial renal glycosuria - it would be interesting to test via urine derived podocytes whether a similar metabolic switch was occurring and its consequences to pave the way for long term treatment regimes.

Reviewer #1 (Public Review):

This manuscript describes the generation and characterisation of a mouse knockout model of Cep78, which codes for a centrosomal protein previously implicated in cone-rod dystrophy (CRD) and hearing loss in humans. Previous work in cultured mammalian cells (including patient fibroblasts) also indicated roles for CEP78 in primary cilium assembly and length control, but so far no animal models for CEP78 were described. Here, the authors first use CRISPR/Cas9 to knock out Cep78 in the mouse and convincingly demonstrate loss of CEP78 protein in lysates of retina and testis of Cep78-/- animals. Next, by careful phenotypic analysis, the authors demonstrate significant defects in photoreceptor structure and function in these mutant animals, which become more severe over a 9 (or 18) month period. Specifically, TEM analysis demonstrates ultrastructural defects of the connecting cilium and photoreceptor outer segments in the Cep78 mutants, which is in line with previously reported roles for CEP78 in CRD and in regulating primary cilia assembly in humans. In addition to a CRD-like phenotype, the authors also convincingly show that male Cep78-/- animals are infertile and exhibit severe defects in spermatogenesis, sperm flagella structure and manchette formation (MMAF phenotype). Furthermore, the authors provide evidence for an MMAF phenotype from a male individual carrying a previously reported CEP78 c.1629-2A>G mutation, substantiating that CEP78 is required for sperm development and function in mammals and supporting previously published work (Ascari et al. 2020).

Finally, to identify the underlying molecular mechanism by which CEP78 loss causes MMAF, the authors perform some biochemical analyses, which suggest that CEP78 physically interacts with IFT20 and TTC21A (an ortholog of Chlamydomonas IFT139) and might regulate their stability. The authors conclude that CEP78 directly binds IFT20 and TTC21A in a trimeric complex and that disruption of this complex underlies the MMAF phenotype observed in Cep78-/- male mice. However, this conclusion is not fully justified by the data provided, and the mechanism by which CEP78 affects spermatogenesis therefore remains to be clarified.

Specific strengths are weaknesses of the manuscript are listed below.

Strengths:

Overall, the phenotypic characterisation of the Cep78-/- animals appears convincing and provides new evidence supporting that CEP78 plays an important role in the development and function of photoreceptors and sperm cells in vertebrates.

Weaknesses:

1) The immunoprecipitation experiments of mouse testis extracts that were used for the mass spectrometry analysis in Table S4 were performed with an antibody against endogenous CEP78 (although antibody details are missing). One caveat with this approach is that the antibody might block binding of CEP78 to some of its interactors, e.g. if the epitope recognized by the antibody is located within one or more interactor binding sites in CEP78. This could explain why the authors did not identify some of the previously identified CEP78 interactors in their IP analysis, such as CEP76 and the EDD-DYRK2-DDB1-VprBP complex (Hossain et al. 2017) as well as CEP350 (Goncalves et al. 2021).

2) Figure 7A-D and page 18-25: based on IPs performed on cell or tissue lysates the authors conclude that CEP78 directly binds IFT20 and TTC21A in a "trimeric complex". However, this conclusion is not justified by the data provided, nor by the previous studies that the authors are referring to (Liu et al. 2019 and Zhang et al. 2016). The reported interactions might just as well be indirect. Indeed, IFT20 is a known component of the IFT-B2 complex (Taschner et al., 2016) whereas TTC21A (IFT139) is part of the IFT-A complex, which suggests that they may interact indirectly. In addition, the IPs shown in Figure 7A-D are lacking negative controls that do not coIP with CEP78/IFT20/TTC21A. It is important to include such controls, especially since IFT20 and CEP78 are rich in coiled coils that tend to interact non-specifically with other proteins.

3) In Figure 7D, the input blots show similar levels of TTC21A and IFT20 in control and Cep78-/- mouse testicular tissue. This is in contrast to panels E-G in the same figure where TTC21A and IFT20 levels look reduced in the mutant. Please explain this discrepancy.

4) The efficiency of the siRNA knockdown shown in 7J-M was only assessed by qPCR (Figure S4), but this does not necessarily mean the corresponding proteins were depleted. Western blot analysis needs to be performed to show depletion at the protein level. Furthermore, it would be desirable with rescue experiments to validate the specificity of the siRNAs used.

5) Figure 7I: the resolution of the IFM is not very high and certainly not sufficient to demonstrate that CEP78, IFT20 and TTC21A co-localize to the same region on the centrosome, which one would have expected if they directly interact.

6) It is not really clear what information the authors seek to obtain from the global proteomic analysis of elongating spermatids shown in Figure 3N, O and Tables S2 and S3. Also, in Table S2, why are the numbers for CEP78 in columns P, Q and R so high when Cep78 is knocked out in these spermatid lysates? Please clarify.

7) Figure 1F and Figure 4K: the data needs to be quantified.

8) Figure 2A: It is difficult to see a difference in connecting cilium length in control and Cep78-/- mutant retinas based on the images shown here.

Reviewer #2 (Public Review):

In this report, the authors have described the generation and characteristics of Cep78 mutant mice. Consistent with the phenotype observed in patients carrying the mutations in CEP78, Cep78 knock-out mice show degeneration in photoreceptors cells as well as defects in sperm. The author further shows the CEP78 protein can interact with IFT120 and TTC21a. Mutation in CEP78 results in a reduction of protein level of IFT120 and TTC21A and mislocalization of these two proteins, offering mechanistic insights into the sperm defects. Overall the manuscript is well written and easy to follow. Phenotyping is thorough. However, improvement of the background section is needed. In addition, some of the conclusion is not sufficiently supported by the data, warranting further analysis and/or additional experiments. The Cep78 KO mice model established by the author will be a useful model for further elucidating the disease mechanism in human and developing potential therapy.

My comments are the following:<br /> 1. Introduction. The statement that "CRD usually exists with combination of immotile cilia defects in other systems" is not correct. CRD due to ciliopathy can have cilia-related syndromic defects in other systems but it is a relatively small portion of all CRDs and the most frequently mutated genes are not cilia-related genes, such as ABCA4, GUCY2D, CRX.<br /> 2. Introduction: Page 4 CNGB1 encodes channel protein and not a cilia gene. It should be removed since it does not fit.<br /> 3. Page 5, given the previous report of CEP78 patients with retina degeneration, hearing loss, and reduced infertility, the statement of "we report CE79 as a NEW causative gene for a distinct syndrome...TWO phenotypes....." Is not accurate.<br /> 4. Figure 1F, the OS of the cone seems shorter, which might be the reason for weaker arrestin staining in the mutant compared to the heterozygous. Also, it would be better to quantify the staining to substantiate the statement.<br /> 5. Figure 1K, panel with lower magnification would be useful to get a better sense of the overall structure defect of the retina. Is the defect observed in the cone as well?<br /> 6. Figure 2A, NPHP1 or other markers specifically label CC would be more useful to quantify the length of CC. Also need to provide a notation for the red arrows in Figure 2. In addition, the shape of CC in the mutant seems differ significantly from the control. It seems disorganized and swollen.<br /> 7. Evidence provided can only indicate direct interaction among CEP78/IFT20/TTC21A.

Reviewer #3 (Public Review):

Authors were aiming to bring a deeper understanding of CEP78 function in the development of cone-rod dystrophy as well as to demonstrate previously not reported phenotype of CEP78 role in male infertility.

It is important to note, that the authors 're-examined' already earlier published human mutation, 10 bp deletion in CEP78 gene (Qing Fu et al., 10.1136/jmedgenet-2016-104166). This should be seen as an advantage since re-visiting an older study has allowed noting the phenotypes that were not reported in the first place, namely impairment of photoreceptor and flagellar structure and function. Authors have generated a new knockout mouse model with deleted Cep78 gene and allowed to convey the in-depth studies of Cep78 function and unleash interacting partners.

The authors master classical histology techniques for tissue analysis, immunostaining, light, confocal microscopy. They also employed high-end technologies such as spectral domain optical coherence tomography system, electron and scanning electron microscopy. They performed functional studies such as electroretinogram (ERG) to detect visual functions of Cep78-/- mice and quantitative mass spectrometry (MS) on elongating spermatids.

The authors used elegant co-immunoprecipitation techniques to demonstrate trimer complex formation.

Through the manuscript, images are clear and support the intended information and claims. Additionally, where possible, quantifications were provided. Sample number was sufficient and in most cases was n=6 (for mouse specimens).

The authors could provide more details in the materials and methods section on how some experiments were conducted. Here are a few examples. (i) Authors have performed quantitative mass spectrometry (MS) on elongating spermatids lysates however, did not present specifically how elongating spermatids were extracted. (ii) In the case of co-IPs authors should provide information on what number of cells (6 well-plate, 10 cm dish etc) were transfected and used for co-IPs. Furthermore, authors could more clearly articulate what were the novel discoveries and what confirmed earlier findings.

The authors clearly demonstrate and present sufficient evidence to show CEP78/Cep78 importance for proper photoreceptor and flagellar function. Furthermore, they succeed in identifying trimer complex proteins which help to explain the mechanism of Cep78 function.

The given study provides a rather detailed characterization of human and mouse phenotype in response to the CEP78/Cep78 deletion and possible mechanism causing it. CEP78 was already earlier associated with Cone-rod dystrophy and, this study provides a greater in-depth understanding of the mechanism underlying it. Importantly, scientists have generated a new knock-out mouse model that can be used for further studies or putative treatment testing.

CEP78/Cep78 deletion association with male infertility is not previously reported and brings additional value to this study. We know, from numerous studies, that testes express multiple genes, some are unique to testes some are co-expressed in multiple tissues. However, very few genes are well studied and have clinical significance. Studies like this, combining patient and animal model research, allow to identify and assign function to poorly characterized or yet unstudied genes. This enables data to use in basic research, patient diagnostics and treatment choices.

Reviewer #1 (Public Review):

The idea that a passive living being can improve the wind dispersal of its seeds by passively changing their drag is enticing. The manuscript shows that high wind events in Scotland are inversely correlated with the ambient humidity. The dandelion pappus morphs with the ambient humidity, being more open in dry conditions, which is associated with stronger wind events. This passive morphing of the shape of the pappi thus leads to a dispersal of the seeds further away from their origin.

The analysis and discussion in the paper is focused on "distance", i.e., how far the pappus will fly. Could the notion of time be relevant too? In wet conditions, perhaps it's better for a seed to hit the ground quickly and start germinating, whereas if its dry, staying up in the air for longer to travel farther might be a better strategy.

Reviewer #2 (Public Review):

I will first state that I am an ecologist that studies wind dispersal, and so my expertise lies in evaluating the determination of their results, the dispersal model, and the ecological significance - I cannot evaluate the PIV methods, although their results seem very reasonable based on other work! I believe this is a very strong paper that will have a lot of interest. In my opinion, it is the perfect demonstration of how to understand wind dispersal from a fundamental and ecological perspective. They first explore the aerodynamics of dandelion diaspores and how they change with the environment. Then they use this information to scale up to how this might affect dispersal across the landscape under different environmental conditions. I think the experiments they have conducted and the models they have included are excellent! I also really enjoyed that this work is the culmination of their body of work, where they have taken a step-by-step approach to convincing readers how dandelion diaspores disperse under different humidity conditions (see Seale et al. 2020 and 2022).

The paper is very well written. The introduction lays out a very clear case as to why the environment should (and ultimately does) influence wind dispersal, and all of the relevant references are cited. It was nice to see them all in one place and is a great summary of the literature for those who are new to the field.

The authors also claim that the environment can have an impact on dandelion dispersal by altering the shape of the diaspore (the pappus closes). This influences the terminal velocity and drag coefficient, and the authors used the appropriate PIV tests to determine that this is the case. They then go on to show that while wet conditions can decrease the terminal velocity which ultimately decreases dispersal distance, under wet/stormy conditions there are often increased wind speeds and this can actually increase dispersal because of increased wind speed. However this last point is a bit confusing to me based on the way the data is laid out.

In all, I really enjoyed this paper! There is a lot to learn from this, and I look forward to reading it in print. I would encourage the authors to make a few updates to their text to make their conclusions crystal clear for readers!

Reviewer #1 (Public Review):

This paper shows that sibling neurons in the zebrafish spinal cord have different inputs and outputs, and do not show interconnectivity - somewhat surprising considering their very similar development. The differences in sibling neuron connectivity are strongly correlated with the level of Notch signaling, suggesting that Notch signaling regulates circuit assembly.

Reviewer #2 (Public Review):

The data presented in this manuscript provide evidence that, in the ventral spinal cord of zebrafish embryos, "sister" V2a excitatory and V2b inhibitory neurons, which arise from common vsx1+ progenitors, extend descending, ipsilateral axons that, although differing in length, remain close to one another. Because of this alignment, V2a and V2b neurons could contribute to a common microcircuit, by receiving inputs from common synaptic circuits, forming synapses on one another, or projecting to common synaptic targets. However, a series of electrophysiological and optogenetic tests exclude these possibilities and indicate that, instead, they receive inputs from distinct sources, they do not engage in synaptic signaling with one another, and they have distinct, downstream synaptic targets. This differs from the mouse cortex, in which clonally related neurons appear to preferentially form connections within a shared microcircuit.

The chief strengths of this work include the imaging data, which nicely reveal the locations and morphologies of sister V2a/b neurons, and the electrophysiological experiments, which provide compelling evidence that sister V2a/b neurons do not function within a shared microcircuit.

The study does have some limitations. First, V2a/V2b sister pairs are not obligate. Instead, about 25% of V2b neurons arise from a vsx1+ progenitor division that also produces a V2s neuron instead of a V2a neuron. To distinguish between these outcomes, the authors use the transient expression of a vsx1:EGFP reporter to label clonal pairs combined with a chx10:Red reporter to label V2a neurons. For the optogenetic experiments, however, the authors were not able to use the V2a neuron marker because they were limited by the reporters available to them. Thus, there is a possibility that some sister neurons tested were V2b/s rather than V2a/b. Second, the electrophysiological data are not paired with examinations of synaptic contacts using light or electron microscopy. Third, the circuits in which V2a and V2b neurons function are incompletely understood, and so knowledge of the synaptic inputs and downstream targets is limited.

On balance, the limitations of the study are rather minor. The manuscript is nicely written, the figures are presented clearly and logically, and the data are sufficient to support the claims and conclusions made by the authors. The results extend our knowledge of developmental strategies used to form neural circuits.

Reviewer #3 (Public Review):

This study asks a simple question and provides a clear and convincing answer. The question is whether sister neurons derived from the same progenitor, using Notch receptor signaling as the underlying cell fate determinant, share pre- and post-synaptic partners. The answer, not in the case of V2a and V2b neurons in the fish spinal cord. Authors show that V2a and V2b neurons derived from the same progenitor are recruited to distinct spinal neural networks.

Reviewer #1 (Public Review):

3' UTR-derived sRNAs are increasingly recognized as post-transcriptional regulators of diverse bacterial processes. While initially assumed to be highly specific regulators with single targets, global RNA interactome approaches challenge this view by suggesting 3'-derived sRNAs can bind and regulate multiple target mRNAs, reminiscent of the regulons of intergenic sRNAs (PMID: 35388892). In Enterobacteriaceae, GlnZ is an sRNA derived from the 3' UTR of the glnA mRNA (PMID: 15718303) that encodes glutamine synthetase. However, the role and function of GlnZ have not previously been determined. In the present work, the authors set out to investigate GlnZ in E. coli and Salmonella enterica. In doing so, they uncover the mechanism of GlnZ biogenesis, namely RNase E-mediated release from the parental glnA mRNA. Additionally, they identify several GlnZ targets (involved in carbon/nitrogen metabolism), some of which are conserved between E.coli and Salmonella while others are species-specific. Downstream mutational characterization of sRNA variants and experiments with target reporter constructs allow them to map sRNA-target interaction sites at nucleotide resolution. Together, their findings further support the idea that 3'-derived sRNAs, too, can act as more global post-transcriptional regulators with multiple direct targets.

This is a thoroughly conducted study and both, important and timely. As the corresponding author points out in the cover letter, this is an instance of similar findings being simultaneously reported by more than one group (see preprint from Gisela Storz' lab: https://www.biorxiv.org/content/10.1101/2022.04.01.486790v1). The results of these two, independently conducted studies largely agree and complement one another.

Reviewer #2 (Public Review):

Miyakoshi et al. investigated the function of the small RNA GlnZ in E. coli and Salmonella. GlnZ originates from the 3'UTR of the glnA mRNA that encodes glutamine synthetase (GS), the central enzyme of nitrogen assimilation in bacteria. It has been confirmed that the processing of glnA and hence GlnZ formation involves Hfq and RNaseE. The authors also reveal that GlnZ regulates the sucA gene encoding the E1o component of 2-oxoglutarate dehydrogenase (OGDH) by complementary base pairing. As 2-OG is part of the TCA cycle as well as a precursor of the GS/GOGAT cycle, GlnZ appears to function as a major element to control carbon flow from the TCA cycle to this nitrogen assimilation pathway. This is an astonishing finding as the glnA gene and many other aspects of nitrogen assimilation via GS are rather well-investigated. Obviously central regulators can still be discovered, even on mRNAs that have been investigated for decades.

The authors present a nice piece of molecular biology work which justifies the major conclusion that GlnZ is formed by processing of glnA and regulates the sucA gene post-transcriptionally. In general, the manuscript is well-written and the data are clearly presented. The figures are great and allow the reader to easily follow the descriptions. Nevertheless, some aspects referring to the actual control of metabolism could be improved. For instance, the authors claim to have proven that "GlnZ represses the expression of SucA TO REDIRECT the carbon flow from the TCA cycle to the nitrogen assimilation pathway". However, the manuscript does not contain data, e.g. of metabolite profiling using glnZ mutants, that really confirm this statement. Even though GlnZ has a significant effect on SucA abundance it is rather weak, especially in E. coli (Fig. 4B). Of course, this is not uncommon for sRNA-dependent expression control and there is no doubt about the importance of the here presented finding. The authors should either include data that indeed show any effect on 2-OG levels and/or metabolic flux through OGDH or at least temper their conclusion and say that their findings only indicate this.

Reviewer #3 (Public Review):

3' UTRs of mRNAs in bacteria have emerged as a reservoir for trans-acting small RNAs (sRNAs) processed from the full-length transcript by endonucleolytic cleavage. Most sRNAs exert their activity through the formation of imperfect base-pairing interactions with cognate target transcripts, and typically either repress or stimulate translation of bound mRNAs. Best studied in enterobacterial species like E. coli and Salmonella, sRNAs oftentimes rely on the presence of an RNA chaperone, Hfq, which facilitates annealing of complementary RNAs.

In the manuscript, Miyakoshi and co-workers report on the enterobacterial sRNA GlnZ which is released from the 3'UTR of glnA mRNA through RNase E cleavage. Miyakoshi and co-workers demonstrate how GlnZ is induced under nitrogen-limiting conditions. Employing a conserved seed sequence, GlnZ post-transcriptionally regulates target mRNAs, including sucA and aceE mRNAs. When inhibiting RNase E-mediated processing (through mutation of recognition sites), SucA regulation is abrogated, suggesting that full-length glnA mRNA is inactive as a post-transcriptional regulator.

A characterization of GlnZ, mainly focusing on the E. coli K12 variant, has recently been published elsewhere (Walling et al., 2022, NAR), and it is important to highlight additional findings of this manuscript.

One strength of the manuscript is the comparison of GlnZ-mediated regulation between two different enterobacterial species, Salmonella and E. coli, however this aspect should be assessed more thoroughly. The authors have identified additional targets through a pulse-expression experiment of GlnZ in Salmonella, but the Salmonella-specific targets await validation.

The mechanism by which GlnZ represses its targets sucA and aceE mRNAs through binding far upstream of the ribosome binding site is interesting but not discussed.

The authors speculate on the role of translation regarding the question why GlnZ but not glnA mRNA are able to engage in target regulation. Given the variation in sequence among different enterobacteria it is an open question whether the distance between the translation stop and the sRNA seed influences the regulatory activity.

Reviewer #1 (Public Review):

In this article, Susswein and colleagues use SafeGraph mobile device location data to characterize seasonal trends in indoor activity in the United States at the county level with relevance for respiratory disease transmission. They find substantial variation in indoor activity over the course of the year, ranging from roughly 25% (summer trough) to 200% (winter peak) of the average/baseline indoor activity in each county. Additionally, they identify two main regions with distinct seasonal trends in indoor activity: one in the north, where indoor activity follows a roughly standard sinusoidal trend, and one in the south where indoor activity may feature an additional summer peak. They also identify a third minor region with spring and fall peaks in indoor activity, corresponding to mountainous areas that are hubs for winter tourism. Using a simple mathematical disease transmission model, they demonstrate that using different seasonal forcing terms as inputs can yield substantially different epidemic curves.

This study's main strength is the volume and resolution of the data. Because of this, the authors are able to provide convincing evidence that seasonal variation in indoor activity exists, that it is substantial, and that it varies geographically across the US. Another important strength is the approach that the authors used to identify regions with similar seasonal trends in indoor activity. By using a network community detection algorithm, they were able to avoid making a priori assumptions about the number, size, and geographic connectivity of the regions, allowing them to make better use of the data itself to inform the delineation between regions.

Despite the volume of the underlying dataset, it is geographically limited to the United States and only captures the locations of mobile devices for which their users have opted in to sharing location data. This calls into question the generalizability of the findings to other countries and to other populations within the US that may have reduced access to mobile devices or may be less likely to share location data. The assessment of between-county differences in seasonal indoor activity trends and the assessment of the impact of the COVID-19 pandemic on indoor activity could benefit from greater detail, as they currently rest mainly on visual inspection of the trends.

Overall, the authors have largely achieved their aims of characterizing indoor seasonal activity in the United States at a fine geographic resolution. This work will be immediately useful for the construction of more evidence-based infectious disease transmission models. The authors have made available their estimates of seasonal deviation in indoor activity at the county level, which can be incorporated directly into disease transmission models. Their descriptions are also sufficient for building models that do not incorporate the full county-level detail but nevertheless account for important regional differences in indoor activity across the US.

Reviewer #2 (Public Review):

Susswein et al. analyze a fine-scale, novel data stream of human mobility, openly available from Safegraph, based on the usage of mobile apps with GPS and sampled from over 45 million smartphone devices. They define a metric $\sigma_{it}$, properly normalized, that quantifies the propensity for visits to indoor locations relative to outdoor locations in a given county $i$ at week $t$. For each pair of counties $i$ and $j$, they compute the Pearson correlation coefficient $\rho_{ij}$ between the corresponding $\sigma$ metrics. This generates a correlation matrix that can be interpreted as the adjacency matrix of a network. They then perform community detection on this network/matrix, effectively clustering together time series that are correlated. This identifies three main clusters of counties, characterized geographically as either in the north of the country, in the south of the country, and possibly in tourism active areas. They then show, via a simple model, how including over-simplified models of seasonality may affect infectious disease models.

This work is very interesting for the infectious disease modeling community, as it addresses a complex problem introducing a new data stream.

This work builds on several strengths, among which:<br /> It is the first analysis of the Safegraph dataset to capture seasonality in indoor behavior.<br /> It provides a simple metric to quantify indoor activity, that thanks to the dataset can be computed with a high level of spatial detail.<br /> It aims at characterizing clusters of counties with a similar pattern of indoor activity.<br /> It aims at quantifying the impact of neglecting finer-scale patterns of seasonality, for example considering seasonality to be homogeneous at the US level.

At the same time, it presents several weaknesses that should be addressed to improve the methodology, its results, and the implication:<br /> There is no quantitative comparison of the newly introduced metric for indoor activity with other proxies of seasonality (e.g. temperature or relative humidity). The (dis)similarity with other proxies may help in assessing the importance of this metric, showing why it can not be exchanged with other data sources (like temperature data) that are widely available and are not affected by sampling issues (more on that later).<br /> A major flow of the analysis is to perform community detection on a network defined by the correlation between time series with an algorithm that is based on modularity optimization. As explained in Macmahon et al.[1], all modularity optimization methods rely on null assumptions that in the case of correlation between time series are violated. Therefore, there is a very strong potential bias in their results that is not accounted for. Possible solutions could be to proceed via the methodology presented in [1] or via a different type of algorithm (e.g. Infomap [2]). In both cases, as the network is thresholded (considering only a correlation larger than 0.9), a more quantitative assessment of the impact of the threshold value should be included.<br /> It is not clear what is the added value of the data on indoor activity, as no fitting to real data is performed. Although this may be considered beyond the scope of this paper, I think it would be crucial to quantify how much a data-informed model would better describe real epidemic data (for example in the case of COVID-19). For now, only the impact of neglecting heterogeneity in indoor activity is shown, comparing a model with region-average parameters vs a model with county-level average parameters. Given that the dataset comes with potential bias in sampling (more on this later) it would be good to assess its goodness in predicting real epidemic spread.<br /> When showing results from different models, no visible errors are shown on the plot. How have the errors been estimated?<br /> The dataset is presented as representative of the US population. However, this has not been assessed over time. As adherence to social distancing is influenced by several socio-economic determinants the lack of representativity in certain strata of the population at a given time may introduce an important bias in the dataset. Although this is an inherent limitation of the dataset, it should be discussed in the paper more thoroughly.

In conclusion, I think that the methodology should be revised to account for the fact that the analysis is performed on a correlation matrix. Capturing seasonal patterns of indoor activity can help in tackling the crucial problem of seasonality in human behavior. This could help in identifying effective strategies of disease containment able to curb disease spread at a lower societal cost than fully-fledged lockdowns.

References<br /> [1] Mel MacMahon and Diego Garlaschelli Phys. Rev. X 5, 021006 (2015).<br /> [2] Martin Rosvall and Carl T. Bergstrom PNAS 105, 1118 (2008).

Reviewer #3 (Public Review):

The authors used smartphone-based mobility data to assess indoor and outdoor activities. By doing so, they were able to show seasonality in the ratio between indoor and outdoor activities and to relate it to a certain extent to seasonality in infectious diseases. They were also able to show that data at the county level is necessary to achieve proper assessment of behavior and that the COVID pandemic considerably impacted behavior patterns.

The major strength of the paper is the simplicity of the concept (proportion of indoor activities compared to outdoor activities), which makes it very straightforward to understand. Another strength is the considerable amount of data (5 million locations) that have been taken into account, and the comparison between the 3 years.

There is nonetheless a limitation in the interpretation of the results, as the definition of indoor and outdoor is not always easy, and most importantly that home is not part of the considered locations. This is a limitation clearly exposed by the authors and their discussion reflects it.

Authors have been able to demonstrate how human behavior could influence seasonality, among others factors, and is not strictly related to climate or weather conditions. Moreover, they used the results to show how COVID impacted behavior (whether because of the disease or non-pharmaceutical interventions), and how precise data are necessary to perform appropriate modelling.

This is an important article, as it shows the potential influence of human behavior on infectious diseases seasonality, but also a very straightforward method that could be reproduced easily.

Finally, it also confirms the necessity to take into account the seasonality of human behavior in future modelling, in order to provide relevant information to public health deciders.

Reviewer #1 (Public Review):

This paper elucidates the developmental-genetic mechanisms that generate the winged and wingless form (morph) of female pea aphids (Acyrthosiphon pisum). Pea aphids reproduce parthenogenetically generating genetically identical offspring, and so the difference between the winged and wingless morphs is environmentally induced (referred to as a polyphenism). Previous studies have shown that the crowding of mothers is sufficient to induce the winged phenotype in the offspring. The authors develop a technique so that they can reliably generate wing-destined (WD) or wingless-destined (WLD) offspring. This allowed them to examine the early development of WD and WLD offspring during the 1st nymphal stage, before wing development can be observed externally, in the 3rd nymphal stage. They find that the wing primordia are apparent in both WD and WLD 1st instars immediately after birth, but that the primordia degrades 30-36h after birth in WLD nymphs. They then demonstrate that this degeneration is due to autophagy rather than apoptosis, evident through the increased expression of autophagy-related genes in WLD nymphs, but not pro-apoptotic genes. The authors next ask what is responsible for inducing autophagy in the WLD nymphs and so transcriptomics to look for genes that are differentially up- or down-regulated in 1st insar WLD versus WD nymphs. One gene, REPTOR2, is markedly down-regulated in WLD versus WD nymphs. REPTOR is an established target of TOR-signaling, which is in turn an established regulator of autophagy. The authors, therefore, focus on REPTOR2 and show that it has arisen through gene duplication of REPTOR in the A. pisum lineages, that it is differentially upregulated in the thorax of WLD versus WD nymphs, and that knock-down of REPTOR2 both reduces levels of the autophagic protein ATG8 in the wing primordia of 1st instar nymph and increases the proportion of winged offspring. Finally, the authors demonstrate that TOR, which canonically represses RAPTOR, negatively regulates autophagy of the wing primordia and positively regulates the generation of the winged morph.

The strength of this paper is that it cleanly implicates a novel gene, REPTOR2, that has arisen through gene duplication, in the generation of alternative morphs in a polyphenism. The paper also provides compelling evidence that the degeneration of the wing primordia in wingless aphids is through autophagy rather than apoptosis. Further, the paper provides another example of how signaling pathways known to be involved in the generation of reaction norms (continuous phenotypic responses to environmental variation) are also implicated in the generation of polyphenisms (discrete phenotypic responses to environmental variation). The paper uses a reliable and reproducible technique to generate wing and wingless forms of aphids upon which developmental studies can be conducted. The results of the paper are straightforward and convincing. A weakness of the paper is that, while it implicates both RAPTOR2 and TOR-signaling in the generation of winged and wingless morphs, it does not provide a causal link between the two. REPTOR (Repressed by TOR) is known to be a regulator of TOR-signaling in Drosophila, activating transcriptional stress response upon TOR inhibition, and the authors argue that REPTOR2 serves to exert a negative effect of TOR signaling on autophagy initiation in wingless aphids. Nevertheless, their data do not unambiguously show this. Specifically, they do not demonstrate that REPTOR2 is downstream of TOR in the signaling pathway that regulates winglessness.

Reviewer #2 (Public Review):

The developmental mechanisms underlying insect polyphenisms are understood for only a few species. Previous studies in pea aphids and planthoppers have shown that insulin signalling is important for differences in wing morphs across environmental conditions. In the pea aphid, mothers that are crowded produce a high proportion of offspring with wings, while mothers housed alone produce offspring without wings. The authors emphasise that in the pea aphid wing loss is a novel trait, and work to identify the developmental processes that lead to wing loss. They find that wing loss is induced by autophagy of the wing disc in the first instar nymphs. Using a transcriptomics approach, they identify a candidate gene, REPTOR2, whose expression is enriched in nymphs destined to become wingless. REPTOR2 is a novel gene that has arisen from duplication in REPTOR. They further demonstrate that reducing REPTOR2 expression by RNAi increases the proportion of winged nymphs. Similarly, reducing the target of rapamycin signalling or feeding mothers on a low-protein diet decreased the proportion of winged nymphs. The authors conclude from these studies that in crowded mothers, high TOR signalling represses REPTOR2 activity leading to reduced autophagy in the wing discs.

Strengths:<br /> 1. The authors have outlined very clear hypotheses and aims, which makes the arguments in the text very easy to follow.<br /> 2. This study is very carefully conducted, and the authors use multiple lines of approach to validate their claims (eg confocal imaging of wing discs to examine ATG8 expression and TUNEL staining to differentiate between autophagy or apoptosis respectively, followed up by qPCR for autophagy and apoptosis genes).<br /> 3. Experiments are appropriately quantified and are backed by statistical tests.<br /> 4. The results lend excellent support to the author's claims.

Weaknesses: The authors do not make a direct link between TOR and REPTOR2 signalling. This seems important since REPTOR2 is a novel gene that arose from the duplication of REPTOR.

Reviewer #1 (Public Review):

In a very interesting and technically advanced study, the authors measured the force production of curved protofilaments at depolymerizing mammalian microtubule ends using an optical trap assay that they developed previously for yeast microtubules. They found that the magnesium concentration affects this force production, which they argue based on a theoretical model is due to affecting the length of the protofilament curls, as observed previously by electron microscopy. Comparing with their previous force measurements, they conclude that mammalian microtubules produce smaller force pulses than yeast microtubules due to shorter protofilament curls. This work provides new mechanistic insight into how shrinking microtubules exert forces on cargoes such as for example kinetochores during cell division. The experiments are sophisticated and appear to be of high quality, conclusions are well supported by the data, and language is appropriate when conclusions are drawn from more indirect evidence. Given that the experimental setup differs from the previous optical trap assay (antibody plus tubulin attached to bead versus only antibody attached to bead), a control experiment could be useful with yeast microtubules using the same protocol used in the new variant of the assay, or at least a discussion regarding this issue. One open question may be whether the authors can be sure that measured forces are only due to single depolymerizing protofilaments instead of two or more protofilaments staying laterally attached for a while. How would this affect the interpretation of the data?<br /> This work will be of interest to cell biologists and biophysicists interested in spindle mechanics or generally in filament mechanics.

Reviewer #2 (Public Review):

Microtubules are regarded as dynamic tracks for kinesin and dynein motors that generate force for moving cargoes through cells, but microtubules also act as motors themselves by generating force from outward splaying protofilaments at depolymerizing ends. Force from depolymerization has been demonstrated in vitro and is thought to contribute to chromosome movement and other contexts in cells. Although this model has been in the field for many years, key questions have remained unanswered, including the mechanism of force generation, how force generated might be regulated in cells, and how this system might be tuned across cellular contexts or organisms. The barrier is that we lack an understanding of experimental conditions that can be used to control protofilament shape and energetics. This study by Murray and colleagues makes an important advance towards overcoming that barrier.

This study builds on previous work from the authors where they developed a system to directly measure forces generated by outward curling protofilaments at depolymerizing microtubule ends. That study showed for the first time that protofilaments act like elastic springs and related the generated force to the estimated energy contained in the microtubule lattice. Furthermore, they showed that slowing polymerization rate did not diminish force generation. That study used recombinant yeast tubulin, including a 6x histidine tag on beta tubulin that created attachment points for the bead on the microtubule lattice. The current study extends that system to show that work output is related to the length of protofilament curls.

Murray and colleagues show this by manipulating curls in two ways - using bovine brain tubulin instead of yeast tubulin and altering magnesium concentration. Previous EM studies indicated that protofilaments on depolymerizing bovine microtubules have similar curvature but are shorter. The authors here use a blend of bovine brain tubulin and bead-linked recombinant yeast tubulin with the 6x histidine tag in their in vitro system and find smaller deflections of the laser-trapped bead than previously observed with pure yeast tubulin. A concern with comparing this heterogeneous bovine/yeast system to the previous work with homogeneous yeast tubulin is that density of 6x histidine-tagged tubulin subunits is likely to be different between the two systems. Also, the rate of incorporation of 6x histidine yeast tubulin into bovine microtubules in the current study may be different from the rate of incorporation into yeast microtubules in the previous study. These differences could lead to changes in the strength of bead attachment to the microtubule lattice and alter the compliance of the bead to deflection by curling protofilaments. These possibilities and lattice attachment strength are not explored in this study, raising concerns about comparing the two systems.

The authors go on to show that magnesium increases bead deflection and work output from the system. The use of magnesium was motivated by earlier studies which showed that increasing magnesium speeds up depolymerization and increases the lengths of protofilament curls. The use of magnesium here provides the first evidence that work output can be tuned biochemically. This is an important finding. The authors then go on to show that the effect of magnesium on bead deflection can be separated from its effect on depolymerization speed. They do this by proteolytically removing the beta tubulin tail domain, which previous studies had shown to be necessary to mediate the magnesium effect on depolymerization rate. The authors arrive at a conclusion that magnesium must promote protofilament work output by increasing their lengths. How magnesium might do this remains unanswered. The mechanistic insight from the magnesium experiments ends there, but the authors discuss possible roles for magnesium in strengthening longitudinal interactions within protofilaments or perhaps complexing with the GDP nucleotide at the exchangeable site, although that seems less likely at the concentrations in these experiments.

The major conclusion of the study is the finding that work output from curling protofilaments is a tunable system. The examples here demonstrate tuning by tubulin composition and by divalent cations. Whether these examples relate to tuning in biological systems will be an important next question and could expand our appreciation for the versatility of depolymerizing microtubules as a motor.

Reviewer #3 (Public Review):

The authors used a previously established optical tweezers-based assay to measure the regulation of the working stroke of curled protofilaments of bovine microtubules by magnesium. To do so, the authors improved the assay by attaching bovine microtubules to trapping beads through an incorporated tagged yeast tubulin.

The assay is state-of-the-art and provides a direct measurement of the stroke size of protofilaments and its dependence on magnesium.

The authors have achieved all their goals and the manuscript is well written.

The reported findings will be of high interest for the cell biology community.

Reviewer #1 (Public Review):

In this work, Li & Meister provide an extensive description of functional cell types within the posterior-medial part of the mouse superior colliculus, corresponding to the upper lateral visual field. Presenting a battery of visual stimuli to head-fixed wild-type mouse lines, they use calcium imaging and subsequent clustering of functional responses to identify 24 functional cell types. Besides the comprehensive sampling of SC cell types, a major strength of the manuscript in my view is the direct comparison with the previously published in vitro RGC data. Overall, the manuscript and the associated data promise to be a valuable resource to experimentalists and computational neuroscientists comparing visual processing across the major processing stages of the mouse visual system. However, in the current form the manuscript still comes with some limitations. In my view, these are related to some parts, where statistical justifications for the conclusions are still missing, where the findings should be more strongly embedded into the current and past literature, and where more efforts should be made to relate the findings in the different mouse lines to those for the overall population.

Reviewer #2 (Public Review):

This paper has collected an impressive data set of the visual response properties of neurons in the visual layers of the mouse superior colliculus. There are 3 main findings of the study. First, the authors identify 24 functional classes of neurons based on the clustering of each neuron's visual response properties. Second, unlike in the retina where each cell type is regularly spaced, functional classes in the superior colliculus appear to cluster near each other. Third, visual representation has a lower dimensionality in the superior colliculus compared to the retina. The dataset has the potential to support the conclusions of the paper, but further analysis is required to make the claims convincing.

Strengths:

The main strength of the paper is its impressive dataset of more than 5000 neurons from the visual layers of the superior colliculus. This data set includes recordings from both an interesting set of genetically labelled classes of cells and from a reasonably large portion of the superior colliculus. This dataset offers the opportunity to support the major claims of the paper. This includes i) the identification of 24 functional classes of neurons, ii) the intriguing possibility that functional classes form local patches within the superior colliculus and iii) that the representation of visual information in the superior colliculus has a lower dimensionality compared to the retina.

Weaknesses:

The weakness of the paper is that its main claims are not adequately supported by the presented data or analysis. First, support for the existence of 24 functional classes is not clear enough. Our major concern is that it is not clear that each class of neurons was distributed across different mice. Are certain cell types overrepresented in individual animals, or do you find examples of each cell type in most animals? In addition, it should be made explicit how the responses of each genetically labeled class of neurons are distributed among the 24 functional clusters. Second, the analysis of the spatial clustering of functional cell types is not complete. Do the same functional clusters sample the same retinotopic locations in different mice? How are clusters of the functional type distributed in visual space? Third, the lower dimensionality of representation in the superior colliculus may be the result of selective projections of retinal ganglion cells, not all retinal ganglion cell types project to the superior colliculus. Please estimate the dimensionality of the visual representation of those retinal ganglion cell types that projects to the superior colliculus.

Reviewer #3 (Public Review):

How parallel retinal outputs are processed in recipient visual areas is largely unknown. The present paper tackles this issue in the mouse superior colliculus - a key target of retinal outputs. Calcium signals of SC neurons were measured in response to a set of stimuli known to differentiate retinal ganglion cells. The resulting responses were then clustered to identify distinct cell types. These measurements were repeated in several transgenic mice with specific subsets of SC neurons labeled. The experiments and analysis generally support the conclusions well. There are several places, however, where the work could be presented more clearly.

Reviewer #1 (Public Review):

The authors have generated a set of seven nanobody tools against two of the largest Drosophila proteins, which are related to vertebrate titin and essential for muscle function. The study of such gigantic proteins is a challenge. They show that each of these nanobodies recognizes their epitope with high affinity (as expected from antibodies), fails to generate a signal after immune-fixation of a mutant for the cognate protein, do not cross-react with each other, and generates a signal in the muscle that makes sense with what one would anticipate for fly titin homologs. In addition, they show that these nanobodies have better penetration and labeling efficiency than conventional antibodies in thick tissues after classical paraformaldehyde fixation. Using these nanobodies, they could deduce the organization of the epitopes in different muscle types and propose a model for Sallimus and Projectin arrangement in muscles, including in larvae which are difficult to label with traditional antibodies due to their impermeable chitin skeleton. Finally, they could fuse the gene encoding one of the nanobodies to the open reading frame of NeonGreen and express the corresponding fusion protein in animals to use the probe in FRAP assays.

The work is very well performed and convincing. However, given its significant redundancy in terms of biological conclusions with the companion study "Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nano-architecture in flight muscles" by the same authors, and other published papers, I recommend the authors further prove the use of their nanobodies in live assays. In particular, the authors should test whether they can use the nanobodies to induce protein degradation either permanently or conditionally.

Reviewer #2 (Public Review):

The data presented in this manuscript are sound but rather descriptive. The contribution - as presented - is mostly of a technical nature. The authors correctly state that anti-GFP nanobodies, while used extensively across many model organisms, have limited utility for in vivo applications when the GFP-tagged protein in question displays abnormal behavior or is non-functional. The creation of nanobodies that are uniquely specific for the protein(s) of interest is therefore a significant improvement, especially since the Sallimus and Projectin-specific reagents reported here react with PFA-fixed material. At least one of these nanobodies, when expressed in vivo, decorates the appropriate target. The source of antigens used for the construction of the nanobody library is Drosophila-derived. The extent of homology of Drosophila Sallimus and Projectin with related proteins in other species is not discussed. Whether the nanobodies reported here would be useful in other (closely related?) species, therefore, remains to be established. For those studying muscle biology in Drosophila, the nanobodies described here will be publicly available as cDNAs. Ease of production implies a readily shared and standardized resource for the field.

Further characterization of these nanobodies by biochemical methods such as immunoblotting would be challenging, given the size of the target proteins. In view of the technical nature of this manuscript, the authors should perhaps critically discuss the distinction between bulky GFP tags versus the much smaller epitope tags and the nanobodies that recognize them, although this was covered in a recent eLife paper from the Perrimon lab. Insertion of small tags, in conjunction with nanobodies that recognize them, would be less perturbing than the much bulkier GFP tag and lend itself to genome-wide applications. Creating nanobodies uniquely specific for each protein encoded in the Drosophila genome is not realistic, and the targeted approach deployed here is obviously valuable.

The authors apply two different approaches to characterize the newly generated Nanobodies: more or less conventional immunohistochemistry with fluorescently labeled nanobodies, and in vivo expression of nanobodies fused to the fluorescent neongreen protein. The superiority of nanobodies in terms of tissue penetration has been shown by others in a direct comparison of intact fluorescently labeled immunoglobulins versus nanobodies. The authors state that in vivo labeling with nanobody fusions "thus far was done only with nanobodies against GFP, mCherry or short epitope tags." There is no fundamental difference between these recognition events and what the authors report for their Sallimus and Projectin-specific reagents. The section that starts at line 304 is thus a little bit of a 'straw man'. There is no reason to assume that a nanobody that recognizes a muscle protein would behave differently than a nanobody that would recognize that same protein (or another) when epitope- or GFP-tagged. What might be interesting is to examine the behavior of these muscle-specific nanobodies in the course of muscle contraction/relaxation: are there conformational alterations that promote dissociation of bound nanobodies? Do different nanobodies display discrete behavior in this regard? The manuscript is silent on how muscles behave in live L3 larvae. The FRAP experiment seems to suggest that not much is happening, but the text refers to the contraction of larval sarcomeres from 8.5 µM to 4.5 µM. Does the in vivo expressed nanobody remain stably bound during this contraction/relaxation cycle? What about the other nanobodies reported in this manuscript? Since the larval motion was reduced by exposure to diethylether, have the authors considered imaging the contractive cycle in the absence of such exposure?

Reviewer #3 (Public Review):

Loreau et al. have presented a well-written manuscript reporting clever, original work taking advantage of fairly new biotechnology - the generation and use of single chain antibodies called nanobodies. The authors demonstrate the production of multiple nanobodies to two titin homologs in Drosophila and use these nanobodies to localize these proteins in several fly muscle types and discover interesting aspects of the localization and span of these elongated proteins in the muscle sarcomere. They also demonstrate that one of these single chain antibodies can be expressed in muscle fused to a fluorescent protein to image the localization of a segment of one of these giant proteins called Sallimus in muscle in a live fly. Their project is well-justified given the limitations of the usual approaches for localizing and studying the dynamics of proteins in the muscle of model organisms such as the possibility that GFP tagging of a protein will interfere with its localization or function, and poor penetration of large IgG or IgM antibodies into densly packed structures like the sarcomere after fixation as compared to smaller nanbodies.

They achieved their goals consistent with the known/expected properties of nanobodies: (1) They demonstrate that at least one of their nanobodies binds with very high affinity. (2) They bind with high specificity. (3) The nanobodies show much better penetration of fixed stage 17 embryos than do conventional antibodies.

They use their nanobodies mostly generated to the N- and C-terminal ends of Sallimus and Projectin to learn new information about how these elongated proteins span and are oriented in the sarcomere. For example, in examining larval muscles which have long sarcomeres (8.5 microns), using nanobodies to domains located near the N- and C-termini, they show definitively that the predicted 2.1 MDa protein Sallimus spans the entire I-band and extends a bit into the A-band with its N-terminus embedded in the Z-disk and C-terminus in the outer edge of the A-band. Using a similar approach they also show that the 800 kDa Projectin decorates the entire myosin thick filament except for the H-zone and M-line in a polar orientation. Their final experiment is most exciting! They were able to express in fly larval muscles a nanobody directed to near the N-terminus of Sallimus fused to NeonGreen and show that it localizes to Z-disks in living larvae, and by FRAP experiments demonstrate that the binding of this nanobody to Sallimus in vivo is very stable. This opens the door to using a similar approach to study the assembly, dynamics, and even conformational changes of a protein in a complex in a live animal in real time.

There are only a few minor weaknesses about their conclusions: (1) They should note that in fact their estimate of the span of Sallimus could be an underestimate since their Nano2 nanobody is directed to Ig13/14 so if all of these 12 Ig domains N-terminal of their epitope were unwound it would add 12 X 30 nm = 360 nm of length, and even if unwound would add about 50 nm of length. (2) They discuss how Sallimus and Projectin are the two Drosophila homologs of mammalian titin, however, they ignore the fact that there is more similarity between Sallimus and Projectin to muscle proteins in invertebrates. For example, in C. elegans, TTN-1 is the counterpart of Sallimus, and twitchin is the counterpart of Projectin, both in size and domain organization. The authors present definitive data to support Figure 9, their nice model for a fly larval sarcomere but fail to point out that this model likely pertains to C. elegans and other invertebrates. In Forbes et al. (2010) it was shown that TTN-1, which can be detected by western blot as ~2 MDa protein and using two polyclonal antibodies spans the entire I-band and extends into the outer edge of the A-band, very similar to what the authors here have shown, more elegantly for Sallimus. In addition, several studies have shown that twitchin (Projectin) does not extend into the M-line; the M-line is exclusively occupied by UNC-89, the homolog of Obscurin.

Reviewer #1 (Public Review):

The authors found that the IDR in Cdc15 gets phosphorylated by multiple kinases, Pom1/Shk1/Pck1/Kin1, and the phosphorylation on IDR inhibits the phase separation of the Cdc15 protein. The phosphorylation was demonstrated in the cell as well as in vitro. Moreover, the phosphorylation sites were identified by mass spectrometry. The phospho-regulation of Cdc15 LLPS was demonstrated by in vitro assay using recombinant proteins. The significance of the phosphorylation on contractile actomyosin ring (CAR) was demonstrated by using a cdc15 mutant carrying 31 Ala-substitutions at the phosphorylation sites (cdc15 31A). The CAR assembled comparable to cdc15+, but maturation and contraction of the ring were faster in the cdc15 31A mutant, suggesting the contribution of the phosphorylation for delaying cytokinesis. This could be one of the mechanisms to ensure the completion of chromosome segregation before the cytokinesis. In this paper, the authors showed over-accumulation of type-II myosin regulatory light chain Rlc1 on CAR in the cdc15 31A mutant during the CAR assembly and its contraction. In addition, the kinases for the Cdc15 IDR phosphorylation are identified as polarity kinases, which restrict the assembly of the CAR formation in the middle. Indeed, inhibition of the kinases increases the ratio of septa formation at the cell tip in the mid1 knockout mutant, which lacks a major positive polarity cue during the mitotic phase. However, in this manuscript, this phenotype is not solely explained by the phosphorylation of the cdc15 31A, because the authors did not show the tip septa formation using cdc15 31A.

Overall, the data supports their conclusion, Cdc15 forms LLPS, and the process is inhibited by the phosphorylation of amino acid residues in the IDR in Cdc15 by polarity kinases. It is still unclear whether LLPS formation is a reversible process regulated by the protein kinases. In vitro experiments showed condensate formation by dephosphorylation of Cdc15 IDR but not diffusion of the LLPS by phosphorylation. I wonder if incubation of the kinases and the Cdc15 IDR condensates induces demolition of the LLPS.

The transition of the Cdc15 IDR phosphorylation and LLPS formation through the cell cycle progression is unclear. In asynchronous cells (most of the cells may be in the G2 phase) and nda3 or cps1 mutants, Cdc15 was still highly phosphorylated. This indicates that the Cdc15 is phosphorylated and the LLPS formation is inhibited throughout the cell cycle. The transition of the phosphorylation status for individual residues could be the next challenge for this research. In addition, currently, there is no approach to monitor the LLPS in wild-type cells. Therefore, it is still unclear if LLPS formation is the physiological mechanism regulating cell division in wild-type cells.

Reviewer #2 (Public Review):

This is a very interesting paper that described how multiple kinases regulate the phase separation of Cdc15 and thus impact its localization and function during cytokinesis. The authors build upon their prior research to show that Cdc15 is phosphorylated in its intrinsically disordered region at multiple sites by different kinases. Molecular simulations suggest that phosphorylation of Cdc15 impacts its F-BAR domain's ability to interact with the membrane. Indeed, the authors show that non-phosphorylatable Cdc15 mutants appear in larger dynamic clusters in the cells and also increase the recruitment of cytokinetic proteins to the actomyosin ring. Furthermore, the authors show that the purified Cdc15 intrinsically disordered region undergoes phase separation when treated with a phosphatase. Also, the phase-separated region can recruit cytokinetic proteins that are known interacting partners of Cdc15. Overall, this is a very well-designed study that provides a deep mechanistic insight into how the Cdc15 scaffold conformation is regulated so that it can bind other proteins and interact with the plasma membrane to facilitate cytokinesis. However, the authors do not show if the sites identified here are specifically involved in phase separation. The authors provide evidence that Cdc15 undergoes phase separation when dephosphorylated by a phosphatase. However, it is not shown if dephosphorylation at the sites identified is indeed responsible for the phase separation. It would be helpful to show whether the purified cdc15-31A mutant protein also undergoes phase separation and increased interaction with cytokinetic proteins even in the absence of phosphatase treatment. This would provide strong evidence that indeed the kinases phosphorylate the identified sites to prevent phase separation.

Reviewer #1 (Public Review):

This paper tests whether people vary their reliance on episodic memory vs. incremental learning as a function of the uncertainty of the environment. The authors posit that higher uncertainty environments should lead to more reliance on episodic memory, and they find evidence for this effect across several kinds of analyses and across two independent samples.

The paper is beautifully written and motivated, and the results and figures are clear and compelling. The replication in an independent sample is especially useful. I think this will be an important paper of interest to a broad group of learning, memory, and decision-making researchers. I have only two points of concern about the interpretation of the results:

1. My main concern regards the indirect indicator of participants' use of episodic memory on a given trial. The authors assume that episodic memory is used if the value of the chosen object (as determined by its value the last time it was presented) does not match the current value of the deck it is presented in. They find that these mismatch choices happen more often in the high-volatility environment. But if participants simply choose in a more noisy/exploratory way in the high volatility environment, I believe that would also result in more mismatched judgments. What proportion of the trials labeled as episodic should we expect to be a result of noise or exploration? It seems conceivable that a judgment to explore could take longer, and result in the observed RT effects. Perhaps it could be useful to match up putative episodic trials with later recognition memory for those particular items. The across-subjects correlations are an indirect version of this, but could potentially be subject to a related concern if participants who explore more (and are then judged as more episodic) also simply have a better memory.

2. The paper is framed as tapping into a trade-off between the use of episodic memory vs. incremental learning, but it is not clear why participants would not use episodic memory in this particular task setup whenever it is available to them. The authors mention that there is "computational expense" to episodic memory, but retrieval of an already-established strong episodic memory could be quite effortless and even automatic. Why not always use it, since it is guaranteed in this task to be a better source of information for the decision? If it is true that RT is higher when using episodic memory, that is helpful toward establishing the trade-off, so this links to the concern above about how confident we can be about the use of episodic memory in particular trials.

Reviewer #2 (Public Review):

This manuscript addresses the broad question of when humans use different learning and memory systems in the service of decision-making. Previous studies have shown that, even in tasks that can be performed well using incremental trial-and-error learning, choices can sometimes be based on memories of individual past episodes. This manuscript asks what determines the balance between incremental learning and episodic memory, and specifically tests the idea that the uncertainty associated with each alters the balance between them in a rational way. Using a task that can separate the influence of incremental learning and episodic memory on choice in two large online samples, several lines of evidence supporting this hypothesis are reported. People are more likely to rely on episodic memory in more volatile environments when incremental learning is more uncertain and during periods of increased uncertainty within a given environment. Individuals with more accurate episodic memories are also more likely to rely on episodic memory and less likely to rely on incremental learning. These data are compelling, even more so because all of the main findings are directly replicated in a second sample. These data extend the notion of uncertainty-based arbitration between different forms of learning/memory, which has been proposed and evaluated in other contexts, to the case of episodic memory versus incremental learning.

The weaknesses in the paper are mostly minor. One potential weakness is the nature of the online sample. Many participants apparently did not respond to the volatility manipulation, making it impossible to test whether this altered their choices. It is unclear whether this is a feature of online samples (where people can be distracted, unmotivated, etc.) or of human performance more generally.

Reviewer #3 (Public Review):

The purpose of this work is to test the hypothesis that uncertainty modulates the relative contributions of episodic and incremental learning to decisions. The authors test this using a "deck learning and card memory task" featuring a 2-alternative forced choice between two cards, each showing a color and an object. The cards are drawn from different colored decks with different average values that stochastically reverse with fixed volatility, and also feature objects that can be unfamiliar or familiar. Objects are not shown more than twice, and familiar objects have the same value as they did when shown previously. This allows the authors to construct an index of episodic contributions to decision-making: in cases where the previous value of the object is incongruous with the incrementally observed value, the subject's choice reveals which strategy they are relying on.

The key manipulation is to introduce high- and low- volatility conditions, as high volatility has been shown to induce uncertainty in incremental learning by causing subjects to adopt an optimal low learning rate. The authors find that the subjects show a higher episodic choice index in the high-volatility condition, and in particular immediately after reversals when the model predicts uncertainty is at a maximum. The authors also construct a trial-wise index of uncertainty and show that episodic index correlates with this measure. The authors also find that at the subject level, the overall episodic choice index correlates with the ability to accurately identify familiar objects, and the reason that this indicates higher certainty in episodic memory is predicting the usage of episodic strategies. The authors replicate all of their findings in a second subject population.

This is a very interesting study with compelling results on an important topic. The task design was a clever way to disentangle and measure different learning strategies, which could be adopted by others seeking to further understand the contributions of different strategies to decision-making and its neural underpinnings. The article is also very clearly written and the results clearly communicated.

A number of questions remain regarding the interpretation of the results that I think would be addressed with further analysis and modeling.

At a conceptual level, I was unsure about the equivalence drawn between volatility and uncertainty: the main experiments and analyses all regard reversals and comparisons of volatility conditions, but the conclusions are more broadly about uncertainty. Volatility, as the authors note, is only one way to induce uncertainty. It also doesn't seem like the most obvious way to intervene on uncertainty (eg manipulated trial-wise variance seems more obvious). The trial-wise relative uncertainty measurements in Fig 4 speak a bit more to the question of uncertainty more generally, but these were not the main focus and also do not disambiguate between trial-wise uncertainty derived from reversals versus within block variation.

Another key question I had about design choice was the decision to use binary rather than drifting values. Because of this, the subjects could be inferring context rather than continuously incrementing value estimates (eg Gershman et al 2012, Akam et al 2015): the subjects could be inferring which context they are in rather than tracking the instantaneous value + uncertainty. I am not sure this would qualitatively affect the results, as volatility would also affect context confidence, but it is a rather different interpretation and could invoke different quantitative predictions. And it might also have some qualitative bearing on results: the subjects have expectations about how long they will stay in a particular environment, and they might start anticipating a context change after a certain amount of time which would lead to an increase in uncertainty not just immediately after switches, but also after having stayed in the environment for a long period of time. Moreover, depending on the variance within context, there may be little uncertainty following context shifts.

Reviewer #1 (Public Review):

Trudel and colleagues aimed to uncover the neural mechanisms of estimating the reliability of the information from social agents and non-social objects. By combining functional MRI with a behavioural experiment and computational modelling, they demonstrated that learning from social sources is more accurate and robust compared with that from non-social sources. Furthermore, dmPFC and pTPJ were found to track the estimated reliability of the social agents (as opposed to the non-social objects).

The strength of this study is to devise a task consisting of the two experimental conditions that were matched in their statistical properties and only differed in their framing (social vs. non-social). The novel experimental task allows researchers to directly compare the learning from social and non-social sources, which is a prominent contribution of the present study to social decision neuroscience.

One of the major weaknesses is the lack of a clear description about the conceptual novelty. Learning about the reliability/expertise of social and non-social agents has been of considerable concern in social neuroscience (e.g., Boorman et al., Neuron 2013; and Wittmann et al., Neuron 2016). The authors could do a better job in clarifying the novelty of the study beyond the previous literature.

Another weakness is the lack of justifications of the behavioural data analyses. It is difficult for me to understand why 'performance matching' is suitable for an index of learning accuracy. I understand the optimal participant would adjust the interval size with respect to the estimated reliability of the advisor (i.e., angular error); however, I am wondering if the optimal strategy for participants is to exactly match the interval size with the angular error. Furthermore, the definitions of 'confidence adjustment across trials' and 'learning index' look arbitrary.

As the authors assumed simple Bayesian learning for the estimation of reliability in this study, the degree/speed of the learning should be examined with reference to the distance between the posterior and prior belief in the optimal Bayesian inference.

Reviewer #2 (Public Review):

Humans learn about the world both directly, by interacting with it, and indirectly, by gathering information from others. There has been a longstanding debate about the extent to which social learning relies on specialized mechanisms that are distinct from those that support learning through direct interaction with the environment. In this work, the authors approach this question using an elegant within-subjects design that enables direct comparisons between how participants use information from social and non-social sources. Although the information presented in both conditions had the same underlying structure, participants tracked the performance of the social cue more accurately and changed their estimates less as a function of prediction error. Further, univariate activity in two regions-dmPFC and pTPJ-tracked participants' confidence judgments more closely in the social than in the non-social condition, and multivariate patterns of activation in these regions contained information about the identity of the social cues.

Overall, the experimental approach and model used in this paper are very promising. However, after reading the paper, I found myself wanting additional insight into what these condition differences mean, and how to place this work in the context of prior literature on this debate. In addition, some additional analyses would be useful to support the key claims of the paper.

(1) The framing should be reworked to place this work in the context of prior computational work on social learning. Some potentially relevant examples:

- Shafto, Goodman & Frank (2012) provide a computational account of the domain-specific inductive biases that support social learning. In brief, what makes social learning special is that we have an intuitive theory of how other people's unobservable mental states lead to their observable actions, and we use this intuitive theory to actively interpret social information. (There is also a wealth of behavioral evidence in children to support this account; for a review, see Gweon, 2021).<br /> - Heyes (2012) provides a leaner account, arguing that social and non-social learning are supported by a common associative learning mechanism, and what distinguishes social from non-social learning is the input mechanism. Social learning becomes distinctively "social" to the extent that organisms are biased or attuned to social information.

I highlight these papers because they go a step beyond asking whether there is any difference between mechanisms that support social and nonsocial learning-they also provide concrete proposals about what that difference might be, and what might be shared. I would like to see this work move in a similar direction.

(2) The results imply that dmPFC and pTPJ differentiate between learning from social and non-social sources. However, more work needs to be done to rule out simpler, deflationary accounts. In particular, the condition differences observed in dmPFC and pTPJ might reflect low-level differences between the two conditions. For example, the social task could simply have been more engaging to participants, or the social predictors may have been more visually distinct from one another than the fruits.

References<br /> (In the interest of transparency: I am not an author on these papers.)

Gweon, H. (2021). Inferential social learning: how humans learn from others and help others learn. PsyArXiv. https://doi.org/10.31234/osf.io/8n34t

Heyes, C. (2012). What's social about social learning?. Journal of Comparative Psychology, 126(2), 193.

Shafto, P., Goodman, N. D., & Frank, M. C. (2012). Learning from others: The consequences of psychological reasoning for human learning. Perspectives on Psychological Science, 7(4), 341-351.

Reviewer #1 (Public Review):

The Voeltz lab in previous work has established a physical connection between the endoplasmic reticulum and mitochondria as an organizing principle in mitochondrial fission and fusion. A key cytoplasmic protein, Drp1 and a mitochondrial outer membrane protein, Mfn1, are known to localize to nodes of interaction between the ER and mitochondria but until now, no ER membrane proteins required in this process have been described. Using a Turbo ID fused to Mfn1, the authors have identified a known ER membrane protein that interacts with functional Mfn. This ER membrane protein, which they call Aphyd1, contains putative acyl hydrolase and acyltransferase domains. Further, in compelling work combining high resolution fluorescence microscopy and gene function analysis they have described the role of this protein in the recruitment of Drp1 and Mnn1 to nodes of interaction between the ER and mitochondria and in the sequential processes of mitochondrial constriction, fission and fusion. The work is clear and nicely quantitative and adds a new molecular element to the important question of how the ER serves to organize the division and fusion of mitochondria.

Reviewer #2 (Public Review):

This study by Nguyen and Voeltz uses proximity biotinylation and advanced imaging to elucidate roles for a lipid-metabolizing enzyme in controlling sites of mitochondrial fusion and fission. Using proximity biotinylation, they identify ABHD16A, which they propose to rename Aphyd, as an ER-resident protein close to mitochondrial fusion and fission sites. They find that knockdown and overexpression of this protein affects mitochondrial morphology and rates of mitochondrial constriction and subsequent fusion and fission. They also find that mutation of two different catalytic domains within ABHD16A have similar but non-identical effects in rescue experiments of siRNA-induced phenotypes. Broadly speaking, the hydrolase domain, known to deacylate phospholipids to form lysophospholipids (primarily phosphatidylserine to lysoPS), was more important for the phenotypes, but roles were also required for the acyltransferase domain. Perturbation of lipid transfer protein activity implicated the PS/PI4P transporter ORP8 in this pathway, strongly suggesting a specific role for ABHD16A in modulating PS metabolism at these sites to promote the mitochondrial constriction, fission, and fusion machineries. This focused study builds a rigorous and compelling story centered around the role a lipid-modifying enzyme in an interesting and important cellular behavior, namely how sites of mitochondrial fission and fusion are defined. Overall, this important study presents a compelling new model for understanding how specific local lipid metabolism at ER-mitochondria contact sites could facilitate mitochondrial fission and fusion events.

Reviewer #3 (Public Review):

In this study, the authors fuse a promiscuous biotin ligase (TurboID) to mitofusin1 to identify new players involved in mitochondrial fission and fusion. They identify an ER membrane protein, ABHD16A, that has been previously established as a phospholipid hydrolase. They rename this protein as Aphyd and go on to study its role in mitochondrial fission and fusion. Using elegant cell biology techniques, their striking images and rigorous analysis convincingly show a key role for Aphyd in recruiting both fission and fusion machineries to ER-associated mitochondrial nodes. Rates of fission and fusion are markedly decreased in the absence of Aphyd. They also show that Aphyd is required for constrictions. The identification of a new player that may regulate mitochondrial fusion and fission is an exciting advance for the field. Going forward, further biochemical analysis of Aphyd's lipid-modifying activities will be needed to shed light on the mechanisms used by Aphyd to deform membranes. In this initial study, the authors provide some tantalizing clues as to how this may occur by showing that versions of Aphyd that have mutations in their lipid-modifying (acyltransferase and hydrolase) domains are impaired in their abilities to generate ER-associated mitochondrial nodes. I look forward to the next chapters of this story to learn more about how Aphyd works.

Reviewer #1 (Public Review):

This manuscript clearly demonstrates that murine malaria infection with Plasmodium chabaudi impairs B cells' interaction with T cells, rather than DCs interaction with T cells. The authors elegantly showed that DCs were activated, capable of acquiring antigens and priming T cells during P. chabaudi infection. B cells are the main APC to capture particulate antigens such as infected RBC (iRBC), while DCs preferentially take up soluble antigens. This study is important to understand how ongoing infections such as malaria may negatively affect heterologous immunizations.

Overall, the experimental designs are straightforward, and the manuscript is well-written. However, there were several limitations in this study.

Specific comments:

1. The mechanism of how the prior capture of iRBC by B cells lead to the impairment of B-T interaction was not understood. It is unclear whether the impairment of B-T cell interaction is due to direct BCR interaction with iRBC, or an indirect response to extrinsic factors induced by malaria infection.

2. Would malaria infection in MD4 mouse that carries transgenic BCR that does not recognize malaria parasite impair subsequent B cell response to HEL immunization? This may clarify whether the impairment of subsequent B cell response is BCR-specific. If malaria impairs subsequent B cell response to HEL in MD4 mouse, it might suggest that other cell types and B cell-extrinsic factors might be involved in causing the impaired B cell responses, instead of malaria affecting B cells directly.

3. MD4 mice were mentioned in the Methods in vitro RBC binding, although none of the figures described the usage of MD4 mice. This experiment data might be important to show whether RBC binding to B cells is mediated through BCR.

4. Does P. chabaudi infection have any effects on B cell uptake of subsequent antigens, such as soluble antigen PE or particulate antigen CFSE-labeled P. yoelii iRBC?

5. Is this phenomenon specific to malaria infection? Does malaria-irrelevant particulate immunization affect T-B interaction of subsequent heterologous immunization?

6. Despite the impaired Tfh and GC 8 days after immunization following malaria infection, Fig. 5F showed GP-specific IgG eventually increased to the same level as the uninfected immunized mice on day 23. Did the authors check whether these mice had a delayed Tfh and GC response that eventually increase on day 23? Are these antibody responses derived from GC, or GC-independent response?

7. Does recovery from malaria infection by antimalarial treatment rescue the B cell response to subsequent heterologous immunization?

8. Fig. 1C shows more nRBC was taken up than iRBC in B cells, but Line 142 states that "B cells bound significantly more iRBC than nRBC. Is there a mistake in the figure arrangement? Why do B cells take up for naïve RBC than iRBC?

9. Fig. S1 C and D are confusing. CD45.1+ CD45.2+ mouse did not receive labeled iRBC, but why iRBC was detected as much as 40% in the spleen of this naïve mouse?

Reviewer #2 (Public Review):

The data presented support the conclusions of the paper, and my concerns are largely conceptional in how we understand this data in the context of malaria infection in vaccination in endemic areas

1) The data is presented based on the idea that antigen uptake and presentation differ between particle and soluble antigens, and that during malaria infection particle uptake is more important due to circulating iRBCs. However, during parasite invasion of RBCs, the parasite sheds large amounts of antigen into the circulation, at least some of which would then be found in a soluble form in the circulation. Can the authors comment on this aspect of infection and if/how this may impact the interpretation of results? Do authors assume that any soluble antigen taken up and presented (via DCs?) during infection would be impacted as for GP66 soluble antigen? Or could an interaction on immune responses where the antigen is presented via both particle and soluble pathways?

2) Impact of particle antigen opsonisation on antigen uptake and presentation. The authors use parasites isolated from mice who have been infected for 6-7 days to investigate the ability of different subsets to update particle antigens. At this time point, have mice developed antibody responses that opsonise these parasites, or are antibody levels low and parasites opsonised? Would opsonised parasites, such as those coated with sera from children in a setting of chronic infection, have a different pattern/ability to be opsonised by different immune cell subsets? And/or would opsonisation change how the DC and other cell types are processing/presenting antigens? While these issues could be addressed experimentally either now or in the future, the manuscript should at least consider this issue because, during a human infection in areas of high exposure, individuals are likely to have reasonable levels of antibodies with opsonised parasites circulating.

3) While authors show that malaria infection disrupts the response to soluble antigens, the relevance directly to vaccination should be considered carefully, specifically because vaccines of soluble antigens are largely given alongside adjuvants which also will modulate DC function. Again, this could be addressed experimentally now or in the future, but definitely should be mentioned and considered when interpreting the results.

Reviewer #1 (Public Review):

This paper details the creation and data behind the website http://pandemics.okstate.edu/covid19/. The authors attempt to explore if there is a cause and effect between the detection of unusually increased mutation activity in the genomic surveillance databases and subsequent near-term surges in SARS-CoV-2 case numbers.

Overall the premise is interesting as other than following case numbers reported to health authorities and observing what is happening in another country, there is no reliable way to predict when a surge is going to occur. Unfortunately, the data demonstrate that there was no reliable metric that could be used to predict surge events. Interestingly, the website has issued a "surge alert" currently for the month of September. It will be interesting to observe whether their model indeed has predictive power or whether the current analysis is merely coincidental with the surges but not necessarily predictive of them.

Reviewer #2 (Public Review):

In this manuscript, Najer et al., perform a comprehensive bioinformatic analysis of SARS-CoV-2 sequences available from public repositories. Through a comparison with the genome sequence of the original Wuhan 2020 strain, they identify the total accumulation of non-synonymous mutations as a predictor of the evolution of new strains. The manuscript provides data for three structural proteins - spike (S), membrane (M), and envelope (E) proteins, as well as data for the non-structural RNA-dependent RNA polymerase (RDRp) protein that serves as a negative control. However, the predictivity of this approach is most marked only for the Omicron variant, with considerable variation in the predictive power of SARS-CoV-2 proteins for other variants. Focusing on a spike, the method does not detect the alpha variant or delta variant surges, which were mostly driven by changes in spike protein, although the level of sequencing data available for the delta variant might have been less. Notably, although the authors conclude that other parameters such as the ratio of non-synonymous to synonymous mutations or the rate of accumulation of non-synonymous mutations are not predictive, they appear to have similar success in predicting the omicron surge.

Reviewer #1 (Public Review):

This interesting manuscript by Goto and Miyamichi analyses calcium dynamics in the kisspeptin neurons of the arcuate nucleus of the hypothalamus during the estrous cycle and during reproductive aging in female mice. In particular, the authors succeed in tracking arcuate kisspeptin calcium activity in the same mice over 10 months, which is quite impressive and brings highly valuable information. The authors demonstrate that the frequency and the amplitude and waveforms of individual synchronous episodes of arcuate kisspeptin neuronal activation vary across the estrous cycle. Unexpectedly, however, aging does not appear to alter markedly calcium dynamics in these neurons.

Reviewer #2 (Public Review):

Goto and Miyamachi aimed to use fiber photometry to chronically record the activity of arcuate kisspeptin neurons, widely accepted as the GnRH pulse generator responsible for reproductive potential, to determine whether changes in their activity occur during the transition to reproductive senescence in female mice. The authors report that reduced estrous cycle regularity in aging mice is accompanied by changes in the amplitude, but not the frequency, of kisspeptin events. They conclude that the reduction in kisspeptin event amplitude may explain prior results showing reduced LH pulse amplitude in aged rats, potentially due to a reduction in kisspeptin expression. The following is a description of the strengths and weaknesses of this study.

Strengths: Fiber photometry recordings of kisspeptin cells in unanesthetized, freely moving mice at multiple time points over a period of months are technically impressive and a strong approach for interrogating changes in kisspeptin cell physiology that may control the reproductive lifespan of mice.

Weaknesses: Although the approach to use chronic imaging of kisspeptin cells from the same animal from 6 months to 15-18 months is impactful; this has only been conducted in two animals. The correlation between LH pulsatile secretion and kisspeptin activity has been well characterized in mice of reproductive ages in prior reports, however, no accompanying LH pulse measurements have been included in this study, and it is possible that the relationship between kisspeptin activity and LH secretion changes during the transition to acyclicity. The paper lacks a clear description of the parameters used to define acyclicity and to affirm that mice have reached reproductive senescence. Finally, the paper lacks histological analysis of the recorded kisspeptin cells, and although the viral vector used in this study has been well characterized, there is a potential for cytotoxicity from repeated imaging and long-term transfection of the vector.

Reviewer #1 (Public Review):

Ferroportin (Fpn) is a Fe2+/H+ antiporter that extrudes Fe2+ from cells and is important for iron homeostasis. Using a combination of proteoliposome assays, mutagenesis and structural studies by cryo EM the authors are aiming to demonstrate that the Fpn-transporter is also capable of Ca2+ influx, indicating a novel route for Ca2+ entry into cells.

Strengthens: The paper combines a number of different methods to robustly demonstrate the interaction of Ca2+ with the iron transporter and to show translocation of Ca2+ is not pH dependent.

Weaknesses: Fpn uses proton-gradient to drive Fe2+ efflux. The proposal is that the antiporter can also passively uptake Ca2+. This means that after Ca2+ release on the inside, Fpn would need to spontaneously rest to the outside again, which it has not evolved to do in the absence of Fe2+. To provide further support for Ca2+ uptake it is important to show that there are mutiple turnovers of the transporter, i.e., more kinetic information is needed,

The impact of this paper is the demonstration that transporters (exchangers) can also operate as facilitative transporters for other substrates. The study also implies that Ca2+ can enter cells by this pathway, but if so the physiological context of this entry route needs further investigation and/or justification.

Reviewer #2 (Public Review):

In this manuscript the authors build upon their previous work describing the structure of the iron efflux pump ferroportin. Here they examine ferroportin's capacity to bind and transport calcium ions. Previous studies indicated a binding site for calcium in Fpn and suggested that calcium binding was needed for iron efflux, while other studies found no requirement for calcium in iron efflux. Here the authors use cryo-EM to structurally characterize a calcium-bound form of Fpn and compare this form to iron- and hepcicin-bound Fpn structures. Using site directed mutagenesis, they functionally characterized the calcium binding site and kinetics of calcium transport and its effects on Fe(II) and Co(II) transport. They report that Ca2+ is transported by Fpn proteoliposomes and Fpn-overexpressing HEK cells, that Ca2+ uptake has little effect on Fe/Co transport, and that Fe/Co efflux inhibits Ca transport.

The data reported here appear to be of high quality and are convincing; the experimental design is excellent and the necessary controls are appropriately employed. A couple of issues need clarification in the text. FPN is clearly an iron efflux pump and these studies make clear that Fpn can also import Ca2+, although it does not appear to function as an Fe2+-Ca2+ antiporter. What is less clear is whether Fpn will transport calcium bi-directionally. A further question that needs explaining is why bind and transport calcium? Cells have a high capacity for calcium flux independent of Fpn. Is there a physiological importance to this activity?

Reviewer #3 (Public Review):

The major strength of this work is that the authors take a complementary approach to understand Ca2+ binding to ferroportin. Importantly, the following lines of evidence are used to establish Ca2+ binding - transport assays, cryo-EM structure, mutagenesis studies (using both transport of Ca2+ and ITC to measure direct binding). These all convincingly indicate that Ca2+ can indeed bind to ferroportin. The authors go on to show that Co2+ can inhibit binding of Ca2+ but not the converse. The authors need to take into account some prior in interpreting their data.

I suggest the following considerations to improve the manuscript:

1) Line 38-39 - the authors state that the S2 site has a more prominent role in iron transport than the S1 site. Billesboelle et al 2020 argues the converse based on the fact that mutations in the S2 site lead to iron overload diseases, suggesting that the S2 site cannot be the key site for iron transport. This is also seen in mutagenesis studies by Bonaccorsi etal FEBS J 2014, which reported that mutation of the S1 site completely abolished iron transport. The authors should consider these alternative models in addition to citing their prior work.

2) It is unclear from the introduction/study why it is important to understand Ca2+ transport by ferroportin. Deshpande et al 2018 established that Ca2+ can regulate Fe2+ transport by ferroportin. While it is clear that Ca2+ binds ferroportin, I am not clear on why this is important from a biological perspective. The authors state that Ca2+ binding may integrate signaling with ferroportin activity, but this is not clearly explored, either with prior studies or in this study. If ferroportin acts as a uniporter, how would it be regulated to prevent inappropriate Ca2+ influx? Is there a clear reason why Ca2+ influx would integrate with iron biology? Overall, the premise of the study seems confusing to me despite the well done biochemistry/structural biology.

3) Can the authors reliably exclude the ability of this new site to bind and/or transport other metals? The CryoEM structure at this resolution cannot reliably distinguish other possibilities (e.g. Zn2+), and it is possible that the observed effects are not specific to Ca2+.

4) The maximal concentration of Ca2+ tested in Figure 4 is 500 micromolar - based on this, the authors indicate that Ca2+ has no effect on Fe2+ transport. This stands in contrast to work by Deshpande et al 2018 and Billesboelle et al 2020 which show that there is a Ca2+ effect on Fe2+ and Co2+ transport (though at higher concentrations). Have the authors tested higher Ca2+ concentrations? Given the extracellular concentration of Ca2+ (2 mM), this seems important.

Reviewer #1 (Public Review):

Here the authors set out to disentangle neural responses to acoustic and linguistic aspects of speech. Participants heard a short story, which could be in a language they understood or did not (French vs. Dutch stories, presented to Dutch listeners). Additional predictors included a combination of acoustic and linguistic factors: Acoustic, Phoneme Onsets, Phoneme Surprisal, Phoneme Entropy and, Word Frequency. Accuracy of reconstruction of the acoustic amplitude envelope was used as an outcome measure.

The use of continuous speech and the use of comprehended vs. uncomprehended speech are both significant strengths of the approach. Overall the analyses are largely appropriate to answer the questions posed.

The reconstruction accuracies (e.g., R^2 values Figure 1) seem lower perhaps than might be expected - some direct comparisons with prior literature would be welcome here. Specifically, the accuracies in Figure 1A are around .002-.003 whereas the range seen in some other papers is about an order of magnitude or more larger (e.g. Broderick et al. 2019 J Neurosci; Ding and Simon 2013 J Neurosci).

One theoretical point relevant to this and similar studies concerns the use of acoustic envelope reconstruction accuracy as the dependent measure. On the one hand, reconstruction accuracy provides an objective measure of "success", and a satisfying link between stimulus and brain activity. On the other hand, as the authors point out, envelope reconstruction is probably not the primary goal of listeners in a conversation: comprehension is. Some discussion of the implications of envelope reconstruction accuracy might be useful in guiding interpretation of the current work, and importantly, helping the field as a whole grapple with this issue.

Overall, the results support the authors' conclusions that acoustic edges and phoneme features are treated differently depending on whether a listener comprehends the language being spoken. In particular, phoneme features contribute to a greater degree when language is comprehended, whereas acoustic edges contribute similarly regardless of comprehension. These findings are important in part because of prior work suggesting that acoustic edges are critically important for "chunking" continuous speech into linguistic units; the current results re-center language units (phonemes) as critical to comprehension.

Reviewer #2 (Public Review):

In this study, the authors used an audiobook listening paradigm and encoding analysis of MEG to examine the independent contributions to MEG responses of putative acoustic and phoneme-level linguistic features in speech and their modulation by higher-level sentence/discourse constraints and language proficiency. The results indicate that:

1) Acoustic and phoneme features do indeed make independent contributions to MEG responses in frontotemporal language regions (with a left-hemisphere bias for phoneme features).<br /> 2) Brain responses to acoustic and phoneme features are enhanced when sentence/discourse constraints are low (i.e. when word entropy is high).<br /> 3) While brain responses to phoneme features are enhanced when the language is comprehended (or word entropy is high), the opposite is observed for acoustic features.

These results are taken to support widely held views on the nature of information flow during language processing. On the one hand, processing is hierarchical, consistent with finding 1 above. On the other hand, information flow between lower and high-levels of language processing is also flexible and interactive (finding 2) and modulated by behavioural goals (finding 3).

This is a methodologically sophisticated study with useful findings that I think will be of interest to the burgeoning community investigating 'neural speech tracking' and also to the wider community interested in language processing and predictive coding. Moreover, the evidence appears convincing.

I thought the impact was somewhat limited by the results presentation, which I think missed some key details and made the study somewhat hard to follow (but this issue can be addressed).

Perhaps more major, I do wonder about the novelty of the study as each of the main findings has precedent in the literature. Finding 1 (e.g. Brodbeck, Simon et al.), Finding 2 (e.g. Broderick, Lalor et al.; Molinaro et al.), Finding 3 (e.g. Brodbeck, Simon et al. although here the manipulation of behavioural goals was through a cocktail party listening manipulation and there were was no opposing modulation of acoustic vs phoneme level representations). Thus, while the study appears well executed, overall I am unsure how significant the advance is. Related to this point, the study's findings and theoretical interpretations (e.g. the brain as a hierarchical 'filter') are consistent with widely held views of language processing (at least within cognitive neuroscience) and so again I question the potential advance of the study.

Reviewer #3 (Public Review):

The manuscript focuses on three central questions (line 64), and having those spelt out explicitly and early on is very helpful. I organize my evaluation around these questions:

"(1) whether phoneme-level features contribute to neural encoding even when acoustic contributions are carefully controlled, as a function of language comprehension":

The manuscript finds that phoneme-level features based on language statistics have a much stronger effect in the native language than the foreign language. The result adds important convergent evidence to a body of work suggesting that such features can isolate brain responses associated with higher-order representations which relate to comprehension.

(2) whether sentence- and discourse-level constraints on lexical information (operationalized as word entropy) impacted the encoding of acoustic and phoneme-level features":

This is a really interesting question, but I have some potential concerns about the method used to analyze it. The Methods section could definitely benefit from a more explicit description (perhaps analogous to Table 8, which is very helpful), so I apologize if I misinterpreted the analysis. The manuscript says "TRFs including all phoneme features were estimated for each condition and language" (260), implying that separate TRFs were estimated for the high and low entropy conditions: One for only high entropy words, and one for only low entropy words. I don't understand how this was implemented, since the continuous speech/TRF paradigm does not allow neatly sorting words into bins (as could be done in trial-based designs). Instead, the response during each word is a mix of early responses to the current word and late responses to the previous word.

My interpretation of the available description (260 ff.) is that two versions of each predictor were created, one for high entropy words setting the predictor to zero during low entropy words, and vice versa. Separate TRFs were then estimated for the low- and high-entropy predictor sets. If this is indeed the case, then I am hesitant to interpret the results, because such a high entropy set of predictors is not just predicting a response in high entropy words, it is equally predicting the absence of a response in low entropy words (and vice versa). This might lead to side effects in the estimated TRFs. Furthermore, such models would estimate responses without controlling for ongoing/overlapping responses to preceding words, which may be substantial (Figure 4 implies that condition changes approximately every 2 words).

"(3) whether tracking of acoustic landmarks (viz., acoustic edges) was enhanced or suppressed as a function of comprehension."

The analysis suggests that in French (foreign language), acoustic neural responses are enhanced compared to Dutch (native language). This is an interesting data-point, and linked to a theoretically interesting claim (that lower-order representations are suppressed when higher-order categories are activated). There is a potential qualification though. Dutch and French are different languages which are probably associated with different acoustic statistics. Furthermore, the audiobooks were most likely read by different speakers (I did not find this information in the Methods section - apologies if I missed it), which, again, might be associated with different acoustic properties. Differences in acoustic responses may thus also be due to confounded differences in the acoustic structure of the stimuli.

Reviewer #1 (Public Review):

In this genetic and imaging based analysis of stem-cell maintenance and organ initiation, two phases important for continued production of shoot organs in plants, the authors tested whether SHR and targets/partners (SCR, SCL23, JWD) provide the circuitry to maintain stem cell pool and contribute to the production of lateral organs. Finding that these factors are indeed expressed in and required for SAM activities, and furthermore, behaviors of SHR and SCR in the root are recapitulated in the meristem, including mobility of SHR (here to epidermis from internal layers), activation of SCR by SHR, and "trapping" of SHR movement by complexing with SCR. Strengths include high quality imaging of reporters and FRET-FLIM measurement to assess in vivo complex formation. The analysis is then extended to link SHR and SCR to shoot-specific factors and auxin, again by testing expression, genetic dependencies and physical interaction. This is repeated for a number of factors and individually, each is well done experiment. Conclusions about causal relationships are somewhat overstated (for example, the idea that SHR-SCR act through CYCD6 to alter cell division is based on expression patterns, not a functional analysis of cycd6).

In general, there are many high-quality studies included in this paper, and the presentation of imaging data (both the images themselves and quantification of data) is excellent. There is also a lot of data, and while each section was presented in a logical way, connections between sections, and the overarching developmental questions were sparse. Because the authors found that many of the relationships defined in the root were recapitulated in the shoot, the present organization leaves one with somewhat of a sense that little new was learned, and yet, the shoot meristem IS different and there are shoot specific inputs into the core regulatory factors. Rewriting to highlight the different activities (and thus expectation about regulation) could make the finding of the same network more interesting and creating a summary figure that highlights the input of shoot specific signals would bring the unique analysis to the forefront.

Reviewer #2 (Public Review):

This study contains a huge amount of data and the images are of high quality. However, the conclusions are not really well supported. The authors may have reached too far from their results. The roles of SHR, SCR and SCL23 in the shoot apex are not really clarified.

The manuscript by Bahafid et al., reports a study of the functions of SHORTROOT (SHR), a well-established root development regulator in the shoot apical meristem (SAM) development with focus on lateral organ initiation. A large amount of data is included in this paper. This study highly depends on imaging, and the images are in general of very good quality. The authors show reciprocal interactions between SHR and SCR with auxin/MP. There are also a large amount of genetic interactions among several genes, including WUS and CLV3. Although the study provides a vast amount of data, the conclusions are not so well supported. There seem to be many interactions, at the protein level, and at the transcriptional regulation level, but the conclusion is nevertheless ambiguous.

Reviewer #3 (Public Review):

This is a comprehensive and extensive investigation of the auxin-dependent role of four GRAS family proteins (SHR, SCR, JKD, and SCL23) in regulating organ initiation and shoot apical meristem (SAM) maintenance. The authors present a detailed phenotypical analysis of the shr and scr mutants, which have fewer cell layers, reduced auxin maximum, and halted proliferation of cells in the G1 phase, indicating SHR and SCR both influence SAM maintenance and organ initiation. Auxin distribution, mediated through MONOPTEROS, was also found to regulate SHR activity in organ initiation. Furthermore, the authors hypothesize and show evidence for a coordinated regulation of CYCD6;1, a known marker for asymmetric cell divisions in the root, by SHR, SCR, JKD, and SCL23 in the SAM. Finally, the roles of SCL23 and WUSCHEL were investigated with respect to SHR-SCR activity to investigate their roles in stem cell maintenance. Overall, the authors presented a thorough and sound analysis of SAM organ initiation and this reviewer applauds the authors for this extensive systematic and comprehensive study, which has produced as well as leveraged new and established material to demonstrate similar mechanisms for organ initiation found in the shoot and root apical meristems.

Reviewer #1 (Public Review):

In this manuscript, the authors present a suite of statistical models that can identify patterns of somatic single base mutational signatures (SBS), insertions, deletions, and structural rearrangements predictive of DNA damage response (DDR) gene deficiency. A similar approach (HRDetect) has already proved successful in identifying BRCA1/BRCA2 deficiency in breast cancers and other tumor types. To generate their models, Sørensen and colleagues consider over 700 DDR gene deficiencies across more than 6,000 patients enrolled through the Hartwig Medical Foundation and PCAWG studies. The authors also consider the full set of COSMIC SBS reference signatures. The models recapitulate known associations between BRCA1/2,TP53, and CDK12 and mutational patterns but also characterize previously undescribed associations involving ATRX, PTEN, CDKN2A, and SMARCA4. Many of these novel models generalize across different tumour types and also primary and metastatic cancers. The authors also consider negative coefficient features in their models which is worthwhile and present hypotheses supporting how negative features might arise. One of the further strengths of the study is its innovative reuse of large-scale cancer genome data sets leading to predictive models with potential use for clinical intervention and demonstrating the potential of using WGS mutational signatures to guide cancer treatment. Many of the findings and observations presented in the paper have the collateral potential to enhance our understanding of the aetiology of SBS signatures. While I don't think the paper presents any major conceptual and technical advances in terms of methodology, the manuscript is important, interesting, and timely.

Reviewer #2 (Public Review):

Sørensen and colleagues performed a comprehensive analysis aiming to find how DNA repair genes shape mutational patterns. They take advantage of the Hartwig Medical Foundation (HMF) and TCGA/ICGC databases which have germline and somatic molecular data. These molecular data layers are used as input features for the predictive models of DNA damage response (DDR) gene deficiency.

Of note, the project is of interest to oncology in the sense of unveiling new genes to be further investigated as a therapeutic candidate target in cancer.

This paper brings statistical modelling based on LASSO regression coupled with appropriate metrics for unbalanced data sets. Their finds recapitulate known DDR-associate genes but novel genes that can be explored in animal models or functional assays with cell lines.

Reviewer #1 (Public Review):

This work by Olesen et al provides a clear and thorough examination of participation in organised colorectal cancer screening in Denmark over 2018-2021, with a particular focus on potential impacts of the COVID-19 pandemic. There is also an analysis by population subgroups that offers additional insight into how the pandemic may have affected participation.

The key strength of this manuscript is access to the presumably complete data from Danish Colorectal Cancer Screening Database, with only a small proportion of individuals being excluded for sensible reasons. Combined with a standard statistical analysis, the results are clear and inarguable.

A weakness is that the manuscript is wholly quantitative. Therefore some of the observations made, particularly for population subgroups, cannot be explained within the scope of this manuscript. The authors have provided hypotheses to explain these, but without further research no firm conclusion can be made.

Another weakness is that, due to the nature of screening, no conclusions can be made on the health impact of COVID-related changes to screening. It is unclear whether small reductions in screening and colonoscopy follow-ups is likely to lead to additional cancers, or later-stage diagnoses.

This manuscript, together with similar analyses in other settings worldwide, provides both an overview of how the COVID-19 pandemic has affected screening, but also potentially guidance for how future potential disruptions should be managed in the cancer screening space. By considering the potential downstream impact of changes to screening, and balancing these against potential harms and resource restrictions associated with (eg) attending health services during a pandemic, policymakers can make informed decisions to manage future shutdowns.

Reviewer #2 (Public Review):

The paper describes the participation in CRC screening in Denmark and compliance to colonoscopy in FIT positive screened people during pandemic.

There are interesting data, particularly in the breakdown by age socioeconomic status and immigrant status. Nevertheless, the study remains very descriptive. When a pandemic occurs and different strategies are put in place in different countries to afford this emergency, probably we also need simple descriptions of what happened, considering anything as a natural experiment to be reported. Furthermore, Denmark is one of the few (or the only) European countries that did not stop CRC screening even during the lockdown. Thus it is worth documenting what happened, with a scientific paper. The consequence is that the paper is not very gripping.

The paper is very well written and the report is rigorous, the methods well documented, tables and figure clear.

Reviewer #1 (Public Review):

Liu, et al. describe an essential role for prostaglandin signaling during neonephrogenesis in the zebrafish kidney following acute kidney injury. They identified that renal interstitial cells are the source of prostaglandin, and demonstrated which components of the prostaglandin biosynthesis pathway as well as which receptor is involved in the signaling. Further, they determined the mechanism by which PGE2 stimulates the proliferation of renal progenitor cells which make the new nephrons, namely to regulate B-catenin levels. This work is systematic, thorough, and well-controlled. The applications of these findings may have a profound impact on the formulation of regenerative medicine treatments for kidney disease.

Reviewer #2 (Public Review):

Previous studies have shown that lhx1 progenitors proliferate upon AKI in adult zebrafish mesonephros. However, these studies have focused primarily on renal progenitor cells (RPCs). This study uses single-cell mRNA sequencing to identify a novel cell type (RICs) in the zebrafish mesonephros that is marked by fabp10a expression within a previously generated GFP transgenic zebrafish line. The authors show that RICs express cox2 to synthesize PGE2 upon gentamicin-mediated AKI, which correlates with PRC proliferation. They demonstrate that PGE2 stimulates RPC proliferation through EP4b receptor activation of PKA, which in turn stabilizes beta-catenin through phosphorylation of both beta-catenin and GSK3beta. They also indicate that beta-catenin stabilization and RPC proliferation is dependent upon wnt4 expression by the RPC itself. The topic of the paper is significant in that it identifies an interstitial in the zebrafish kidney and suggests several mechanisms by which it supports nephron regeneration.

Reviewer #1 (Public Review):

Tan and colleagues examine the role of additional genetic removal of Munc13 in murine cultured synapses deficient for RIM and ELKS. They utilize a comprehensive set of morphological, ultrastructural and functional experiments to conclude that Munc13 is a nonredundent factor in synaptic vesicle priming. In addition, the results contribute to the ongoing discussion whether synaptic vesicle docking represents synaptic vesicle priming.

The main strength of the work is the use of a sophisticated genetic model, and the quality of the performed experiments. The results strongly support the conclusion of the nonredundant role of Munc13 in synaptic vesicle priming.

The weakness of the paper is that the findings from these study has limited impact, as the genetic and functional interaction of Munc13 and RIM has been extensively analyzed on a qualitative and quantitative level (Kaeser 2021, Zarebidaki 2020). While this paper benefits from the additional deletion of ELKS, the specific contribution of ELKS in comparison to the older studies is not in the focus of the study.

While the genetic removal of all 6 genes involved clearly require the use of conditional KO mice, the resulting outcome of the experimental design is a hypomorphic phenotype, as neurotransmitter release is still detected. This complicates the interpretation of the findings and weakens the strength of the conclusions.

Reviewer #2 (Public Review):

Tan et al. have used state-of-the-art methodology (mouse genetics, superresolution microscopy and synaptic electrophysiology) to further delineate the role of Munc13 proteins by investigating their function within a scenario in which the presynaptic active zone is deprived of major protein scaffolds. The authors have transduced Cre-expressing lentiviruses into hippocampal neuronal cultures from mice with floxed alleles to remove six fundamental components of the presynaptic active zone: RIM1, RIM2, ELKS1, ELKS2, Munc13-1, Munc13-2 and Munc13-3. The first part of the study comprises a comparison between neurons lacking RIM1, RIM2, ELKS1, ELKS2, on one side, and neurons lacking Munc13-1 and Munc13-2 on the other. Within the first group of neurons the levels of Munc13-1 at the nerve terminals are already reduced (assessed by confocal and STED microscopy and western blots) and the residual amount left is located far away from the active zone. Remarkably synapse formation occurs normally upon the hextuple knock-out of active zone proteins, however, vesicle docking is disrupted and single-action potential evoked and spontaneous release is reduced at glutamatergic and GABAergic synapses. A key finding is that the single-action potential evoked release still detected in the RIM/ELS cuadruple knock-out is almost completely abolished upon the additional knock-out of Munc13-1 and Munc13-2. This is a major observation of the study that support, as the authors concluded, that Munc13 promotes the fusogenicity of synaptic vesicles even when Munc13 is not properly located at the active zone. Careful electrophysiological measurements show that in the absence of Munc13 the size of the readily releasable pool (RRP) of synaptic vesicles is further reduced without specific changes in the vesicular release probability. Overall, the electrophysiological data support well the notion that Munc13 is specifically responsible for the remaining RRP and therefore reinforce the notion that Munc13 acts at the priming stage and can do it in part independently of RIMs and ELSs. Importantly, the results further support the notion that synapse formation is a remarkably resilient process that occurs even under strong perturbation of presynaptic function.

As a secondary conclusion, the authors point out that postsynaptic response is intact, this specific point should be further discussed and analyzed.

The study is very clearly written and presents very relevant findings of interest for readers in the field of the molecular mechanisms of synaptic operation.

Reviewer #3 (Public Review):

In the present study, Tan and colleagues studied synaptic transmission, presynaptic protein levels, and synaptic ultra-structure in hippocampal cultures of mice lacking the key active-zone proteins RIM (1, 2), ELKS (1, 2), and Munc13 (1, 2). Compared to cultures lacking only RIM and ELKS, additional loss of Munc13 results in a further decrease of synaptic Munc13-1 levels, a similar reduction of the number of docked synaptic vesicles, and a more pronounced decrease of total synaptic vesicle number. At the physiological level, these RIM-ELKS-Munc13 hextuple KO cultures display a further decrease in the pool of release-ready synaptic vesicles with largely unchanged release probability compared with RIM-ELKS quadruple KO cultures.

The data presented in the study are of high quality, and the generation of RIM-ELKS-Munc13 hextuple KO mouse cultures further demonstrates the feasibility of complex KO mouse models. A major question that remains to be addressed is if the release that remains in the absence of RIM and ELKS indeed mostly depends on Munc13.

Reviewer #1 (Public Review):

This manuscript addressed an important question regarding an obstacle to hair reprogramming in older mice that is not present in newborn mice. The conclusions were justified by experimental results. However, the scientific novelty is limited, and there is a lack of functional characterization of the newly formed hair cells.

There are several strengths of this study: 1. It addressed a significant question as hearing loss is an important public health issue. 2. Well-designed genetic approaches. 3. Experiments were well designed and justified. 4. Experimental results are convincing. 5. Conclusions were well justified by experimental results. There are also several weaknesses:

1. The scientific novelty is limited. It is known that overexpression of several transcription factors simultaneously can reprogram hair cells and non-hair cells with hair cell characteristics.

2. Transcription factor Atoh1 and downstream GFI1and POU4F3 have been used to reprogram embryonic stem cells and chick otic epithelial cells in vitro to cells expressing several hair cell genes and displaying key hair cell features.

3. There is no functional characterization of newly reprogramed hair cells in adult mice although FM 1-43 dye was used for characterizing reprogramed hair cells in neonatal mice.

4. It is not understood why the changes in transduction channel protein expression were not highlighted in gene analysis.

5. It will be nice if hair cell-like electrophysiological properties can be found in newly reprogramed hair cells.

Reviewer #2 (Public Review):

In this article, Iyer et al discuss the mechanics of reprogramming challenges encountered by supporting cells towards their potential pathway of dedifferentiating into hair cell types. Previous literature has shown the ability of ATOH-1, GFl-1, and POU4F3 to transform supporting cells into hair-like cell types. Here authors suggest that the combinatorial expression of these TFs can enhance the efficiency of the transcriptional remodeling of supporting cells to initiate the reprogramming toward hair-like cell lineage. It is a well-conducted study. Please see my comments/concerns below.

1. In the representative images, the effect of GFl-1 seems to be less efficient or has no effect on reprogramming the lineage of supporting cells to hair cell-like cells in comparison to two other groups ATOH-1 alone or ATOH-1, GFl-1, and Pou4F3 combined (Figure 1, 1- S2, 2B, 4A) and even the single-cell RNA seq can be interpreted similarly (Figure 3C, 6C. According to authors and previous literature, GFl1 is supposed to be acting in concert to enhance the efficiency of this lineage conversion at least in older animals. The representative images and single-cell UMAPs show that either GFl-1 is not efficient or less efficient than ATOH-1 alone or ATOH-1, GFl-1, and Pou4F3 combined. Hence, why authors chose not to explore ATOH-1 and Pou4F3 without GFl-1.

2. In Figure 3C, the authors find the most reduction in cell numbers in lateral GER during transcriptional reprogramming. Can authors comment on why the cells in this region are more susceptible to lineage reprogramming into hair cell-like cells?

3. In figure 5A, how can the existing hair cells be distinguished from newly formed hair cell-like cells.

4. Authors cited previous literature showing that existing hair cells can affect lineage reprogramming of supporting cells through Notch signaling. So would it not be a better experimental design when the hair cells were depleted prior to transcriptional reprogramming.

5. Genetic mutations that lead to functional disruptions in supporting cells are also linked to hearing loss. Can authors predict how feasible would be the idea of in vivo conversion of one important cell type to another important cell type?

6. Are reprogrammed hair cell-like cells transcriptionally similar to outer hair cells, inner hair cells, or none?

Reviewer #1 (Public Review):

The authors investigate whether and how PFA fixation affects the structures formed by some proteins that undergo LLPS. They do that by over-expressing a number of constructs in cells and imaging them by live cell fluorescence microscopy, after which they fix the cells and image the same cell after fixation. Their results clearly show that, for different proteins and with different tags, there is a non-systematic alteration of the LLPS structures.

In parallel to this experimental work, the authors also present and analyze a dynamic computational model in which they investigate how different fixation rates for those proteins inside and outside the condensates can lead to alterations in the overall condensate organization after fixation. Their model shows that if the fixation rate inside the condensate is different than outside the condensate, and if the dynamics of protein exchange in/out of the condensates are fast enough, fixation artifacts are to be expected.

It remains to be seen whether the alterations in condensate structures after fixation (as seen experimentally) are caused by different fixation rates (as shown computationally).

Overall, this manuscript puts forward an important observation, on how chemical fixation can alter cellular structures, such as those of membraneless organelles.

Reviewer #2 (Public Review):

The manuscript entitled "Fixation Can Change the Appearance of Phase Separation in Living Cells" discussed the different fixation artefacts that can change the appearance of LLPS. The manuscript points out a fundamental question in the field of phase separation which is rarely discussed. The authors found that PFA fixation can both enhance and diminish putative LLPS behaviors; in some cases, it can also create condensates that did not exist in living cells. Using a simple but elegant model, they found that protein localization in fixed cells depends on an intricate balance of protein-protein interaction dynamics, the overall rate of fixation, and notably, the difference between fixation rates of different proteins. They conclude that less dynamic interactions are better captured by PFA fixation. The text is clearly written, the experiments are well designed and the simulations give an interesting explanation of the different artefacts observed after fixation.

To describe LLPS or to distinguish between polymer-polymer phase separation and LLPS, recent studies have used single particle tracking, a technique allowing to follow the dynamics of individual proteins in living cells (https://doi.org/10.7554/eLife.60577; https://doi.org/10.7554/eLife.69181; https://doi.org/10.7554/eLife.47098). The authors should mention that such an approach can be a good alternative to avoid the artefact of fixation.<br /> Using techniques such as single particle tracking or FCS, it is possible to estimate the effective diffusion coefficient of protein-living cells. When a liquid phase separation is formed, it is also possible to estimate the diffusion coefficient of the protein of interest (POI) inside versus outside of the LLPS. The authors say that less dynamic interactions are better captured by PFA fixation. In the simulation part, would it be possible to predict from the diffusion coefficients of the POI inside a condensate the effect of the PAF fixation?

Finally, the authors propose that in the future, it will be important to design novel fixatives with significantly faster cross-linking rates than biomolecular interactions to eliminate fixation artifacts in the cell. It would be even more interesting if the authors could propose some ideas of potential novel fixatives. Did they test several concentrations of PFA, for example? Did they test different times of PFA incubation? Did they test cryofixation and do they know what would be their effect on LLPS? Do they have novel fixatives in mind?

Adding some precisions about these points in the simulation and in the fixation protocol would increase the impact of the manuscript. Otherwise, the study is interesting and thought-provoking.

Reviewer #3 (Public Review):

The authors compare the detection of biomolecular condensates in living cells overexpressing fluorescently tagged IDR proteins and upon fixation with paraformaldehyde (PFA). Given that they observe differences in the number and size of the condensates in the fixed versus living cells the authors conclude that the fixation method can introduce an artifact in the visualization of these condensates. Next, through kinetic modeling simulations, the authors propose a model in which the extent of the artifact introduced by PFA fixation correlates with the strength of the protein-protein interaction: artifacts are lower when the protein‐protein interactions are stable and less dynamic compared with the overall fixation rate. Based on their comparative analysis of PFA fixation and the kinetic modeling the authors strongly recommend caution in the interpretation of data obtained in PFA-fixed cells and suggest that parallel studies with living cells should be performed.

Understanding whether/how fixation methods affect the detection of biomolecular condensates is of broad interest given the importance of LLPS in regulating different aspects of cell biology. However, in this manuscript, the authors use only paraformaldehyde as a fixation method and study only fluorescently-labelled IDR proteins. The work would benefit from a comparison between living cells and cells fixed with other fixation methods; in addition, it would be useful to test the impact of these fixation methods on the detection of endogenous proteins or IDR proteins without fluorescent tag.

Reviewer #1 (Public Review):

There are five major claims. First is a replication of lower spatial frequency representation in V4. This is based on two examples shown in Fig. 3. The differences look clear but should be analyzed statistically.

The second claim is that, on the large scale of the visual field representation in V2 and V4, spatial frequency is mapped from high to low going from fovea to periphery, here estimated as lateral to medial, as in V1. The analysis for this is to plot the geometric centroids of 6 different spatial frequency band responses, for orientation contrasts (Fig. 4A) and color contrasts (Fig. 4B), and show that they progress in position from lateral to medial for high to low frequencies. This seems like an unusual analysis that obscures most of the original data concerning the relationship of spatial frequency response profiles to the two-dimensional imaging area. And, the centroids do not show a continuous map, but rather a bunching of points except for the extremes. The additional data are 4 supplemental examples with three, different frequency ranges. These data are not analyzed statistically.

The third and most important claim is that spatial frequency and orientation are mapped orthogonally (and recursively) in V2 and V4, as seen in Fig. 5 and the Fig. 5 supplement. Together these figures present two regions in V4 and two regions in V2. If these are the only analyzed regions, the authors need to specify more clearly how they were selected. Presumably, though, other regions were analyzed, and the authors should present results from all analyzable regions, and use statistical analyses to establish significance.

The fourth claim is that color-sensitive regions in V4 are more associated with low spatial frequencies. The one significant example (the analysis and statistical tests need to be explained), shown in Fig. 6, shows a weak relationship to color for both spatial frequency bands, and the other examples presented in the supplementary are not significant and have even lower absolute relationships. These results, if presented, should be considered inconclusive.

The fifth claim is for stripe-like periodicity of spatial frequency representation in V2, related to color tuning. This is supported by ostention to binary maps of spatial frequency tuning in Fig. 7 and supplement. Establishing this periodicity would require statistical analysis, and in any case, seems impossible since only a sliver of V2 is visible in these brain surface images, so stripes orthogonal to the V1/V2 boundary (i.e. CO stripes) cannot be distinguished from other patterns of spatial frequency tuning. In fact, Fig. 5E and S5I do not appear to have iso-frequency contours biased toward that orientation.

Reviewer #2 (Public Review):

This paper reports the results of optical imaging experiments on areas V4, V2, and V1 in anesthetised macaque monkeys. The experiments were designed to reveal details of the representation of spatial frequency (SF), orientation (OR), and color in these areas. Evidence for gradients of SF selectivity across the areas is presented. It is also shown that SF and OR maps in V2 and V4 have iso-parameter contours that intersect at right angles, in agreement with, and extending, observations made in V1 and visual areas in cats and other species. Color domains tend to be located in low-SF domains and avoid the higher SF regions. Relationships between V2 stripes and SF preference are also established.

These findings are a potentially valuable contribution to understanding the maps that exist in V4, which have received less attention than those in areas V1 and V2. However, I have some serious concerns about the validity of the results.

Reviewer #3 (Public Review):

This is a potentially fundamental study in which the authors used intrinsic signal optical imaging to characterize orientation, spatial frequency (SFs), and color maps in area V2 and V4 of macaques. They show that foveal regions have higher SF preferences and V1 has a preference for higher SFs compared to V2 and V4. They also show that color regions prefer lower SFs. Strikingly, they show that orientation and SFs are mapped orthogonally in V2 and V4. Finally, they show evidence of periodicity in SF preference in V2.

The data look convincing, but I would like the authors to clarify/discuss certain aspects of the analyses, as detailed below. Overall, I think this study is well done and adds to our understanding of the architecture of the primate visual cortex.

Major:

1. I found the proposed hypercolumn architecture in Figure 1B very difficult to understand. SFs vary in a continuum, so why are only two levels (low SF and high SF) shown in two different colors? Iso-orientation and Iso-SF lines could have been shown in different colors also (say HSV colors for orientation to show the circular mapping and gray colormap for SF going from low to high). Similar to what has been done for iso-hue and iso-brightness lines in the color region. Perhaps it may be worthwhile to show the same proposed architecture in V1 as well, in which orientation maps form pinwheels and colors are in separate blobs. It was unclear to me how this architecture could have pinwheels/blobs as well.

2. It is not clear to me how the details of the functional maps depend on the choice of stimuli. In single unit studies, typically a large number of orientations and SFs are used to independently map the SF and orientation tuning preferences. In contrast, here only 2 orientations are used in one case to map the color space. Even for mapping the orientation space, only 4 orientations are used. For mapping the color space also, only the hues along the red-green axis are varied (L-M pathway). I understand that some of these choices could be due to the recording modality (imaging), but it would be very useful if the authors could discuss how/if these stimulus choices can affect their results. More details of the stimuli, such as the drift rate of the gratings, and the cie (x,y, Y) coordinates of red & green hues would be useful.

3. Can you show the iso-contour lines for orientation on the orientation maps also as a supplementary figure to see how well the algorithm works? Figure 5A shows iso-orientation lines on the SF map. The iso-SF contours shown in Figure 5B easily correspond to the colors in the SF map shown in 5A, but I had difficulty mapping the orientation. Also, I was wondering whether the way the comparisons are done to get the maps (for example, in Figure 4, the same 4 stimuli are compared in two different ways to get orientation and color maps) can potentially impose some constraints on those maps. I say this because it is striking to me that almost every red and blue line shown in 5C and 5G appears to intersect orthogonally (as also shown in 5D and 5H).

4. To me the orthogonality of SF and Orientation contours in Figure 5 was the most striking result. Can you show how this analysis looks for V1? The supplementary figure also shows only V2 and V4.

5. The claim about periodicity is not well quantified. If the authors wish to make this claim, they need to show the Fourier transform of the activation pattern as a function of space and show clear peaks in the spectrum. Also, the authors can perhaps clarify what is the spatial resolution of the imaging technique itself.

Reviewer #1 (Public Review):

Auwerx et al. have taken a new approach to mine large existing datasets of intermediary molecular data between GWAS and phenotype, with the aim of uncovering novel insight into the molecular mechanisms which lead a GWAS hit to have a phenotypic effect. The authors show that you can get additional insight by integrating multiple omics layers rather than analyzing only a single molecular type, including a handful of specific examples, e.g. that the effect of SNPs in ANKH on calcium are mediated by citrate. Such additional data is necessary because, as the authors' point out, while we have thousands of SNPs with significant impact on phenotypes of interest, we often don't know at all the mechanism, given that the majority of significant SNPs found through GWAS are in non-coding (and often intergenic) regions.

This paper shows how one can mine large existing datasets to better estimate the cellular mechanism of significant, causal SNPs, and the authors have proven that by providing insight into the links between a couple of genes (e.g. FADS2, TMEM258) and metabolite QTLs and consequent phenotypes. There is definitely a need and utility for this, given how few significant SNPs (and even fewer recently-discovered ones) hit parts of the DNA where the causal mechanism is immediately obvious and easily testable through traditional molecular approaches.

I find the paper interesting and it provides useful insight into a still relatively new approach. However, I would be interested in knowing how well this approach scales to the general genetics community: would this method work with a much smaller N (e.g. n = 500)? Being able to make new insights using cohorts of nearly 10,000 patients is great, but the vast majority of molecular studies are at least an order of magnitude smaller. While sequencing and mass spectrometry are becoming exponentially cheaper, the issue of sample size is likely to remain for the foreseeable future due to the challenges and expenses of the initial sample collection.

Reviewer #2 (Public Review):

Auwerx et al. present a framework for the integration of results from expression quantitative trait loci (eQTL), metabolite QTL (mQTL) and genome-wide association (GWA) studies based on the use of summary statistics and Mendelian Randomization (MR). The aim of their study is to provide the field with a method that allows for the detection of causal relationships between transcript levels and phenotypes by integrating information about the effect of transcripts on metabolites and the downstream effect of these metabolites on phenotypes reported by GWA studies. The method requires the mapping of identical SNPs in disconnected mQTL and eQTL studies, which allows MR-based inference of a causal effect from a transcript to a metabolite. The effect of both transcripts and metabolites on phenotypes is evaluated in the same MR-based manner by overlaying eQTL and mQTL SNPs with SNPs present in phenotypic GWA studies.

The aim of the presented approach is two-fold: (1) to allow identification of additional causal relationships between transcript levels and phenotypes as compared to an approach limited to the evaluation of transcript-to-phenotype associations (transcriptome-wide MR, TWMR) and (2) to provide information about the mechanism of effects originating from causally linked transcripts via the metabolite layer to a phenotype.

The study is presented in a very clear and concise way. In the part based on empirical study results, the approach leads to the identification of a set of potential causal triplets between transcripts, metabolites and phenotypes. Several examples of such causal links are presented, which are in agreement with literature but also contain testable hypotheses about novel functional relationships. The simulation study is well documented and addresses an important question pertaining to the approach taken: Does the integration of mQTL data at the level of a mediator allow for higher power to detect causal transcript to phenotype associations?

Major Concerns<br /> 1. Our most salient concern regarding the presented approach is the presence of multiple testing problems. In the analysis of empirical datasets (p. 4), the rational for setting FDR thresholds is not clearly stated. While this appears to be a Bonferroni-type correction (p-value threshold divided by number of transcripts or metabolites tested), the thresholds do not reflect the actual number of tests performed (7883 transcripts times 453 metabolites for transcript-metabolite associations, 87 metabolites or 10435 transcripts times 28 complex phenotypes). The correct and more stringent thresholds certainly decrease the overlap between causal relationships and thus reduce the identifiable number of causal triplets. Furthermore, we believe that multiple testing has to be considered for correct interpretation of the power analysis. The study compares the power of a TWMR-only approach to the power of mediation-based MR by comparing "power(TP)" against "power(TM) * power(MP)" (p. 12). This comparison is useful in a hypothetical situation given data on a single transcript affecting a single phenotype, and with potential mediation via a single metabolite. However, in an actual empirical situation, the number of non-causal transcript-metabolite-phenotype triplets will exceed the number of non-causal transcript-phenotype associations due to the multiplication with the number of metabolites that have to be evaluated. This creates a tremendous burden of multiple testing, which will very likely outweigh the increase in power afforded by the mediation-based approach in the hypothetical "single transcript-metabolite-phenotype" situation described here. Thus, for explorative detection of causal transcript-phenotype relationships, the TWMR-only method might even outperform the mediation-based method described by the authors, simply because the former requires a smaller number of hypotheses to be tested compared to the latter. The presented simulation would only hold in cases where a single path of causality with a known potential mediator is to be tested.

2. A second concern regards the interpretation of the results based on the empirical datasets. For the identified 206 transcript-metabolite-phenotype causal triplets, the authors show a comparison between TWMR-based total effect of transcripts on phenotypes and the calculated direct effect based on a multivariable MR (MVMR) test (Figure 2B), which corrects for the indirect effect mediated by the metabolite in the causal triplet. The comparison shows a strong correlation between direct and total effect. A thorough discussion of the potential reasons for deviation (in both negative and positive directions) from the identity line is missing. Furthermore, no test of significance for potential cases of mediation is presented. Due to the issues of multiple testing discussed above, the significance of the inferred cases of mediation is drawn into question. The examples presented for causal triplets (involving the ANKH and SLC6A12 transcripts) feature transcripts with low total effects and a small ratio between direct and total effect, in line with the power analysis. However, in these examples, the total effects are also quite low. Its significance has to be tested with an appropriate statistical test, incorporating multiple testing correction. Furthermore, the analysis of the empirical data indicates that the ratio between direct and indirect effect of a transcript on a phenotype is in most cases close to identity, except for triplets with low total effects. This fact should be considered in the power analysis, which assigned the highest gain in power by the mediation analysis to cases of low direct to total effect ratio. The empirical data indicate that these cases might be rare or of minor relevance for the tested phenotypes.

3. Related to the interpretation of causal links: horizontal pleiotropy needs to be considered. The authors report the identification of causal links between TMEM258, FADS1 and FADS2, arachidonic acid-derived lipids and complex phenotypes. However, they also mention the high degree of pleiotropy due to linkage disequilibrium at the underlying eQTL and mQTL region as well as the network of over 50 complex lipids known to be associated with the expression of the above transcripts. Thus, it seems possible that the levels of undetected lipid species may be more important for the phenotypic effect of variation in these transcripts and that the reported "mediators" are rather covariates. Such horizontal pleiotropy would violate a basic assumption of the MR approach. While we think that this does not invalidate the approach altogether, it does affect the interpretation of specific metabolites as mediators. This is aggravated by the fact that metabolic networks are more tightly interconnected than macromolecular interaction networks (assortative nature of metabolic networks) and that single point-measurements of metabolites may not be generally informative about the flux through a specific metabolic pathway.

Reviewer #1 (Public Review):

The paper studied spatial-temporal characteristics of dominant T cell clones in juvenile idiopathic arthritis. The authors found that the composition and functional characteristics of immune infiltrates are strikingly similar between joints within one patient, and observed a strong overlap between dominant T cell clones, especially Treg. Moreover, in localized autoimmune disease there is auto-antigen driven expansion of both T effector and Treg clones, that are highly persistent and are (re)circulating.

Reviewer #2 (Public Review):

In the manuscript, Mijnheer et al mainly exploited CyTOF Helios mass cytometer and TCRβ repertoire sequencing to investigate the T cell composition and distribution in peripheral blood and synovial fluid, and further explored the temporal and spatial dynamics of regulatory T cells (Tregs) and non-Tregs in the inflamed joints of Juvenile Idiopathic Arthritis (JIA) patients. Their results indicate that the activated effector T cells and hyper-expanded Treg TCRβ clones found at the inflamed joints are highly persistent in the circulation, and the dominant of high degree of sequence similarity of Treg clones could serve as the novel therapeutic targets for the JIA treatment. Overall, the research design is appropriate, and the methods are adequately described in the study. However, several issues are required to be addressed.

(1) The criteria for the JIA patient's recruitment should be clearly presented in the method section. For example, what is the specific included criteria and excluded criteria? Or did the patients take medicines for the treatment during the study?<br /> (2) As for the correlation analysis of the entire spectrum of node frequencies, the SFMCs and PBMCs isolated from 3 patients were conducted in the study. The sample size is too limited to obtain robust results and to make a convincing conclusion from the correlation analysis. And it is shown that a total of 9 JIA patients have been involved in the study. Could the author clarify it?<br /> (3) The results of the study indicate that the hyper-expanded T cell clones are shared between left and right knee joints. Since JIA may affect one or more joints, did the author check other joints to see if the same expanded T cell clones infiltrate multiple joints, such as hand or wrist?<br /> (4) For Fig.2B, the Treg CD25+FOXP3+ population was significantly enriched in synovial fluid (SF). Is it from the left knee joints or the right knee joints?<br /> And in the context of Line 144-148, it indicated the SF, however, the title of axis in Fig.2B indicated Synovial Fluid Mononuclear Cells (SFMCs). Please keep consistent.<br /> (5) For the longitudinal sampling timelines of JIA patients shown in Supplementary Fig.3, the interval of PB and SF sample collection is not consistent. And only 1 patient completed 4 visits and the sample collection. It is hard to make any conclusion from 1 patient.

Reviewer #1 (Public Review):

Tafenoquine is an important 8-aminoquinoline antimalarial, mostly aimed at the management of Plasmodium vivax malaria. Through the retrospective analysis of several previously performed efficacy trials, the authors aimed to better understand the drugs mechanism of action, while exploring the possibility of improved efficacy through dose increment.

Strengths: robust analysis approaches unlocked three main messages with the potential of improving the clinical practice:<br /> i. P. vivax recurrency is positively associated with tafenoquine terminal half-life and D7 methemoglobin levels.<br /> ii. The methemoglobin levels support the current view that tafenoquine, acts through its metabolites, similar to what is believed for primaquine.<br /> ii. Most importantly, the therapeutic window of tafenoquine is wider than previously considered, allowing the suggestion of a significant increase in dosing, from 300 mg to 450 mg, leading to significantly increased efficacy.

Weaknesses: being a retrospective analysis, the work is limited to the available data. In particular, and as referred by the authors, no drug levels are reported. Additionally, there are some aspects that in my view need more detailed analysis and discussion, in particular, what seems to be a lack of exploration as to the importance (or lack of it) of the patient CYP2D6 status in Tafenoquine T1/2, methemoglobin levels, and overall efficacy. These mild weaknesses do not change the overall conclusions of the study.

Reviewer #2 (Public Review):

The current treatment for the radical cure of Plasmodium vivax malaria is primaquine, it was first made available in the 1950s and there is a need for better treatments. Recently a new drug was licensed, tafenoquine. Tafenoquine is a single-dose treatment due to the drug's long half-life. The expected increase in treatment adherence is an important advantage, however, the drug's slow elimination has also a drawback. Patients must be tested for a ubiquitous enzyme (glucose-6-phosphate dehydrogenase) deficiency prior to treatment, as the use of this drug in the G6DP deficient population could lead to life-threatening haemolysis. Implementing accurate quantitative testing in remote malaria-endemic areas is challenging. Providing point-of-care test equipment, supplies and training may not be cost-effective as the efficacy of tafenoquine has not been proven non-inferior to primaquine.

Thus strategies to increase tafenoquine efficacy are of paramount importance to raise the public health relevance of the first new drug developed for the radical cure of vivax malaria in the last 70 years. This paper polled together and analysed the clinical information of participants of the tafenoquine clinical trials, and using models they evaluated the influence of dose per weight on the recurrence rate of the disease. They predict that the tafenoquine efficacy would surpass the primaquine one if the tafenoquine dose was increased. They also correlated the levels of methaemoglobin production and the reduction of relapses, implying that methaemoglobin can be a surrogate marker of efficacy.

As with any model prediction, these results should be confirmed in clinical trials, especially the safety profile of the suggested regimen. The surrogate marker of oxidative drug activity is a very interesting indirect efficacy measurement, although it is limited to any indirect outcome. Even the vivax recurrence rate itself has limitations as vivax relapse and reinfection cannot be differentiated. Still, these results provide a solid support for future clinical trials that might reinforce the public health relevance of tafenoquine.

Reviewer #3 (Public Review):

By assembling the vast majority of global tafenoquine pharmacology data from clinical treatment studies that led to the 8-aminoquinoline's registration in 2018, the authors of this manuscript have convincingly made their argument that the currently recommended treatment dosage of 300mg (in combination with chloroquine) is too low and needs to be increased by at least 50%. Access to the multiple data sets is thorough, the modelling reasonable and the conclusion reached is sound.

How did we get here (again) under-dosing malaria patients with a class of drugs we have been working on for a century? Speaking as someone who was associated with tafenoquine development over two decades, it seems that worry about adverse events, specifically hemolysis in G6PD deficient persons, overcame the operational requirement to give enough drugs in a single dose regimen. However, tafenoquine is very safe in G6PD normal persons who by definition were the ones entered into the clinical treatment trials. Risk-benefit judgments cannot always be weighted towards "safety" especially when the real concern was that a single severe adverse event would derail the entire development program. Real-world effectiveness matters and should now result in the studies the authors state are needed to certify the higher dose regimen.

The schizophrenic nature of tafenoquine development needs to be mentioned. This manuscript discusses malaria treatment and includes nearly all the relevant data, but extensive work was also done to support the chemoprophylaxis indication largely sponsored by the US Army. These prophylaxis efforts were often separate from the parallel efforts on treatment indication to the loss of both groups who were ostensibly working on the same drug. 450mg tafenoquine is not a large dose; 600mg (over 3 days) is routinely given at the beginning of malaria chemoprophylaxis. Up to twice that amount was given in phase 2 studies done in Kenya in 1998 which resulted in the only described severe hemolytic reaction when one G6PD deficient heterozygote woman was given 1200mg over 3 days due to incorrect recording of her G6PD status. It is not easy to hemolyze even G6PD-deficient erythrocytes due to the slow metabolism of tafenoquine. Nearly all clinical trials of both primaquine and tafenoquine have experienced similar hemolytic events when there were errors in the determination of G6PD status. This does not mean that all 8-aminoquinolines are dangerous drugs, only that a known genetic polymorphism needs to be accounted for when treating vivax malaria.

The authors point out the utility of 7-day methemoglobin concentrations in predicted drug success/failure in the prevention of subsequent relapses. This is important and stresses the requirement of drug metabolism to a redox-active intermediate as being a common property of all 8-aminoquinolines. Tafenoquine and primaquine are similar but not identical and the slow metabolism of tafenoquine to its redox-active intermediates explains its main advantage of being capable of supporting a single-dose cure. The main reason this was not appreciated much earlier is we were looking in the wrong place. Metabolic end-products (5,6 orthoquinones) are in very low concentrations after single-dose tafenoquine in the blood, but being water-soluble they are easily located in the urine. Such urine metabolites indicative of redox action are very likely to be complementary to methemoglobin measurements which mark the redox effect on the erythrocyte. Despite earlier simplifying assumptions made during tafenoquine development (no significant metabolites exist), metabolism to redox-active intermediates must be embraced as the explanation of drug efficacy and not a cause of undesirable adverse events.

Another dark cloud over tafenoquine mentioned by the authors was the very disappointing results of the INSPECTOR trial in Indonesia whose full results are yet to be published. The failure of P vivax relapse prevention using 300mg tafenoquine with dihydroartemisinin-piperaquine in Indonesian soldiers was largely ascribed to under-dosing. Although this may have been partially true, another aspect indicated in figure 15 of the appendix is the nature of the partner drug. Artemisinin combinations with tafenoquine do not produce the same amount of methemoglobin (indicative of redox metabolism) as when combined with the registered partner drug chloroquine. We do not understand tafenoquine metabolism, but it is increasingly clear that what drug is combined with tafenoquine makes a very substantial difference. Despite the great operational desire to use artemisinin combination therapy for all malaria treatment regimens, this may not be possible with tafenoquine. Chloroquine likely is driving tafenoquine metabolism as it has no real effect on latent hypnozoites in the liver by itself. Increased dose studies with tafenoquine need to be done with chloroquine, not artemisinin.

Treatment of P vivax malaria to prevent relapse by tafenoquine is the first but not the only indication of this long-acting 8-aminoquinoline. Besides chemoprophylaxis, tafenoquine has also been recently shown in controlled human challenges and field studies in Africa to block transmission at very low dosage regimens. If we are to realize tafenoquine's potential to block transmission in a population to eliminate malaria, we first have to get the treatment regimen and its combination partner right. This paper is another good step along the road to really understanding how to use this new antimalarial drug.

Reviewer #1 (Public Review):

This paper is a continuation of other research by this group and represents another step back in time for peptide preservation in eggshells. It is exciting to see Miocene age peptides and that they overlap so completely with both extant ostrich struthiocalcin as well as the previously described Pliocene peptides. The biggest weakness is the lack of tables showing both the de novo peptides as well as those detected by database searching.

Reviewer #2 (Public Review):

In this manuscript, the authors use tandem mass spectrometry to identify peptides from the eggshell protein struthiocalcin-1 preserved in a fossilized eggshell ~6.5 Ma years old. They report multiple peptide spectral matches (peptide identifications) to a small section of the struthiocalcin-1 protein. The high-resolution, well-annotated spectral matches provided by the authors show that that region of struthiocalcin-1 may be preferentially preserved in ancient ostrich shell tissue.

These findings are important because Cenozoic tissues greater than 1 Ma in age are drastically unexplored in terms of protein preservation. Thus, these data represent a big step forward in the investigation of proteomic preservation throughout the Cenozoic.

Reviewer #1 (Public Review):

The manuscript by Heckman and Doe describes a nice set of experiments that extend previous studies of the plasticity in wiring and growth of a sensory axon terminal (Dbd) and its connection to a partner interneuron (A08a) in Drosophila embryonic/larval ventral nerve cord. The authors confirm and extend prior studies in the lab that showed misrouting of Dbd axons cause changes in its site of connection on medial versus lateral dendrites of A08a. The authors show using misrouting and ablation of Dbd that the site of axonal innervation plays a role in promoting dendrite outgrowth in that specific domain of the A08a dendritic field, suggesting a contact-dependent dendrite growth mechanism regulates early connections. The authors then describe a second mechanism where activity of the Dbd sensory neuron regulates a separate aspect of early connectivity, whereby reduced activity leads to increase A08a dendrite growth globally and increased activity suppresses overall A08a dendrite growth. This study fits with other work in the field on the role of activity in synaptic wiring while highlighting the opposing roles of contact versus activity in establishing early connection patterns. They also identify a brief developmental window where Dbd ablation causes A08a dendritic undergrowth, suggesting an early critical period for contact-dependent dendritic growth modulation, similar to that observed for activity-dependent plasticity. Overall, this is a nice study that provides important advances in our understanding of the plasticity of early neuronal wiring.

Reviewer #2 (Public Review):

The authors report a series of genetic experiments that allow for the rigorous evaluation of the role of pre-synaptic contact and activity on dendrite morphogenesis and/or stabilization between a model pair of connected sensory and interneurons in the Drosophila larval CNS. Experiments to mis-position the presynaptic arbors of the DBD neuron reveal contact-dependent effects on post-synaptic dendrite growth. Ablation, silencing and activation experiments support the interesting model that neuronal activity in the sensory afferents act to globally constrain post-synaptic dendrite growth. Thus, coordination of presynaptic contact and activity act in opposition to sculpt post-synaptic dendrites. One weakness/limitation of the study is the inability of the authors to evaluate whether the observed effects are due to changes in the initiation of dendrite growth as opposed to maintenance or stabilization effects. The authors adequately acknowledge and discuss this limitation. Overall, this is a beautifully conducted study that adds new insights into synaptic partner matching.

Reviewer #3 (Public Review):

In this manuscript, Emily Heckman and Chris Doe outline their investigation of how two specific partner neurons interact during the development of the Drosophila larval nerve cord, a specific proprioceptive sensory neuron (called 'dbd') and one of its postsynaptic partners (called 'A08a').

Experiments were executed that ask three questions:<br /> 1. How might dendrites of the A08a neuron postsynaptic to dbd change when this sensory neuron is silenced or over-activated?<br /> 2. How might those A08a dendrites change when the dbd presynaptic partner is experimentally removed?<br /> 3. Is there are critical period when dbd killing has maximal effect on changes to A08a dendrites?

The aim was to reveal some of the cellular mechanisms that principally shape the development of postsynaptic dendrites during nervous system development.

Overall, the paper is well written and the figures beautifully presented. However, I have reservations about the manuscript as it stands.<br /> Foremost is the question as to what new insights this work reveals that have not already been clearly demonstrated by a number of other studies across a range of model systems, including those cited in the discussion? This work might distinguish itself at the level of detail achieved by precision made possible through the genetic tools available, yet it does not make use of other aspects, such as the connectome also available to probe more deeply into changes that the above manipulations provoke.

The final experiment of inducing dbd cell killing at different stages of embryonic and larval development reveals what might be a critical period for cell contact-based regulation of postsynaptic dendritic growth regulation. This is a nice touch and could be the basis for interesting future work. However, much stronger would have been to have had a more robust sample size, clear demonstration of the dynamics of dbd cell killing itself (as potentially relevant to the A08a response) and, ideally, an independent verification via a separate method or on a different cell pair.

Reviewer #1 (Public Review):

This study examines how the COVID-19 pandemic impacted cervical cancer screening participation to invitations sent through the organized cervical cancer screening program of Denmark. I think the results are particularly enlightening in the context of pandemic recovery, as they show that while the short-term participation (90 days) dropped due to public health messaging emphasizing staying at home, the long-term participation (365 days) did not drop; this suggests that women did not completely miss the opportunity to screen during the pandemic, but simply postponed their screening to a later point in time. I think this has implications, especially for modeling the impact of the pandemic on cancer incidence, as many screening models have made the assumption that screenings missed during the pandemic would not be "caught up" later leading to higher cancer incidence in the long term; however, this study suggests that this is not the case and that there is a natural 'catch up' of screening that occurs over time. This is reassuring, as a short delay in cervical cancer screening would not be expected to lead to overly important long-term negative health outcomes.

Particular strengths of this study include the population-based registry covering the whole target population, and the ability to link the data to socioeconomic variables of interest to examine whether there were particular groups of women which were more impacted than others. The models also accounted for seasonal and long-term trends in cancer screening participation, which bolsters the confidence that their results are not the result of trends in cervical screening participation over time and are most likely attributable to the COVID-19 pandemic. However, as the statistical methods do not include an interaction test for the overall effect of each socioeconomic variable, it is not clear whether the differences that are observed between women by age and socioeconomic status are significant.

Reviewer #2 (Public Review):

The study included all women aged 23-64 years invited for cervical cancer screening in Denmark in 2015-2021 (n=2,220,00). The Danish registries provide an ideal setting for the study. Classification of explanatory covariates followed Danish and international standards. The authors estimated the prevalence ratios using a generalised linear model with a log link for the Poisson family. Material and statistical methods are appropriate for the study's aims.

As the authors write, several studies have demonstrated lower participation among immigrants and women with lower socioeconomic status. However, the authors wanted to evaluate whether divergence may have been exacerbated during the pandemic. Unfortunately, they do not provide any justification for why they would hypothesize that to happen.

The authors write that women who unregistered from the screening programme within 1 year since invitation (n=56,920) were excluded. If those who are at higher risk of cancer and with lower participation rates unregister themselves, the compliance to screening could be overestimated.

The authors find that some age groups i.e. women aged 40-49 and those aged 60-64 years had a lower participation rate and conclude that it could indicate that the restrictions within a society affect different age groups disproportionally. The authors do not try to explain the finding and it should be scrutinized to rule out a chance. Comorbidity is strongly associated with age so if this is attributed to self-isolation, there should be a gradient. Why 50-59 years old would be different from 60-64 years?

In general, study results support the conclusions. The authors consider the inconsistent health messages as a reason for women not to participate. What about fear? In several countries, there was a clear decrease in emergency admissions to the hospital which suggest that people were avoiding hospital because they were nervous about catching COVID-19.

Reviewer #1 (Public Review):

This study explores the mechanisms responsible for reduced steroidogenesis of adrenocortical cells in a mouse model of systemic inflammation induced by LPS administration. Working from RNA and protein profiling data sets in adrenocortical tissue from LPS-treated mice they report that LPS perturbs the TCA cycle at the level of succinate dehydrogenase B (SDHB) impairing oxidative phosphorylation. Additional studies indicate these events are coupled to increased IL-1β levels which inhibit SDHB expression through DNA methyltransferase-dependent DNA methylation of the SDHB promoter.

In general, these are interesting studies with some novel implications. I do, however, have concerns with some of the author's rather broad conclusions given the limitations of their experimental approach. The paper could be improved by addressing the following points:

1. The limitations of using LPS as the model for systemic inflammation need to be explicitly described.<br /> 2. The initial in vivo findings, which support the proposed metabolic perturbation, are based on descriptive profiling data obtained at one time point following a single dose of LPS. The author's conclusion that the ultimate transcriptional pathway identified hinges critically on knowledge of the time course of this effect following LPS, which is not adequately addressed in the paper. How was this time and dose of LPS established and are there data from different dose and time points?<br /> 3. Related to the point above, the authors data supporting a break in the TCA cycle would be strengthened direct biochemical assessment (metabolic flux analysis) of step kin the TCA cycle process impacted.<br /> 4. The proposed connection of DNMT and IL1 signaling to systemic inflammation and reduced steriodogenesis could be more firmly established by additional studies in adrenal cortical cells lacking these genes.

Reviewer #2 (Public Review):

The present manuscript provides a mechanistic explanation for an event in adrenal endocrinology: the resistance which develops during excessive inflammation relative to acute inflammation. The authors identify disturbances in adrenal mitochondria function that differentiate excessive inflammation. During severe inflammation the TCA in the adrenal is disrupted at the level of succinate production producing an accumulation of succinate in the adrenal cortex. The authors also provide a mechanistic explanation for the accumulation of succinate, they demonstrate that IL1b decreases expression of SDH the enzyme that degrades succinate through a methylation event in the SDH promoter. This work presents a solid explanation for an important phenomenon. Below are a few questions that should be resolved experimentally.

The authors should confirm through direct biochemical assays of enzymatic activity that steroidogenesis enzyme activity is not impaired. Many of these enzymes are located in the mitochondria and their activity may be diminished due to the disturbed, high succinate environment of the cortical cell as opposed to the low ATP production.

What is the effect of high ROS production. Is steroidogenesis resolved if ROS is pharmacologically decreased even if the reduction of ATP is not resolved?

Does increased intracellular succinate (through cell permeable succinate treatment) inhibit steroidogenesis even if there is not a blockage of OXPHOS?

It should be demonstrated the genetic loss of IL1 signaling in adrenal cortical cells results in a loss of the effect of LPS on reduced steroidogenesis and increased succinate accumulation.

It should be demonstrated the genetic loss of IL1 signaling in adrenal cortical cells results in a loss of the effect of LPS on SDH activity and ATP production and SDH promoter methylation

It should be shown that the silencing of DNMT eliminates or diminishes the effect of LPS on reduced steroidogenesis and increased succinate accumulation.

Does silencing of DNMT reduce OXPHOS in adrenal cortical cells?

The effects of LPS on reduced adrenal steroidogenesis are not elaborated at the physiological level. The manuscript should demonstrate the ramifications of the adrenal function decreasing after LPS. Does CORT release become less pronounced after subsequent challenges? Does baseline CORT decrease at some point? No physiological consequences are shown. Similarly, these physiological consequences of decreased adrenal function should be dependent on decreased SDH activity and OXPHOS in adrenal cells and this should be demonstrated experimentally.

Reviewer #3 (Public Review):

The authors attempted to elucidate mechanisms underlying adrenal dysfunction in severe inflammation.

Utilizing transcriptomic, proteomic and metabolomic analyses of adrenocortical cells in male mice after lipopolysaccharid induced systemic inflammation is a major strength of this study.

The use of sophisticated methods and the results support the conclusion of the authors that the Interleukin 1beta - DNA methyltransferase 1 - succinate dehydrogenase b axis with increased succinate and reduced ATP levels disrupts steroid production in lipopolysaccharid induced systemic inflammation.

Various inflammatory conditions in humans are treated with steroids and this animal based study may help identify future therapeutic targets besides the administration of glucocorticoids.

Reviewer #1 (Public Review):

Neverov and colleagues present a large-scale computational investigation of epistatic interactions between substitutions in the spike protein. The analysis is based on an improved version of their previous approach that has been applied to other organisms to the Influenza A virus. They find several sets of interacting sites that tend to change in concert.

The approach is sensible and the work seems well executed. A systematic investigation of epistatic interactions is important to better understand the constraints and drivers of future SC2 evolution. This work is hence an important contribution to the field and a nice complement to experimental work by Jesse Bloom's group and others.

The authors uncover several groups of residues that seem to change in concert. The identified groups make sense, but further validation and comparison with experimental or other computational approaches would strengthen the conclusions.

Reviewer #2 (Public Review):

The authors use the phylogeny of SARS-CoV-2 to find signals of functional interactions among the evolving amino acids of the spike protein. They do this by looking for pairs of substitutions that either tend to appear consecutively on branches, indicating positive interactions, or to appear on separate branches, indicating negative interactions. Although a massive number of SARS-CoV-2 sequences have been collected, many of these sequences have errors in them or are similar to each other. This affects the accuracy of the reconstructed phylogeny and the placement of mutations on it, creating difficulties for this approach. Still, the authors are able to identify several sets of sites with clear signals of interaction, and where the interaction makes sense given the structure of the protein. Some of these sites are carried by the Omicron variant, indicating that positive epistasis likely played a role in its evolution.

Reviewer #3 (Public Review):

Neverov et al. conduct an analysis of the SARS-CoV-2 phylogeny to identify pairs of sites in the rapidly evolving spike protein that fix concordant mutations more or less frequently than expected, reflective of epistasis between spike mutations. The authors modify an existing method to this end, making some updates to their algorithm that I find logically intuitive. I find this to be an interesting question that is important for understanding the molecular forces that influence future SARS-CoV-2 evolution. I find the study uncovers some valuable examples of epistasis, but have some key questions about the Methods that make it unclear to me how efficiently the method is performing.

Reviewer #1 (Public Review):

Summary:

It is widely known that lesioning the vHP produces anxiogenic effects, and cells in the vHP increase their firing rates in the anxiogenic location. This paper aims to investigate the neural dynamics of the vHP when a non-anxiogenic location changes to an anxiogenic one during spatial navigation. For testing, the authors removed half of the side walls of the elevated linear maze or track in the middle of the session. They reported that cells in the vHP remapped and overrepresented anxiogenic places as the walls were removed. Also, the authors claim that single-cell activities recorded before entering the open portion of the track could be used to predict how far the rat would explore along the open segment of the track.

Strength and weakness:

The experimental paradigm was well-designed to examine the main research question. It is novel in that the authors recorded single cells electrophysiologically in the vHP, as this has been done only in very few studies. However, in the current version of the manuscript, the main argument (tied to Figure 5) is not supported by detailed neural and behavioral data. Specifically, the authors did not provide the basic firing properties of the electrophysiological data to verify the quality of single-cell recording data. Also, they did not compare the velocity and position data before entering the open arm between the proximal and distal exploration. Thus, it is hard to reject the alternative hypothesis that the difference in neural activities between the exploration types stems from behavioral differences, not necessarily based on prediction signals.

Significance of the work:

This study should contribute to the single-cell-level understanding of the vHP with only very few experimental data available in the literature on the topic. Since some of the previous studies that claimed to record the ventral hippocampus actually targeted the intermediate portion of the hippocampus, this study would set a new standard for investigating the true ventral hippocampus.

Reviewer #2 (Public Review):

The authors examined the neural activity of the ventral hippocampus (vH) during exploration of anxiogenic environments. They first recorded vH neuronal activity when animals explored the elevated plus maze (EPM). Although they observed that peak firing activity increased when rats explored anxiogenic locations, this effect was difficult to quantify since rats did not often explore these locations. In order to resolve this issue, they developed a novel type of elevated linear maze (ELM). In the anxiogenic location of the ELM, they observed anxiety-related neuronal activity and demonstrated that the direction-dependent activity of vH neurons became homogenized. Additionally, the authors demonstrated that the activity of the vH neurons reflected and predicted, using a support vector machine (SVM), the exploration of an anxiogenic location, suggesting that vH neurons do not only code for anxiogenic environments, but also may reflect the intention to explore anxiogenic locations.

Strength:<br /> S1. In their study, the authors introduced a modified ELM task that can instantly reconfigure side walls in the anxiogenic environment while rats are being recorded on the maze. This method was intended to overcome the low-sampling issue observed in the anxiogenic environments where animals usually avoid entering. In fact, this modification allowed them to study between non-anxiogenic and anxiogenic conditions within the same maze and in a single recording session.

S2. Also, it is known that recording large number of cells from vH has been quite challenging in the field. The authors successfully examined more than 130 neurons from the vH area across six rats and determined remapping effect when animals were exposed to the anxiogenic environment.

S3. The authors tried to examine the neural population carefully to exclude any other factors to focus solely on the effect of anxiety, although it has been shown that abrupt changes in the environment can cause the hippocampus to remap.

Weakness:<br /> Despite the fact that the authors are trying to answer potentially important and intriguing questions in the anxiety field, some important details are missing from their description of the data.

W1. It is remarkable and impactful that the authors found that the vH neurons overrepresent, remap, and lose directionality under anxiogenic conditions. Conceptually, such dramatic changes as well as prospective biased memory 'replays' have been reported in the dorsal hippocampus under anxiogenic task settings, such as using electrical foot shocks, for example, Wu et al, Nat.Neuro, 2017. Also, another paper (Girardeau et al.., 2017, Nat Neuro) reported that an aversive trajectory is more reactivated in the dorsal hippocampus.

W2. Technically, they used tetrodes in vH and were able to collect more than 130 units, with histological data indicating that recording sites ranged from CA1 to CA3 of vH (Figure 1B). They used a semi-automated clustering method to isolate individual units but did not subdivide them into CA1, CA3 and/or pyramidal cells or interneurons. It appears that the representative examples in Figure 1C contain both pyramidal cells and interneurons, which are well characterized in terms of remapping in the dorsal area.

W3. Readers may find Figure 5 difficult to follow. They are not intuitive to understand how to read/interpret the figure panels.

Reviewer #3 (Public Review):

The authors provide interesting data showing that ventral hippocampal (vH) cells show rapid remapping when an open area appears in the environment, displaying a concentration of place field center in the new open area. Additionally, distinct direction-dependent neural activity is lower in the open areas and activity in the closed area can be used to predict the extent of exploration in the open area.

Though the authors provide some interesting new findings, several key classic place cell-related metrics were not evaluated, decreasing the potential impact of the work. For example, What percent of vH cells are place cells? What is are the place field size, information content, and peak and mean firing rate of open and closed preferring cells? Is there any characteristic in common among cells that show a shift in their place field towards the open space before the open space is shown? What is the stability of spatial representation of the same cell across days and across the same session?

There are not many hippocampal remapping papers related to threat exposure, but the authors fail to cite the few relevant papers that exist. The authors should include in their discussion the results from Wang et al., 2012 and Wang et al., 2015 (PMID: 26085635 and PMID: 23136419). The authors also should discuss Kong et al., 2021 (PMID: 34533133) and Schuette et al., 2021 (PMID: 32958567). These papers have related results on hippocampal remapping during exposure to threatening environments. The absence of these papers being cited provides a misleading view that the results are more novel than they actually are when considering the relevant literature.

Reviewer #1 (Public Review):

The authors set out to develop an in vitro model of multiple species representing diversity in the CF airway as a platform for a range of studies on why polymicrobial communities resist therapy. The rationale for their design is sound and the methods appear justifiable and reproducible. The major strength of this work is in producing a method for a range of future work, ideally for multiple groups in the field. The primary findings are interesting but not groundbreaking. One weakness in the method of reporting interspecies interactions and another in evaluating alternative causes of lasR advantages present opportunities for a stronger research contribution beyond this terrific method.

Reviewer #2 (Public Review):

Differences between the infection environment and in vitro model systems likely contribute to disconnects between the antimicrobial susceptibility profile of bacterial isolates and the clinical response of patients. The authors of this paper focus on a specific aspect of the infection environment, the polymicrobial nature of some chronic infections like those in people with Cystic Fibrosis (CF), as a factor that could impact antibiotic tolerance. They first use published genomic datasets and computational techniques to identify a clinically relevant, four-member polymicrobial community composed of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus spp., and Prevotella spp. They then develop a high throughput methodology in which this community grows and persists in a CF-like environment and in which antibiotic susceptibility can be tested. The authors determine that living as a member of this community decreases the antibiotic tolerance of some strains of biofilm-associated P. aeruginosa and increases the tolerance of most strains of planktonic and biofilm-associated S. aureus and planktonic and biofilm-associated Streptococcus. They focus on the decreased tolerance of P. aeruginosa and determine that a ΔlasR mutant of P. aeruginosa does not display increased tobramycin susceptibility in the mixed community. One of the phenotypes associated with a ΔlasR mutant is an overproduction of phenazines. The authors find that by deleting the phenazine biosynthesis genes from ΔlasR, they can restore community-acquired susceptibility. They further investigate this phenomenon by showing that a specific type of phenazine, PCA, is significantly increased in mixed communities with the ΔlasR mutant compared to WT. Finally, they demonstrate that adding a specific phenazine, pyocyanin, to mixed communities can restore the tolerance of WT P. aeruginosa.

Strengths:

With this study the authors address a very important problem in infectious disease microbiology - our in vitro drug susceptibility assays do a poor job of mimicking the infection environment and therefore do a poor job of predicting how effective particular drugs will be for a particular patient. By demonstrating how an infection-relevant community modifies tolerance to a clinically relevant drug, tobramycin, the authors identify specific interactions that could be targeted with therapeutics to improve our ability to treat the chronic infections associated with CF. In addition, this study provides a framework for how to effectively model polymicrobial infections in vitro.

The experiments in the paper are very rigorous and well-controlled. Statistical analysis is appropriate. The paper is very well-written and clear.

The authors do an admirable job of using in silico analysis to inform their in vitro studies. Specifically, they provide a comprehensive rationale for why they chose and studied the specific community they did.

The authors provide a very robust dataset which includes determining how strain differences of each of their four community members affect community dynamics and antibiotic tolerance. These types of analyses are laborious but very important for understanding how broadly applicable any given result is.

Weaknesses:

The authors very clearly and convincingly demonstrate that WT P. aeruginosa becomes more susceptible to tobramycin in their mixed community. Our ability to turn these types of observations into therapeutic development depends on mechanistic insight. That said, it is unclear if the authors can make any solid conclusions about what specific aspects of the polymicrobial environment cause WT P. aeruginosa to become more susceptible. The authors make a compelling case that increased phenazine production by the ΔlasR mutant restores tolerance in the mixed community and that exogenous phenazine addition increases the survival of WT P. aeruginosa in the mixed community. However, it remains a plausible explanation that the effects of phenazines on tobramycin susceptibility are independent of the initial observation that WT. P. aeruginosa becomes susceptible to tobramycin in the mixed community.

Some aspects of the methodology are unclear. Specifically, the authors note that they use a specific sealed container system to grow their strains in anoxic conditions, which mimic portions of CF sputum. However, it is unclear how the authors change medium over the course of their experiments, or how they test susceptibility to tobramycin, without exposing the cells to oxygen. It is well understood that oxygen exposure impacts the susceptibility of P. aeruginosa to tobramycin, so it is very important that the methodology involving oxygen deprivation and exposure is described in detail.

Reviewer #3 (Public Review):

This manuscript by Jean-Pierre et al. describes the creation and experimentation with a model CF lung community in an artificial sputum medium. The group uses data from 16S rRNA sequencing studies to select organisms for creating the model and then performs experiments to determine outcomes of growth competition and antibiotic tolerance in a community context. The main finding of the manuscript is that P. aeruginosa, notorious for its antimicrobial resistance phenotypes, is more susceptible to tobramycin in the community context than when grown alone. The manuscript is well prepared and follow-up experiments with mutant strains and phenazines greatly strengthen the project overall. The initial results paragraph where the authors go through the rationale for selecting the different organisms is perhaps a bit overkill, the organisms selected make sense based on their prevalence in CF airways, which in and of itself is a strong enough rationale. This aspect of the manuscript could be minimized to focus more on the exciting culture experiments in the latter parts of the results. Overall, this is a strong and well-crafted manuscript that will have a broad interest in the CF and microbial ecology fields.

Major Critiques:

I have two major critiques of this study.

1. Prevotella growth in monoculture. After reading the methods section it appears that the cultures were extensively washed and prepped prior to the inoculation into ASM. Prevotella did not grow alone, is this due to oxygen penetration of the cells during preparation? Perhaps oxygen is present in ASM prior to placement in an anaerobic bag? It is interesting, and perhaps worth exploring, whether the mixed community draws down oxygen from the media explaining the ability of Prevotella to grow. I suspect this is the case, but more detail is needed in the methods and this experiment would help us understand this interesting result.

2. Dilution of the community reproducing toby tolerance of P. aeruginosa. In supplemental figures, the replication of the 1:1000 dilution of the mixed community with P. aeruginosa shows poor replication and very large error bars. This experiment should be repeated to ensure it is reproducible.

Reviewer #1 (Public Review):

The authors sought to explore the brain age paradigm in the early stages of Alzheimer's disease, focusing on the combination of different MRI modalities (brain structure derived from T1-weighted MRI and functional connectivity derived from resting state fMRI). Their goal was to understand how different unimodal brain ages and a combined multi-modal brain age related to risk factors related to Alzheimer's disease, namely hippocampal volume, cognitive performance, amyloid or tau positivity from PET scans or CSF data and neurofilaments. As part of this, they aimed to ascertain which brain age models performed more accurately.

The major strength of the methods is the novel combination of different MRI modalities using Gaussian Processes and stacking to predict brain age. Another strength is the use of multiple data sources for both model training and testing, reducing the reliance on a single site and decreasing the likelihood of overfitting, which should improve generalisability. A weakness is the poor fit of the functional connectivity model to the data, whereby the vast majority of test participants were shown to have younger appearing brains, even those with cognitive impairment. This indicates that an alternative fMRI processing pipeline could have been beneficial, however, no experimentation on this important facet of the analysis was included. Another weakness is the relatively limited sample size compared too much of the brain age literature and the failure to report the R^2 metric, which is important for the comparison of this study with previously published reports. Potentially, more accurate models would have led to clearer results, as there are a number of borderline findings which hinder clear interpretation.

In general, the study did meet its stated goals and was able to generate a multi-modality brain-age model and this model did show older appearing brains in people with cognitive impairment. This model also showed that people with older appearing brains had poorer cognition, lower hippocampal volume, and greater amyloid deposition. In people who met the criteria for being cognitively impaired, greater tau deposition on PET scans was associated with an older appearing brain. One claim of the study is that the multi-modality brain-age model was more accurate than the brain-volume model, however, it is unclear from the report whether appropriate statistics were used for this. The authors need to clarify exactly what procedure they undertook to compare the models, as they potentially employed an erroneous method (determining statistical significance based on the number of bootstraps instead of the number of observations) which may have led them to mistakenly claim better performance.

Given the relatively poor or equivocal performance of the brain age models and the relatively small sample sizes available, it is not clear that the modelling or dataset will have a big impact on the field. More accurate modelling methods are openly available, as are larger datasets. Nevertheless, the study is well-motivated and scientifically rigorous, so the results themselves are informative regarding the interrelationships of key Alzheimer's biomarkers and risk factors.

Reviewer #2 (Public Review):

Millar et al. have used multimodal brain magnetic resonance imaging (MRI) data from subjects with preclinical AD (cognitively normal with amyloid pathology), cognitive impairment, and matched cognitively normal (CN) subjects to predict the subject's age (i.e. brain age). To do so they have trained a Gaussian Process Regression (GPR) model using data from 3 datasets. The predicted age was then compared to the chronological age to calculate the brain age gap (BAG). Using resting-state functional MRI (rsfMRI) they calculated functional connectivity across 300 brain regions. Similarly, T1w MRI images have been used to calculate volumetric measures across 68 cortical and 33 subcortical regions. The results were then used as features in two separate models resulting in FC-BAG and Vol-BAG measures, respectively. A third model is then devised by "stacking" the previous model's predictions as features in a new model resulting in Vol+FC-BAG. The models were then applied to the test dataset and BAG measures were calculated for subjects in CN, preclinical AD, and cognitively impaired (CI) groups. All models show significantly higher BAGs for subjects with cognitive impairments. Finally, the authors have examined the relationship between BAGs measures and Amyloid Markers, Tau Markers, Neurodegeneration Markers, and Cognition.

Strengths:

The manuscript is very clearly written. The study is well designed and for the most parts the method section contains all the necessary information to replicate the steps. The sample size is comparable to similar studies investigating brain age in clinical populations and the inclusion and exclusion criteria are clearly stated. In order to avoid bias and overfitting in the predictive models the authors have (1) used a separate training (+validation) set and test set and (2) removed any subjects with potential pathology or impairment from the training set. Using data points on the plots as well as a combination of boxplot+violin plots makes the results clear and data distributions are provided when necessary. Furthermore, chronological age has been used as a covariate to correct the relationship between BAG and age, making the results more interpretable and reliable. Focusing on the stronger section of the results, the study shows a higher Vol-BAG (or Vol+FC-BAG) in subjects with CI, which is significantly related to higher Amyloid PET, higher PET and CSF-related Tau measures, and lower global cognition. In conclusion, the Vol-BAG results are clear and clinically relevant, based on a model with a reasonable prediction performance.

Weaknesses:

The manuscript follows authors' recently published work on FC-BAG in symptomatic and preclinical Alzheimer disease (Millar et al, Neuroimage 2022) by adding T1w volumetric measures from Freesurfer. Based on the results the additional value of rsfMRI connectivity is at best marginal. The FC-BAG model has a weak performance and is outperformed by Vol-BAG. The marginal benefit of adding FC-BAG to the Vol-BAG model is around 10% which comes with the additional cost of a new and more computationally demanding modality as well as making the biological relevance of the model almost untraceable. The preclinical findings reported as "Specifically, FC-BAG may capture a unique biphasic response to preclinical AD pathology" while potentially interesting are based on an unreliable model (FC-BAG) and can be a spurious finding. These results need further validation both robustness analysis within the current sample and in independent datasets. The other findings related to preclinical AD are based on the hippocampal volume which as the authors have mentioned in the discussion limitation is part of the features included in the Vol-BAG model and absent from FC-BAG.

In conclusion, the manuscript has clear findings based on the Vol-BAG model differentiating the cognitively impaired subjects from other groups and these results relate to the clinical severity of the disease as measured by Amyloid Markers, Tau Markers, and Cognition.

Reviewer #3 (Public Review):

In this manuscript, the authors aim to study the relationship between the Brain Age Gap (BAG) measure - based on functional connectivity and structural features - and different AD biomarkers such as amyloid, tau, cognition, and neurodegeneration in cognitively healthy and demented individuals. The main results showed increased BAG in cognitively impaired individuals. In this subgroup of individuals BAG models based on structural data were associated with more advanced AD pathology and lower cognitive performance. The BAG models based on fMRI data seem to show a U-curve in the health-disease continuum. The authors discuss the results in terms of a biphasic response of fMRI - while structural-based BAG would capture progression as well as highlight the advantages of multimodal data to understand health and disease in healthy aging.

While the study has its merits such as the use of novel metrics, a decent sample with biomarkers and fMRI, etc., I believe some of the main conclusions of this paper are not fully substantiated by the results. The results based on the structural BAG model are solid (i.e., CI participants have older BA compared to healthy controls). However, I find the conclusions regarding the fMRI-BAG and the multimodal-BAG models are not fully supported by the results. The biphasic response of fMRI-BAG results - and the subsequent advantage of multimodal BAG - is based on p-values between .05 and .10 which have very low evidential value (e.g., Benjamin et al, 2018). I strongly discourage reporting these results as "marginal" and drawing assertive interpretations on this basis. Further, the poor performance of fMRI seems to add little information (in the stacked model) to the structural-only BA model.

The aim of the authors is to be commended, that is to take advantage of powerful machine learning methods and multimodal imaging to better understand the health-disease continuum in aging. This path is promising and can lead to both, better predictive tools and a better understanding of the aging brain. Further, the sample is good, from a single cohort, with multiple MRI modalities and biomarker information and the manuscript is easy to read and includes a very informative introduction. Also, it has some interesting findings such as in Fig. 2C and 2E where the graphs seem to show how BAG seems to be most useful at younger ages if used to predict dementia. Having said that, the "marginal" effects are central to the conclusions of this paper and are a critical caveat. Other methodological limitations of this paper are the parcellation used for structural BAG, which is relatively gross, a possible effect of motion on preprocessed functional connectivity, and the lack of multiple comparisons correction. Finally, the lack of a detailed description of the higher-level statistical analysis is detrimental to the clarity of the manuscript and leads to some confusion regarding the carried analyses.

Reviewer #1 (Public Review):

This is an important, well-written and easily comprehended quantitative imaging study that analyzes the motion of endo-lysosomal compartments within axons in vivo using simultaneous multiphoton imaging in the mammalian brain. The simultaneous dual two-photon imaging is well-executed and represents a substantive advance in a field that relies heavily on in vitro neuronal culture preparations. This work opens the door to neurons that have aged appropriately and done so in the context of normal synaptic and neuromodulatory input, without an excess of added factors that occurs with in vitro cell culture. The authors solve an issue of cell polarity, providing strong support for their ability to determine directional movement (anterograde versus retrograde). In principle, this could become a generalized approach, opening this type of experiment up to other investigators. Finally, interesting differences in motion are observed, including activity-dependent and calcium-dependent changes that differ from measurements made in vitro. This is a significant technical advance with interesting observations that substantively move the field forward.

Reviewer #2 (Public Review):

Here the authors used viral expression and two-photon imaging, a very demanding approach, to explore the transport dynamics of three membrane markers (Neuropeptide Y-dense core vesicles, LAMP1-endolysosomes and RAB7-late endosomes) in vivo in the mouse brain. This allowed deciphering for the first time anterograde and retrograde velocities in vivo rather than in cultured neurons. The authors showed that the different vesicular compartments have different anterograde and retrograde velocities, pausing at synapses. They further used brain slices to explore the effect of increased calcium levels.

Major strengths reside in the novelty of the approach (in vivo!).

The main weakness relates to the lack of novel mechanisms and the difficulty of using such a sophisticated setup on a routine basis.

This is a technical 'tour-de-force', a clear reference article for future studies addressing vesicular transport in vivo.

Of course, one would be curious to see many more markers studied in this setup. Also, the same study in mouse mutants would be extremely interesting.

Reviewer #3 (Public Review):

The authors use two-photon imaging to visualize various axonal organelle populations that they have virally labeled with fluorescent proteins, including DCVs and late endosomes/ lysosomes. The latter topic is a bit contentious, as the authors use two labels that tag potentially overlapping and not highly specific markers so that the nature of the tagged organelle populations remains unclear. Notably, the authors also have previously published a detailed account of how DCVs traffic in vivo, so the novelty is mostly in comparing the behavior of different organelles and the potential influence of activity.

Overall, the reported results mostly corroborate the expectations from previous in vitro and in vivo work on these organelles and other cargoes, performed by the authors and their collaborators, as well as in many other laboratories:<br /> (i) Different organelles have different transport behaviors regarding speed, the ratio of anterograde to retrograde moving organelles, etc.<br /> (ii) Organelles move in different ways when they pass specific anatomical landmarks in the axons, such as presynaptic terminals.<br /> (iii) Activity of a neuron (here measured by calcium imaging) can impact the measured transport parameters, albeit in a subtle and mechanistically not well-defined manner. The chosen experimental design precludes a more detailed analysis, for example of the precise movement behavior (such as defining the exact pausing/movement behavior of organelles, which would require higher imaging speeds) or of a correlation of different organellar behavior at synaptic sites or during activity (which would require three-channel simultaneous imaging of two organelle classes plus a synaptic or activity marker).

In summary, this publication uses sophisticated in vivo labeling and imaging methods to corroborate and complement previous observations on how different axonal organelles move, and what influences their trafficking.