Reviewer #1 (Public Review):

Tomanek and Guet describe the results of an evolution experiment where they allowed the bacterium E. coli to adapt to various concentrations of galactose as an additional carbon source. These conditions impose different degrees of demand for the galK enzyme, whose expression level depends on the promoter sequence and on the number of copies of the galK locus. Given that the initial promoter is random and weak, both amplifications of the locus and mutations in the promoter are expected to be adaptive. The experimental strains of E. coli were equipped with a fluorescent reporter system designed to discriminate between these two types of mutations. Furthermore, two strains, IS+ and IS-, were engineered with high and low rates of duplication around the galK locus, respectively. The main result is that at higher concentrations of galactose, where the demand for galK is high, E. coli adapts by acquiring combinations of both types of adaptive mutations, amplifications, and promoter mutations. In contrast, at low concentrations of galactose, where the demand for galK is low but not zero, E. coli appear to adapt by acquiring either an amplification or a promoter mutation but not their combinations. The observation of apparent interference between the acquisition of these two types of mutations is interesting and novel. The authors provide an intuitive explanation for it: when one mutation is sufficient to achieve the optimal expression of the gene, the mutation that is acquired first makes the other mutation obsolete, i.e., there is negative epistasis (possibly even sign epistasis) between these mutations, in the sense that the second mutation is much less adaptive (or possibly even deleterious) in the presence of the first one, in the low-demand environment. The authors discuss the possible implications of this finding for our understanding of molecular evolution and propose a new Amplification Hindrance hypothesis. This hypothesis states that, since amplifications occur at much higher rates than individual point mutations, they can slow down or even prevent sequence divergence. The amplification hindrance hypothesis stands in contrast to the Innovation-Amplification-Divergence hypothesis which is currently the default paradigm and states that amplifications generally accelerate sequence divergence.

STRENGTHS:

The authors designed a powerful reporter system that allows them to monitor the evolutionary dynamics of amplifications and promoter mutations. They ask an important question: how do early evolutionary dynamics of adaptation to environments with different demands for gene expression look like? The phenotypic data they present looks very interesting and shows the existence of interference between amplifications and point mutations in low-demand but not in high-demand conditions. The Amplification Hindrance hypothesis is a novel and useful intellectual contribution to the field.

WEAKNESSES:

In my opinion, the main weakness of the paper is that, while the interference between amplifications and point mutations in the low-demand condition clearly happens (most convincingly shown in Figure 5), its causes remain unclear. In particular, the authors claim that this interference is caused by negative epistasis, but the possibility of clonal interference without epistasis has not been decisively ruled out. The authors mention clonal interference tangentially in the Discussion, but they do not seriously address this alternative explanation. Yet, understanding the cause of this phenomenon is important because clonal interference and negative epistasis have different implications for long-term evolution.

The authors' main hypothesis is that, in the low-demand conditions, expression-increasing point mutations in the promoter provide much lower fitness benefits (or even incur fitness costs) in strains with galK amplifications compared to the ancestral strain without amplifications. The most direct way to test this hypothesis would be to measure the fitness effects of a point mutation in genetic backgrounds with and without amplifications in conditions with low and high demand for galK. This decisive experiment has unfortunately not been done. Instead, the authors construct an indirect argument, whose essence is as follows.

They show that, over the course of the experiment in the low-demand environment, the IS+ populations have acquired fewer point mutations than IS- populations (Figure 5). In addition, the phenotypic data in Figures 2 and 4 demonstrate that IS+ mutations in the low-demand environment contain three phenotypic classes of cells: ancestral, YFP+ and YFP+CFP+. The YFP+ clones are shown to have only one or two promoter mutations. The YFP+CFP+ cells must have duplications, and it is likely (although not quite certain, see below) that they do not have any promoter mutations. These data demonstrate quite convincingly that, whenever adaptation by duplications is possible, the rate at which point mutations segregate and accumulate declines. These data are consistent with the authors' hypothesis based on negative epistasis. However, they also seem to be consistent with the idea that amplifications and point mutations exhibit clonal interference without negative epistasis.

It may be possible to construct an argument against this alternative hypothesis based on the comparison between different environments, but such an argument would have to take into account the fact that clonal interference depends not only on the rates of mutations (which are presumably the same in all environments) but also on their fitness effects which vary across the environment. Another possibility to argue against clonal interference might be by carrying out simulations, although this approach also seems challenging without knowing some key population genetic parameters. The most direct way to resolve this ambiguity would be to demonstrate negative epistasis as discussed above.

Another, less critical but still important, issue mentioned above concerns the authors' claim that the YFP+CFP+ cells have only duplications but no promoter mutations (e.g., LL. 276-277). This is certainly consistent with intuition since these cells have an increased level of both YFP and CFP relative to the ancestor. However, as far as I can tell, there is no evidence to support this claim directly. My understanding is that the authors base this claim on the fact that YFP+CFP+ cells form a cluster of points on the YFP vs CFP plots that is distinct from the cluster of "mixed" cells, which are shown to have both an amplification and a promoter point mutation (Figure 3). But it is still logically possible that the YFP+CFP+ cells have an amplification and a promoter mutation other than the one found in the "mixed" cells (e.g., weaker). The most direct way to show that YFP+CFP+ cells have no promoter mutations would be to sequence a few of them. Another possibility would be to calibrate the YFP/CFP fluorescence measurements against galK copy number.

Reviewer #1 (Public Review):

Dectin-1 is a known C-type lectin receptor that has a role in recognizing pathogen glycans, particularly beta-glucan. Haji et al. present evidence that Dectin-1 also has an endogenous ligand, another C-type lectin that is enriched on platelets called CLEC-2. Using a Dectin-1 reporter line, they identify human platelets as a source for the ligand, raising a panel of mAbs to human platelets and identify one (6D11) that prevents signalling and use this to identify CLEC2 as the Dectin-1 receptor by immunopurification. The authors go on to further characterise this interaction by showing that it occurs between the human but not mouse orthologues, showing that it is the stalk region of human Dectin-1 that contains the ligand which consists of sialylated core 1 O-linked glycans displayed on Thr 105 and adjacent amino acids. Finally, the authors over-express human Dectin-1 in mice within the context of a null background for another CLEC-2 ligand (podoplanin) and show that this rescues embryonic lethality. They show that Dectin-1-dependent CLEC-2 signalling is not sufficient to induce platelet aggregation but can rescue perinatal lethality in podoplanin-deficient mice.

There is a large amount of work in this manuscript and the experiments are well controlled and the results unequivocal and usually verified by several independent techniques. The paper is very well-written making the volume of data accessible. There is novelty here in that this unusually finds that 2 C-type lectin receptors directly interact and the biochemical characterisation of the glycan binding determinants is very well performed. While the authors show that the interaction occurs between the human but not mice orthologues (because mouse Dectin-1 lacks the critical EDxxT motif that is necessary for displaying the o-linked glycan in the correct context) they were able to investigate the functional role of the interaction by generating mice that overexpressed human Dectin-1 in a podoplanin-deficient background. Normally, podoplanin-deficient mice die at birth due to defects in lymphatic vessel development, but this lethality is rescued by overexpression of human Dectin-1. This xeno-overexpression data can be difficult to interpret but suggests that while human Dectin-1 signalling to mouse platelet CLEC-2 is insufficient to drive platelet activation and thrombus formation, it is sufficient to rescue some aspects of platelet function involved in lymph vessel development. They conclude that human Dectin-1 (which is broadly expressed on different tissues) provides a basal level of tonic signalling through platelet-expressed CLEC-2 to establish platelet signalling thresholds.

I thought the manuscript was comprehensive in its approach, well written, and that the experimental data supported the conclusions.

Reviewer #1 (Public Review):

The authors tackle an interesting problem: how do ant colonies regulate foraging in response to their collective hunger? In previous work, the authors related the colony's response to individual ants sensing their own food levels and its temporal dynamics. Looking more carefully at the spatial dynamics of ants, the authors now find that foragers tend to move toward the depth of the nest when their food load is high and toward the nest exit when it is low. This is an elegant and computationally inexpensive set of rules that explains the spatiotemporal dynamics of the system.

Overall, the paper is written clearly, the methods are sound, and I agree with the interpretation of the results.

I do have a few comments and suggestions:

1) How exactly are the inward outward directions defined? Is it simply, away, or towards the entrance? It is not clear from the text, and since this system is not symmetric (cubic with entrance at one of the corners) the authors should clarify.

2) To analyze the biased random walk analysis of the ants, the authors "coarse-grained" the steps as being "inwards" "outwards" and "stay". It's not clear how this level of granulation is justified. Since the authors have access to the actual trajectories and all trophallaxis events, why not just calculate the actual turning angles between consecutive steps the ants take? This would give an actual assessment of both the bias and the noise imposed on the random walks, which the authors could then use directly in their models.

3) It would be important to better connect the author's previous mechanism (relating the colony's response to individual ants sensing their own food levels and its temporal dynamics) to the new mechanism (spatial-temporal dynamics). Are they mutually exclusive? It would be useful to elaborate on this in the Discussion.

4) It would be useful to add a few supplementary movies from the experiments, showing ants moving toward the entrance with low food loads, and moving away from the entrance with high food loads.

Reviewer #1 (Public Review):

This paper addresses an important issue of how humans select foot placement when running on uneven terrain. The authors examine empirical and model-based data to determine whether runners specifically opt for more level locations on such surfaces. The manuscript suggests potential strategies of how humans mitigate fore-aft impulses during foot contact so as to maintain stability in response to changes in terrain.

Overall, the manuscript provides additional insight into lower-limb mechanics of runners on natural surfaces. The model-based analysis of lower limb compliance is especially useful in the context of stability and understanding human motor control. In addition, participant foot placement analysis using empirical and statistical models is very compelling and provides insight into how much planning occurs during running on uneven terrain. However, there are a few concerns of note:

- A central motivation of the study appears to be that past research did not incorporate height and slope variations when studying gait on uneven terrain. Although some of the past work cited by the authors does focus on step-like terrain, the (Voloshina, Ferris 2015) study had both height and slope variations. In that particular study, the terrain consisted of blocks that were smaller than the dimensions of the average human foot. This means that, during each foot-flat phase, the foot had to span at least two blocks of different heights, placing it at a slope in the fore-aft direction. Similarly, the columns of that terrain layout provided slope variations in the medio-lateral direction. Referring to this surface as "step-like" is inaccurate and potentially misleading. Considering that the terrains in the present study and the study from 2015 likely cause very similar types of perturbations to the runner, the motivation behind the current study is not strongly validated. The authors should consider re-evaluating their results in the context of past studies.

- A primary outcome of the study is the analysis of empirical and model-based fore-aft impulses experienced by runners during foot contact. The authors suggest that this measure is related to stability but do not provide extensive explanations. It would be helpful to include additional background information on how impulse analyses have been used previously and why they are particularly fitting in this context.

- The study evaluates participants running back and forth on a 24m track for up to 10 minutes. This means that participants had to perform many turns during each trial. The authors present metabolic energy expenditure data but do not address how these data may be skewed due to the large instances of directional changes. Measuring metabolic data during such tasks is generally noise-prone, potentially leading to an inaccurate representation of energy expenditure. Considering that this is a comparative study and participants had to perform such turns for each terrain trial, this issue could be minor. However, the authors are encouraged to provide more detail on experimental protocol. Addressing whether or not participants stepped off the terrain to switch directions and providing insight into how this experimental approach could potentially affect outcomes could be particularly helpful.

Reviewer #1 (Public Review):

This is a fascinating paper that takes up an important question with a creative and new approach. We have a few suggestions that we hope are constructive for the authors.

1. One nagging concern is that the category structure in the CNN reflects the category structure baked into color space. Several groups (e.g. Regier, Zaslavsky, et al) have argued that color category structure emerges and evolves from the structure of the color space itself. Other groups have argued that the color category structure recovered with, say, the Munsell space may partially be attributed to variation in saturation across the space (Witzel). How can one show that these properties of the space are not the root cause of the structure recovered by the CNN, independent of the role of the CNN in object recognition?

2. In Figure 1, it could be useful to illustrate the central observation by showing a single example, as in Figure 1 B, C, where the trained color is not in the center of the color category. In other words, if the category structure is immune to the training set, then it should be possible to set up a very unlikely set of training stimuli (ones that are as far away from the center of the color category while still being categorized most of the time as the color category). This is related to what is in E, but is distinctive for two reasons: first, it is a post hoc test of the hypothesis recovered in the data-driven way by E; and second, it would provide an illustration of the key observation, that the category boundaries do not correspond to the median distance between training colors. Figure 5 begins to show something of this sort of a test, but it is bound up with the other control related to shape. Similarly, if the claim is that there are six (or seven?) color categories, regardless of the number of colors used to train the data, it would be helpful to show the result of one iteration of the training that uses say 4 colors for training and another iteration of the training that uses say 9 colors for training. The text asserts that Figure 2 reflects training on a range of color categories (from 4 to 9) but doesn't break them out. This is an issue because the average across these iterations could simply be heavily biased by training on one specific number of categories (e.g. the number used in Figure 1). These considerations also prompt the query: how did you pick 4 and 9 as the limits for the tests? Why not 2 and 20? (the largest range of basic color categories that could plausibly be recovered in the set of all languages)?

3. Regarding the transition points in Figure 2A, indicated by red dots: how strong (transition count) and reliable (consistent across iterations) are these points? The one between red and orange seems especially willfully placed.

4. Figure 2E and Figure 5B are useful tests of the extent to which the categorical structure recovered by the CNNs shifts with the colors used to train the classifier, and it certainly looks like there is some invariance in category boundaries with respect to the specific colors uses to train the classifier, an important and interesting result. But these analyses do not actually address the claim implied by the analyses: that the performance of the CNN matches human performance. The color categories recovered with the CNN are not perfectly invariant, as the authors point out. The analyses presented in the paper (e.g. Figure 2E) tests whether there is as much shift in the boundaries as there is stasis, but that's not quite the test if the goal is to link the categorical behavior of the CNN with human behavior. To evaluate the results, it would be helpful to know what would be expected based on human performance.

5. The paper takes up a test of color categorization invariant to luminance. There are arguments in the literature that hue and luminance cannot be decoupled-that luminance is essential to how color is encoded and to color categorization. Some discussion of this might help the reader who has followed this literature. Related, the argument that "neighboring colors in HSV will be neighboring colors in the RGB space" is not persuasive. Surely this is true of any color space?

6. The paper would benefit from an analysis and discussion of the images used to originally train the CNN. Presumably, there are a large number of images that depict man-made artificially coloured objects. To what extent do the present results reflect statistical patterns in the way the images were created, and/or the colors of the things depicted? How do results on color categorization that derive from images (e.g. trained with neural networks, as in Rosenthal et al and presently) differ (or not) from results that derive from natural scenes (as in Yendrikhovskij?).

7. It could be quite instructive to analyze what's going on in the errors in the output of the classifiers, as e.g. in Figure 1E. There are some interesting effects at the crossover points, where the two green categories seem to split and swap, the cyan band (hue % 20) emerges between orange and green, and the pink/purple boundary seems to have a large number of green/blue results. What is happening here?

8. The second experiment using an evolutionary algorithm to test the location of the color boundaries is potentially valuable, but it is weakened because it pre-determines the number of categories. It would be more powerful if the experiment could recover both the number and location of the categories based on the "categorization principle" (colors within a category are harder to tell apart than colors across a color category boundary). This should be possible by a sensible sampling of the parameter space, even in a very large parameter space.

9. Finally, the paper sets itself up as taking "a different approach by evaluating whether color categorization could be a side effect of learning object recognition", as distinct from the approach of studying "communicative concepts". But these approaches are intimately related. The central observation in Gibson et al. is not the discovery of warm-vs-cool categories (these as the most basic color categories have been known for centuries), but rather the relationship of these categories to the color statistics of objects-those parts of the scene that we care about enough to label. This idea, that color categories reflect the uses to which we put our color-vision system, is extended in Rosenthal et al., where the structure of color space itself is understood in terms of categorizing objects versus backgrounds (u') and the most basic object categorization distinction, animate versus inanimate (v'). The introduction argues, rightly in our view, that "A link between color categories and objects would be able to bridge the discrepancy between models that rely on communicative concepts to incorporate the varying usefulness of color, on the one hand, and the experimental findings laid out in this paragraph on the other". This is precisely the link forged by the observation that the warm-cool category distinction in color naming correlates with object-color statistics (Gibson, 2017; see also Rosenthal et al., 2018). The argument in Gibson and Rosenthal is that color categorization structure emerges because of the color statistics of the world, specifically the color statistics of the parts of the world that we label as objects, which is the same approach adopted by the present work. The use of CNNs is a clever and powerful test of the success of this approach.

Reviewer #1 (Public Review):

This paper uses 2 cohorts from the UK and links a) risk factors associated with low antibody levels after vaccination and b) risk factors for infection. The paper makes the important point that following the third vaccination, risk factors associated with low antibody response after the first vaccination, are less likely to lead to sub-protective levels. This highlights the importance of obtaining a booster shot. Though it is not a primary finding of the paper, the observed discordance between self-reported infection and anti-nucleocapsid positivity is an important finding. While these findings are potentially useful, the presentation of the data is somewhat unfocused, and the message is presented in a diffuse fashion. Moreover, certain key components of the analysis such as the assay threshold and timing of samples after the 2nd vaccine are a bit confusing and require clarification. The use of univariate analyses can be misleading. Finally, the relevance of the findings relative to our current stage of the pandemic with multiple new VOCs requires a clearer explication.

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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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.

DOI: 10.7554/eLife.82094

Resource: RRID:ZFIN_ZDB-ALT-170110-1

Curator: @MufeiL

SciCrunch record: RRID:ZFIN_ZDB-ALT-170110-1

What is this?

DOI: 10.7554/eLife.82094

Resource: RRID:ZFIN_ZDB-GENO-191211-1

Curator: @MufeiL

SciCrunch record: RRID:ZFIN_ZDB-GENO-191211-1

What is this?

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.

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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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.

Water is seen as an economic resource and because of its scarcity is considered to be priced at full economic and environmental cost. Additionally, it is considered that it should be set aside for its highest valued users and managed by private companies for profit.

Claim 1: Water is no longer a human right or those without it would be able to appeal for access to water. Evidence for Claim 1

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 #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 #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 #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 #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 #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 #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 #1 (Public Review):

The authors develop and freely disseminate the THINGS-data collection, a large-scale dataset incorporating MRI, MEG, eye-tracking, and 4.7 million similarity ratings for 1,854 object concepts. Demonstrating the reliability of their data, the authors replicate nearly a dozen previous neuroimaging papers. This "big data" approach significantly advances our ability to link behavioral measures with neuroimaging at scale, with the potential to spark future insights into how the mind represents objects.

I thought that the article was well-written, with a sound methodological approach, high-quality results, and well-supported conclusions. I am overall enthusiastic about this work, and I think THINGS will provide an important benchmark for future big data approaches in cognitive and computational neuroscience.

However, I thought it was also important to articulate more directly the potential insights this dataset can offer to the field. Although the authors mentioned that they "provided five examples for potential research directions", it was not clear to me what these new research directions were, given that the authors entirely describe replications in the results.

Reviewer #1 (Public Review):

The authors of this paper have used emerging environmental DNA (eDNA) methods to profile depth-biodiversity in two deep ocean trenches.

Strengths:<br /> Working in deep ocean habitats is challenging and this paper provides data from not one but two deep ocean trenches and provides new perspectives on biodiversity distributions in these habitats. The most interesting finding is that deep ocean habitats appear to contribute much more biodiversity, as measured using relatively novel eDNA techniques than previously thought. The comparison of these two trenches also illustrates the variable nature of these biodiversity patterns and suggests trench-specific features (e.g. unique habitats and/or productivity) influence deep ocean biodiversity.

Weaknesses:<br /> While eDNA methods are becoming more established, there remains skepticism by many in the scientific community about the origins of the detected DNA (e.g. does it drift in from other areas or water layers?). If these concerns aren't addressed (i.e. by citing supporting literature on the fate of eDNA), the different biodiversity profiles between trenches could possibly be explained by differing oceanography. There is also some important methodological information that is missing from this manuscript. For example, sampling volumes will affect the amount of biodiversity detected, but it is not clear if sample volumes are consistent across depths and study areas. It was also not indicated whether field controls (blanks) were taken to assess the potential contamination of samples. Lastly, the literature in the eDNA field is progressing rapidly and there are some missing papers (e.g Thomsen et al. 2016, Canals et al. 2021, McClenaghan et al. 2020, Govindarajan et al. 2021, etc.) that are relevant to the technique used in this manuscript and the habitat studied.

Reviewer #1 (Public Review):

This group previously demonstrated that trisomy 21 causes an increase in PCNT levels, and this increase leads to pericentrosomal crowding and inhibition of ciliogenesis in fibroblasts. The authors here use trisomy and tetrasomy 21 retinal pigment epithelium cells generated by microcell-mediated chromosome transfer (MMCT) and previously generated mouse models of human trisomy 21. The well-quantified data and well-reasoned paper compellingly demonstrate that modestly increased PCNT levels can attenuate ciliogenesis and may result in trisomy 21-associated phenotypes such as cerebellar growth defects.

Reviewer #1 (Public Review):

The authors describe a feeding system for killifish that allows high precision control of feeding amount and schedule on a per-tank basis. The system permits automation of this task using open-source and affordable components and software. Due to this emphasis, the system appears amenable to manufacture by individual research groups and the approach appears very scalable (although more detailed build, programming and assembly instructions and videos might be useful for groups with little experience with microcontrollers and manufacturing). An exciting aspect of the system is the possibility to modify the system for different purposes. For example, it might be possible to reduce the minimum feeding amount, thereby allowing more fine grained exploration of effects related to feeding shedule. I am very enthusiastic about the open-source "maker" aspects of this work.

The authors next explore two interesting applications of the system. First, they show that precise control of food allows automated investigation of lifespan extension under calorie restriction (CR) conditions. This is an important use case for a system of this type and showing that it is fit for this application is important.

Secondly, the authors show an exciting modification of the system that involves only addition of a simple red light LED. This modification allows use of the system in a associative learning / conditioning paradigm.

Finally, they show that there is an age-dependent decline in learning as evaluated by this conditioning paradigm. I am very enthusiastic about this additional function and, again, this example demonstrates the flexibly and open nature of the technology, suggesting that others can likely modify and expand the system to suite their own questions and applications. In summary, I am enthusiastic about the technology described and about the approach by which the system was developed.

However, at the current stage, the biological applications are essentially validation experiments - e.g. showing that CR can be implemented and that the system can be used for learning and memory experiments. Neither of these aspects is pursued beyond the basic validation experiments (showing that lifespan extension can be achieved and that there is age-dependent decline in associative learning).

Reviewer #1 (Public Review):

Leukemic cells are known to remodel bone marrow niche to promote their expansion and to suppress normal hematopoiesis. However, molecular mechanisms remain largely unknown. In this manuscript, authors developed new experimental models in mice to address this issue, using mouse BCR-ABL-driven ALL cells marked with YFP, or DOX-inducible MLL-AF9 AML cells. After transplantation of either of these cells, authors discovered suppression of host hematopoiesis. Using these systems, authors tested their hypothesis on lymphotoxin receptor-mediated interaction of the leukemic cells and stroma cells.

The main conclusions here are: 1) lymphotoxin signaling through its receptor mediates IL7 down regulation and alters gene expression related to inflammation etc. in stroma cells, 2) IL7 down regulation leads to reduction in B lymphoid cells but not myeloid cells, 3) lymphotoxin expression in leukemic cells is induced by DNA damage response, and 4) CXCR4, which is known to be induced in B cells in response to stroma cells, collaborates with DNA damage in induction of lymphotoxin in leukemic cells. Taken together, authors suggest that a positive feedback loop of leukemic cells and stroma cells for leukemic cell proliferation and normal hematopoietic suppression, involving lymphotoxin and CXCR4 in leukemic cells and lymphotoxin receptor in stroma cells. Generally, these conclusions and the model of the positive feedback regulation are supported, to a reasonable level, by the experimental results provided in the manuscript. However, some of the results show small effects of manipulations, leaving the pathological significance of the feedback model as a future issue.

Reviewer #1 (Public Review):

Polarization in cells and organs is often dictated by opposing polarity domains. In grass subsidiary cells, several proteins (including PAN1) were previously found to polarize in a discrete patch prior to asymmetric division. Zhang et al. identify POLAR via transcriptional profiling of Bdmute, a mutant that lacks subsidiary cells The authors effectively show that Bdpolar mutants have defective subsidiary cells. A distinctive and exciting localization pattern of POLAR is demonstrated, which is opposite to PAN1. This localization pattern is further contextualized by showing that PAN1 and MUTE are both required for POLAR's distinctive localization; however, PAN1 polarization is unaffected in both polar and mute. The integration of MUTE, POLAR, and PAN1 is particularly important as it integrates how polarity proteins and fate factors interact with each other.

Bdpolar mutants have defects in subsidiary cells that lead to defects in stomatal function. The authors carefully and quantitatively compare the phenotypes of pan1 and polar and conclude distinct roles for the two proteins based on differences in phenotypes including nuclear polarization, division site specification, and repeated rounds of cell division. The discovery and localization of POLAR are very exciting, but the comparison between single alleles of pan1 and polar and the extrapolation requires scrutiny. In particular, the data on division site specification in pan1 seem inconsistent with the % defective subsidiary cells and nuclear migration defects. However, these are addressable and given the exciting nature of the localization and pathway determination, the paper's impact stands.

Reviewer #1 (Public Review):

The research investigates the genetic basis for resistance to high CO2 levels in the human pathogenic fungus Cryptococcus neoformans. Screening collections of over 5,000 gene deletion strains revealed 96 with impaired growth, including a set of genes all related to the same RAM signaling pathway. Further genetic dissection was able compellingly to place where this pathway lies relative to upstream inputs and through the isolation of suppressor mutants as potential downstream targets of the pathway. Given the high levels of CO2 encountered by fungi in the human host, this work may provide new directions for the control of disseminated fungal disease.

The research presents both strengths and weaknesses.

Strengths include:

(1) One of the largest scale analyses of genes involved in growth under high CO2 concentrations in a fungus, revealing a set of just under 100 mutants with impaired growth.<br /> (2) Elegant genetic epistasis analysis to show where different components fit within a pathway of transmission of CO2 exposure. For example, over expression of one of the kinases, Cbk1, can overcome the CO2-sensitivity of mutations in the CDC24 or CNA1 genes (but not in the reciprocal overexpression direction).<br /> (3) Isolation of suppressor mutations in the cbk1 background, now able to grow at high CO2 levels, was able to lead to the identification of two genes. Follow up characterization, which included examining in vitro phenotypes, gene expression analysis, and impact during mouse infection was able to reveal that the two suppressors restore a subset of the phenotypes impacted by mutation of CBK1. Indeed, one conclusion from this careful work is that the reduced virulence of the cbk1 mutant is not due to its sensitivity to high levels of CO2, perhaps an unexpected finding given the original goals of the study towards linking CO2 sensitivity with decreased virulence.

Weaknesses include:

(1) What is the rationale for examining gene expression using the NanoString technology of 118 genes rather than a more genome-wide approach such as RNA-sequencing?<br /> (2) Without additional species examined, some of the conclusions about differences in impact between ascomycetes and basidiomycetes might instead reflect differences between species. For example, RAM mutants in other strains of C. neoformans do not exhibit so strong a temperature sensitive phenotype. Or to extend the comparison further, one might assume given the use of CO2 for Drosophila manipulations that the RAM pathway components in an insect would not be required for surviving high CO2.<br /> (3) Given the relative ease of generate progeny of this species, it would have been informative to explore if the suppressors of cbk1 also suppressed the loss of genes like CDC24, CNA1, etc, equivalent to the experiment performed of overexpression of CBK1 in those backgrounds.

Reviewer #1 (Public Review):

This paper makes an important contribution to the current debate on whether the diversity of a microbial community has a positive or negative effect on its own diversity at a later time point. In my view, the main contribution is linking the diversity-begets-diversity patterns, already observed by the same authors and others, to genomic signatures of gene loss that would be expected from the Black Queen Hypothesis, establishing an eco-evolutionary link. In addition, they test this hypothesis at a more fine-grained scale (strain-level variation and SNP) and do so in human microbiome data, which adds relevance from the biomedical standpoint. The paper is a well-written and rigorous analysis using state-of-the-art methods, and the results suggest multiple new experiments and testable hypotheses (see below), which is a very valuable contribution.

That being said, I do have some concerns that I believe should be addressed. First of all, I am wondering whether gene loss could also occur because of environmental selection that is independent of other organisms or the diversity of the community. An alternative hypothesis to the Black Queen is that there might have been a migration of new species from outside and then loss of genes could have occurred because of the nature of the abiotic environment in the new host, without relationship to the community diversity. Telling the difference between these two hypotheses is hard and would require extensive additional experiments, which I don't think is necessary. But I do think the authors should acknowledge and discuss this alternative possibility and adjust the wording of their claims accordingly.

Another issue is that gene loss is happening in some of the most abundant species in the gut. Under Black Queen though, we would expect these species to be most likely "donors" in cross-feeding interactions. Authors should also discuss the implications, limitations, and possible alternative hypotheses of this result, which I think also stimulates future work and experiments.

Regarding Figure 5B, there is a couple of questions I believe the authors should clarify. First, How is it possible that many species have close to 0 pathways? Second, besides the overall negative correlation, the data shows some very conspicuous regularities, e.g. many different "lines" of points with identical linear negative slope but different intercept. My guess is that this is due to some constraints in the pathway detection methods, but I struggle to understand it. I think the authors should discuss these patterns more in detail.

Finally, I also have some conceptual concerns regarding the genomic analysis. Namely, genes can be used for biosynthesis of e.g. building blocks, but also for consumption of nutrients. Under the Black Queen Hypothesis, we would expect the adaptive loss of biosynthetic genes, as those nutrients become provided by the community. However, for catabolic genes or pathways, I would expect the opposite pattern, i.e. the gain of catabolic genes that would allow taking advantage of a more rich environment resulting from a more diverse community (or at least, the absence of pathway loss). These two opposing forces for catabolic and biosynthetic genes/pathways might obscure the trends if all genes are pooled together for the analysis. I believe this can be easily checked with the data the authors already have, and could allow the authors to discuss more in detail the functional implications of the trends they see and possibly even make a stronger case for their claims.

Reviewer #1 (Public Review):

This is an interesting paper that presents a novel idea for the identification of risk factors amongst highly correlated traits in a Mendelian randomization paradigm - a previous investigation (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8438050/) has considered PCA, but not sparse PCA. There are clear conceptual reasons why sparse PCA may be an improvement, as detailed in this paper. Overall, the paper does a good job in terms of motivating this work and comparing the methods. A large chunk of the motivation for the method is conceptual (rather than empirical), and it's unlikely that any method would outperform others in all circumstances, but the authors do a good job of illustrating differences and giving a clear and qualified recommendation.

Reviewer #1 (Public Review):

There were two parts to this paper. The first was to build a network model with parameters carefully adjusted to match those seen in the turtle cortex. The second was to simulate the circuit, and show that it could produce reasonably repeatable patterns of activity in response to a single, externally added, spike.

As a model of the turtle cortex, the paper was pretty convincing. And the explanation for the repeatable patterns of activity - a small number of very strong connections and a very low background firing rate - seemed eminently reasonable. This paper should serve as a very good starting point for understanding computing in the turtle cortex.

However, average firing rates in the turtle are extremely low - 0.1 Hz, at least in these simulations. Their model is unlikely, therefore, to account for activity in the mammalian cortex, which exhibits a much higher background firing rate, and for which there's not a lot of evidence for the extremely strong connections seen in the turtle.

Reviewer #1 (Public Review):

This paper describes detailed experiments to characterize the morphology and deformability, of red blood cells (RBCs) from COVID patients as compared to healthy individuals. Deformability is characterized by the visualization of cell shapes during flow in a microfluidic channel at high strain rates. One important feature of the study is that it considers the changes in patient RBCs when placed in healthy plasma and vice versa. An important observation is that the changes to RBCs properties appear, from this report, to be reversible - diseased cells revert to normal morphology and deformability upon immersion in healthy plasma. It also reports metabolics and proteomics analyses to shed light on the connections between the biochemical environment and RBC properties. One important question with regard to the changes in COVID-RBC properties with respect to plasma composition is whether the effect is simply due to dilution - are the factors responsible for the pathological morphology just diluted away when the cells are immersed in plasma that does not contain them? The studies are performed at very low hematocrit, so the composition equilibrium established here will not correspond to physiological conditions. This issue needs further discussion.

Reviewer #1 (Public Review):

This paper estimates the selective effects of loss-of-function mutations in each gene, ultimately providing an estimate of the overall distribution of fitness effects, and point estimates for each gene. Unlike some measures of intolerance such as pLI, the parameter the authors estimate (effectively the compound parameter hs) is interpretable in terms of evolutionary fitness. The most comparable analysis is by Weghorn et al (2019) which estimates the same parameter, but on a smaller sample and using a different approach.

The point estimates will be broadly useful for future analyses, and the overall distribution is an interesting result. The enrichment in various disease cohorts is unexpected but nice to demonstrate. Overall, I found the approach to be elegant and it has the nice property that it can be easily generalized to more complicated models. The data cleaning and filtering is quite extensive but all seems well done and appropriate. Qualitatively, the results clearly make a lot of sense (Figure 3 is an excellent figure) My only major questions are around how quantitatively robust this analysis is to the choice of parameters and hyperparameters including priors, mutation rates, and demography. I don't think that extensive work is required, but it would be helpful to see some quantification of this uncertainty.

Reviewer #1 (Public Review):

The authors note contradictory clinical data on the effects of functional FAAH mutations on body weight in clinical samples. They aim to resolve this issue via animal modes and genetic approaches combined with endocrine manipulations to vary "context". Major strengths are comprehensive evaluation of FAAH variant in several models of neuroendocrine changes in body weight (CORT, leptin, gherlin) and provide some mechanistic insight at the signal transduction level. Localization of FAAH modulation to AGRP neurons is a strength. Weaknesses include lack of cellular mechanisms, i.e. how AEA release from AGRP neurons affects ongoing cellular/synaptic activity to regulate behavioral/physiological phenotypes. The work is impactful as it potentially reconciles contradictory clinical data, is comprehensive and rigorous in many ways. These data will provide insight into how FAAH activity regulates body weight in the context of distinct hormonal signals and will likely have a major impact on the field.

Reviewer #1 (Public Review):

When we tilt our heads, we do not perceive objects to be tilted or rotated. In this study, the authors investigate the underlying neural underpinnings by characterizing how neurons in monkey IT respond to objects when the entire body is tilted. They performed two experiments. In the first experiment, the authors record single neuron responses to objects rotating in the image plane, under two conditions - when the animals were tilted +20{degree sign} or -20{degree sign} relative to the gravitational vertical. Their main finding is that neural tuning curves for object orientation were highly correlated under these conditions. This high correlation is interpreted by the authors as indicative of encoding of object orientations relative to an absolute gravitational reference frame. To control for the possibility that the whole-body tilt could have induced compensatory torsional rotations of the eyes, the authors estimated the eye torsional rotation between the {plus minus}20{degree sign} whole-body tilt to be only {plus minus}6{degree sign}. In the second experiment, the authors recorded neural responses to objects rotated in the image plane with no whole-body tilt but with a visual horizon that could be tilted by the same {plus minus}20{degree sign} relative to the gravitational vertical. Here too they find many neurons whose tuning curves were correlated between the two horizon tilt conditions. Based on these results, the authors argue that IT neurons represent objects relative to the gravitational or absolute vertical.

The question of whether the visual system encodes objects relative to the gravitational vertical is an interesting and basic one, and I commend the authors for attempting this question through systematic testing of object selectivity under conditions of whole-body tilt. However, I found this manuscript extremely difficult to read, with important analyses and controls described in a very cursory fashion. I also have several major concerns about these results.

First, the high tuning correlation in the {plus minus}20{degree sign} whole-body tilt conditions could also occur if IT neurons encoded object orientation relative to other fixed contextual cues in the surrounding, such as the frame of the computer monitor. The authors ideally should have some experiment or analysis to address this potential confound, or else acknowledge that their findings can also be interpreted as the encoding of object orientation relative to contextual cues, which would dilute their overall conclusions.

Second, I do not fully understand torsional eye movements myself, but it is not clear to me whether this is a fixed or dynamic compensation. For instance, have the authors measured torsional eye rotations on every trial? Is it fixed always at {plus minus}6{degree sign} or does it change from trial to trial? If it changes, then could the high tuning correlation between the whole-body rotations be simply driven by trials in which the eyes compensated more? The authors must provide more data or analyses to address this important control.

Third, I find that when the objects were presented against a visual horizon, different object features are occluded at each orientation. This could reduce the correlation between the neural response in the retinal reference frame, thereby biasing all results away from purely retinal encoding. The authors should address this either through additional analyses or acknowledge this issue appropriately throughout.

Reviewer #1 (Public Review):

This is a brief set of experiments that tells a nice story that is relevant to a very important area of biology, namely senescence. The authors identify a role for lncRNA H19 in senescence and delve into the upstream and downstream factors that could describe the phenomenon. They identify CTCF and p53 as upstream regulators of H19 in senescing cells and propose that sponging of let-7 could be a contributing factor to H19's effects via altered regulation of EZH2.

The work is backed up by strong data in cell models. However, the work could benefit from additional mechanistic data to support the most important conclusions. For example, does H19 sponge let-7 in these cells? What are the relative levels of expression of H19 compared to let-7 in these cells? Is the let-7 binding site on H19 required for the effects of H19? And does let-7 directly regulate EZH2 in these cells? Can a direct role for H19 in affecting EZH2 be ruled out in these cells?

Reviewer #1 (Public Review):

In the work by Van Eyndhoven et al., the authors aim to determine if the cell state present in the cells that first produce Type I Interferon (IFN-I, an antiviral cytokine) is stochastically regulated or may be epigenetically inheritable. This work builds from previous studies demonstrating that IFN-I responses occur in two waves: a small proportion of early responding "precocious" cells which induce population-wide responses through autocrine and paracrine signaling. The authors contextualize their study well within the literature, and discuss the hypotheses of stochasticity or determinism driving early responding cell fate. Within this context, the authors set out to characterize and model the nature of these "first responder" cells during IFN-I antiviral signaling. Developing a quantitative imaging approach to measure IRF7 translocation, the authors measure the proportion of first responder cells as defined by higher ratios of nuclear/cytosolic IRF7 expression. Transfection of Poly(I:C) induces IFN-I signaling and leads to ~2% first responders, in line with previously published work. The authors then show that responder frequencies increase following treatment with a DNA methyltransferase inhibitor, suggesting a relationship between epigenetic regulation and responder potential. To test the hypothesis that the first responder cell state occurs stochastically, the authors adapted the Luria-Delbruck fluctuation test by evaluating responder frequency as a function of cell division or generation. First witnessing high variability of responder frequencies using limiting dilution clonal expansion followed by low stable frequencies after 100 divisions (similar to regular cultures), the authors suggest that the first responder state may be partially heritable and develop a mathematical model of transient heritability. Finally, to assess whether cell density and quorum sensing contribute to this transient heritability, cells plated at different densities were interrogated for responder frequencies after a fixed number of divisions; only low density seeding led to high and variable responder frequencies.

The interrogation of IFN-I early responding cells by Van Eyndhoven et al. is well executed and supports the claim that first responder events are non-stochastic. However, the use of transgenic reporter cells in vitro may limit the findings reported in the manuscript to this system, and awaits further experimentation to assess the generalizability of these findings to overall cellular decision-making during inflammatory responses. Identifying the mechanisms responsible for transient heritability and the density-dependent regulation will be of high interest.

1) Context and definitions for stochasticity and heritability: The authors provide well-referenced introductions and explanations throughout the manuscript. However, key understanding of concepts for their central hypothesis on transient heritability are not shared until well into the results sections (Lines 215-227), leaving the introduction somewhat unclear on the authors thinking and motivation. The manuscript would benefit by including clear definitions of "stochastic", "transiently heritable", and "heritable" and their relationships to "intrinsic" and "deterministic" in the introduction.

2) Generalizability of findings to other cell types, systems, and triggers: The cell line and Poly(I:C) delivery method used by the authors lacks sufficient characterization to extend the conclusions derived from its use. Notably, the NIH3T3-IRF7-CFP cell line expresses IRF7 constitutively and thus may only be a good model for cells with similar expression levels; many primary cells only express IRF7 at low levels or not at all until stimulated (PMID: 2140621). The conclusions would be greatly strengthened by demonstrating similar first responder dynamics/heritability in other cell types. The experiments measuring the efficiency of Poly(I:C) delivery by transfection lack sufficient resolution to determine if the Poly(I:C) is intracellular or membrane bound. IFN-I response kinetics, and potentially quality, would likely be distinct between cytosolic and endosomal sensing and may impact the likelihood of becoming a first responder.

3) Epigenetic regulation of transient heritability: To test the contribution of epigenetic regulation on first responder fate, the authors treat their cells with DNMTi. While treatment with this drug does increase the proportion of first responder cells, the authors don't provide evidence that the mechanism of action is mediated by inhibiting DNA methylation. This is further confounded by the reduced responder frequencies in DNMTi treated cells transduced with Poly(I:C) (Fig 4g). The authors offer an explanation for this observation, but their reported data (Fig 4h) doesn't measure whether DNMTi, leads to latent retrovirus activation, broader demethylation, or a combination of the two.

4) Temporal experimental data to validate and extend transient heritability and quorum sensing: Developing a model for cellular-decision making during early IFN-I responses, the authors formalize and test the hypothesis of transient heritability. While the data largely fit the model proposed (Fig 6D-F), the reported data points lack sufficient temporal resolution to validate the model during the earlier and more variable generations. Given that by generation 9 variability in first responder frequency has almost stabilized, there is only one data point (generation 6) to evaluate the fit of the ODE described. More densely sampled data points below generation 10 are necessary to validate the model. Moreover, a discussion of Kon calculation/observation, meaning, and validation is missing. To partially test their claim that Kon is a function of density (i.e., quorum sensing), the authors plate cells at different densities and measure the responder frequency at generation 6. This analysis lacks contextualization of other autocrine and paracrine signals potentially impacting IFN-I response. Moreover, these signals will be diverse in different cell types and could impact Kon and/or the overall model.

Reviewer #1 (Public Review):

The study by Lehmann et al. reports novel structures of the human ferroportin (SLC40A1), which is responsible for iron transport in the body. Specifically, ferroportin controls the plasma concentration of iron by transporting Fe2+ out of the cell. To regulate plasma iron concentrations, the liver releases hepcidin, a peptide-based hormone that inhibits ferroportin activity. Specific inhibitors of ferroportin are being developed to treat thalassemia and sickle cell disease, which are diseases that result in reduced red blood cell function.

The present study reports the structure of human ferroportin in complex with one such inhibitor, vamifeport, which is currently in clinical trials for sickle cell disease. The authors use their structures to suggest a mechanism for vamifeport binding to ferroportin and support the structural data with in vitro binding assays to study the specific interactions made in the binding site. In addition, one of the structures obtained was a novel protein conformation, an occluded state. This is the first occluded state observed for ferroportin, enabling the authors to discuss the implications for understanding the transport mechanism. However, this appears to have resulted in a slightly confusing analysis.

Overall the study is well presented, although in several places appears overly wordy and might benefit from being edited to focus on the main points the authors wish to highlight. For example, the title focuses on the new insights gained from the vamifeport complex. Yet, the discussion section focuses almost entirely on the transport mechanism, with little additional analysis of the mechanism of vamifeport inhibition. In my view, the paper suffers from this disconnect, as the functional data support the vamifeport structure, not the transport mechanism. Yet, the discussion focuses heavily on the transport mechanism, with little reference to the results. Rather, the discussion relies on an in-depth understanding of secondary active transport literature (MFS, NRAMP, etc.).

The data is high quality, and the conclusions drawn about the orientation of the drug in the binding site are sound. This study represents an important advance in understanding iron homeostasis in the human body and current methods to modulate iron transport to treat human disease.

Reviewer #1 (Public Review):

In this manuscript, Vandrey et al characterize axonal projections from fan cells in the lateral entorhinal cortex (LEC) to the medial entorhinal cortex (MEC). Their findings are important and the manuscript is well-written.

Reviewer #1 (Public Review):

Andreyeva et al. developed a novel purification/mass spec approach to identify SuUR-associated proteins. From this biochemical tour de force, they identify a complex consisting of the insulator-associated protein Mod(Mdg4) and SuUR that they term, SUMM4. They show that this complex (at least SuUR) has ATPase activity, which is an exciting result was no known biochemical activity associated with SuUR. Given SuUR's function in the under-replication of Drosophila salivary glands, the authors show that SuUR and Mod(Mdg4) at least partially localize on polytene chromosomes and that SuUR displays at least a partial dependence on Mod(Mdg4) for localization to IH, but not PH regions. Finally, using two independent genetic reporters, they show that SuUR itself has an insulator function, which is a new function for SuUR and exciting as it is likely a diploid cell-specific function for SuUR. The authors then attempt to show the Mod(Mdg4) functions in under-replication. Unfortunately, under-replication is minimally, if at all, changed in the Mod(Mdg4) mutant. While the authors bring up several possible scenarios of why this could be, it is still uncertain whether Mod(Mdg4) has a direct effect on under-replication.

Strengths:<br /> The authors developed a very useful strategy to identify protein interactions through multiple purification steps using mass spectrometry. This approach can be applied to different systems and will be generally useful to the community. Through this approach, they provide very compelling data that SuUR and Mod(Mdg4) form a complex. Furthermore, the experiments all have been rigorously performed and the data is of high quality.

Weaknesses:<br /> The way the paper is written, its main focus is on under-replication. What the authors were not able to conclusively demonstrate is whether Mod(Mdg4) functions in under-replication.

Reviewer #1 (Public Review):

This is a relatively straightforward manuscript describing an r package that attempts to address issues in color-blindness in the interpretation of multicolor overlapping plots. The demonstration of its usefulness is solid and the findings will be significant in that they should become one of the standards that the scientific community strives to achieve for greater inclusiveness.

Reviewer #1 (Public Review):

The layered costs and benefits of translational redundancy by Raval et al. aim to investigate the impact of gene copy number redundancy on E. coli fitness, using growth rate in different media as the primary fitness readout. Genes for most tRNAs and the three ribosomal RNAs are present in multiple copies on the E. coli chromosome. The authors ask how alterations in the gene copy number affect the growth rate of E. coli in growth media that support different rates of growth for the wild type.

While it was shown before that mutants with reduced numbers of ribosomal RNA operons grow at reduced rates in rich medium (LB), this study extends these findings and reaches some important conclusions:

1) In a poor medium (supporting slow growth rates), the mutants with fewer rRNA operons actually grow faster than the wild type, showing that redundancy comes at a cost.

2) The same is true for mutants with reduced gene copy number of certain tRNAs and correlates with slower rates of protein synthesis in these mutants.

3) That rRNA operon gene copy number is more decisive for growth rate than any tRNA gene copy number (>1).

In addition, measurements of strains with deletions of genes encoding tRNA-modification enzymes that affect tRNA specificity are included. While interesting, no unifying conclusion could be reached on the impact of these mutations on growth rate.

The well-known "growth law" relationships between growth rate and macromolecular composition (RNA/protein ratio, for example) specifically concern steady-state growth rates. It is concerning that all growth rates in this work were measured on cultures that were only back-diluted 1:100 from overnight LB precultures. That only allows 6-7 doubling times before the preculture OD is reached again. The exponential part of growth would end before that, allowing perhaps only 3-4 generations of growth in the new medium before the growth rate was measured. Thus, the cultures were not in balanced growth ("steady state") when the measurements were made, rather they were presumably in various states of adapting to altered nutrient availability.

A second concern is the use of the term "tRNA expression levels" in the text in Figure 4. I believe the YAMAT-seq method reports on the fractional contribution of a given tRNA to the total tRNA pool. Thus, since the total tRNA pool is larger in fast-growing cells than in slow-growing cells, a given tRNA may be present at a higher absolute concentration in the fast than in the slow-growing cells but will be reported as "higher in poor" in figure 4, if the given tRNA constitutes a smaller fraction of the total tRNA pool in rich than in poor medium. For this reason, the conclusions regarding the effect of growth medium quality on tRNA levels are not justified.

Reviewer #1 (Public Review):

In this paper from Geisberg et al., the authors examined cleavage site usage in yeast and human cells that express Pol II mutants with faster or slower elongation rates. The authors focused on two types of alternative cleavage sites, one being multiple sites clustered within a short range (called within cluster sites) and the other being multiple sites that are distant from one another (called between cluster sites). The authors identified polarity site usage of within a cluster in cells expressing mutant Pol II. Slower Pol II leads to more proximal site usage whereas faster Pol II mutant to distal site usage. In contrast, these trends were not observed with sites between clusters. The authors made four conclusions based on these observations. Overall this is a very well-written paper revealing some fundamental features associated with cleavage site choice. Most conclusions are supported by their data. I do, however, have some concerns about their between-cluster analysis.

Reviewer #1 (Public Review):

Gao et al developed various genetic permutations of mouse models of kindlin-2 deficiency in the hepatocytes to explore its function. Hepatocyte-specific loss of kindlin-2 results in severe inflammatory liver injury, accelerated fibrosis/portal hypertension, and massive hepatocyte cell death by apoptosis. These effects are reversed by ablation of TNF signally or by caspase 8 deletion. AAV-mediated replacement of kindlin-2 protects the mice from chemically induced acute liver injury.

Reviewer #1 (Public Review):

Ruesseler and colleagues combine careful paradigm design, psychophysical and EEG analyses to determine whether information leakage during decision formation is strategically adjusted to meet changing task demands. Participants made motion direction judgments that required monitoring a continuous stream of dot motion for 'response periods' characterised by a sustained period of coherent motion in a leftward or rightward direction. Coherence was modulated on a frame-to-frame basis throughout the task furnishing a parametric regressor that could be used to interrogate the longevity of sensory samples in the decision process and their influence on corresponding EEG signals. Participants completed the task under varying conditions of response period length and frequency. Psychophysical kernel analyses suggest that sensory samples had a more short-lived impact on the participants' choices when response periods were rare, suggestive of greater information leakage. When the stimulus perturbations were regressed against the EEG data, it highlighted a centro-parietal component that showed increased responsiveness to large shifts in evidence when those shifts were more rare, suggestive of a role in representing surprise. An additional triphasic component was found to correlate with the time constant of integration as estimated from the kernel analyses.

This is a very timely paper that addresses an important and difficult-to-address question in the decision-making field - the degree to which information leakage can be strategically adapted to optimise decisions in a task-dependent fashion. The authors apply a sophisticated suite of analyses that are appropriate and yield a range of very interesting observations. The paper centres on analyses of one possible model that hinges on certain assumptions about the nature of the decision process for this task which raises questions about whether leak adjustments are the only possible explanation for the current data. I think the conclusions would be greatly strengthened if they were supported by the application and/or simulation of alternative model structures.

The behavioural trends when comparing blocks with frequent versus rare response periods seem difficult to tally with a change in the leak. The greater leak should result in a reduction in the rate of false alarms yet no significant differences were observed between these two conditions. Meanwhile, false alarms did vary as a function of short/long target durations which did not show any leak effect in the psychophysical kernel analyses. Are there other models that could reproduce such effects? For example, could a model in which the drift rate varies between Rare and Frequent trials do a similar or better job of explaining the data? This ties in to a related query about the nature of the task employed by the authors. Due to the very significant volatility of the stimulus, it seems likely that the participants are not solely making judgments about the presence/absence of coherent motion but also making judgments about its duration (because strong coherent motion frequently occurs in the inter-target intervals). If that is so, then could the Rare condition equate to less evidence because there is an increased probability that an extended period of coherent motion could be an outlier generated from the noise distribution? Note that a drift rate reduction would also be expected to result in fewer hits and slower reaction times, as observed.

Some adjustment of the language used when discussing FAs seems merited. If I have understood correctly, the sensory samples encountered by the participants during the inter-response intervals can at times favour a particular alternative just as strongly (or more strongly) than that encountered during the response interval itself. In that sense, the responses are not necessarily real false alarms because the physical evidence itself does not distinguish the target from the non-target. I don't think this invalidates the authors' approach but I think it should be acknowledged and considered in light of the comment above regarding the nature of the decision process employed on this task.

The authors report that preparatory motor activity over central electrodes reached a larger decision threshold for RARE vs. FREQUENT response periods. It is not clear what identifies this signal as reflecting motor preparation. Did the authors consider using other effector-selective EEG signatures of motor preparation such as beta-band activity which has been used elsewhere to make inferences about decision bounds? Assuming that this central ERP signal does reflect the decision bounds, the observation that it has a larger amplitude at the response on Rare trials appears to directly contradict the kernel analyses which suggest no difference in the cumulative evidence required to trigger commitment.

P11, the "absolute sensory evidence" regressor elicited a triphasic potential over centroparietal electrodes. The first two phases of this component look to have an occipital focus. The third phase has a more centroparietal focus but appears markedly more posterior than the change in evidence component. This raises the question of whether it is safe to assume that they reflect the same process.

Reviewer #1 (Public Review):

The authors provide a comprehensive series of experiments to show that IF promotes rapid hepatocyte proliferation driven by the dual action of systemic FGF15 (intetinally-derived) and localized WNT signaling. Hepatocyte proliferation during periods of IF maintains a steady liver-to-body-mass ratio. This study provides the first example of the dietary influence on adult hepatocyte proliferation and is highly relevant to the putative beneficial effects of IF in multiple chronic diseases. Additionally, it challenges the view that liver tissue is quiescent except in patholgical injury.

Reviewer #1 (Public Review):

This study elucidates a role of EHD2 as a tumor/metastasis promoting protein. Prior work has found varying results indicating that high expression of EHD2 is either associated with good or poor outcomes. In this work the authors find that EHD2 is expressed in both the nucleus and cytoplasm, and that high cytoplasmic to nuclear expression is associated with a poor prognosis. Using WT and either shRNA knockdown or CRISPR KO cells, they show that EHD2 promotes 3D growth, migration and invasion in vitro, and tumor growth and metastasis in vivo. Importantly, re-expression of EHD2 in KO cells rescues the loss of function phenotype. Mechanistically, the investigators show that the loss of EHD2 decreases the calveoli and that this decreases the Orai1/Stim induced calcium influx. Finally, they show that inhibitors of store operated calcium entry (SOCE) phenocopies the loss of EHD2. Together the data support a protumorigenic role for EHD2 via store-operated calcium entry and reinforce the utility of targeting calveoli and SOCE in tumors with high cytosolic EHD2. This study provides a rationale for using SOCE inhibitors in a subset of breast cancers, and a potential predictive biomarker for using SOCE inhibitors based on high expression of EHD2.

Reviewer #1 (Public Review):

The authors convincingly show directionally tuned signals in AD and RSC. RSC is found to have a lower proportion of HD cells than AD, and RSC HD cells are more sensitive to angular velocity than AD HD cells. Importantly, HD responses are shown to be tightly correlated between the two areas. Population decoding of head direction, performed on AD neuron ensembles or RSC ensembles, revealed similar shifts following visual cue rotation and also similar HD drift in darkness, indicating that the HD representation across both areas is coordinated. The study further finds that AD-to-RSC connections are relatively frequent, while RSC-to-AD connectivity is very sparse. This asymmetry in functional connectivity is matched by viral tracing results. Together, the results lead the authors to the conclusion that this corticothalamic connection is likely not driving visual landmark updating of the global head direction system.

This is a welcome piece of work, providing the first assessment of the high degree of coherence between AD and RSC HD representations, using pairwise and population-level analysis methods, which had not been accessible before. It will be a valuable reference for researchers interested in inter-area interactions in the head direction system, leaving the question of how and where visual reference updates are fed into the HD circuit open for further investigations.

Reviewer #1 (Public Review):

This manuscript by the Karakas lab reports on new structures of the volume regulated LRRC8 anion channels. These ubiquitously expressed channels play key roles in cell volume regulation and in allowing efflux of organic osmolytes, neurotransmitters, and drugs. In addition to regulating cell volume LRRC8 channels might play roles in signal transduction, cell migration, apoptosis, tumor drug resistance, and stroke. Thus, elucidating their architecture and structure is of critical importance. LRRC8 channels are obligate multimers of variable stoichiometry, with the LRRC8A subunit being absolutely required for assembly of functional channels. Structures of homomeric LRRC8A and LRRC8D channels revealed a hexameric assembly with closed pores. However, the functional properties of these homomeric channels differ from those of recorded in cells, raising questions on the physiological relevance of these conformations. The authors here determine the structure of a LRRC8C-LRRC8A chimera (termed 8C-8A(IL125)) with functional properties that closely resemble those of native channels. Remarkably, the 8C-8A(IL125) chimera assembles as a heptamer with a large pore. Unexpectedly, in the structures the channel's pore is occupied by density that could correspond to lipids.

Reviewer #1 (Public Review):

In 2020, Sugisawa et al. reported that Piezo1ion channels can be activated by ssRNAs, both synthetic and derived from fecal matter, suggesting that these may be the first identified natural ligands to agonize Piezo channels. Nickolls et al., provide a careful and rigorous investigation of the effect of ssRNAs and fecal extracts on Piezo channel activity in three cell lines, using both calcium imaging and electrophysiology. They find that Piezo1 is not responsive to ssRNAs nor responsible for calcium flux in response to fecal extracts in HEK293 and RIN14b cells. Overall, this study addresses the question of ssRNAs as a Piezo ligand clearly and thoroughly, with rigorous, well-controlled experiments. Overall, I am excited about this study as a necessary clarification for the field of Piezo mechanosensation.

Reviewer #1 (Public Review):

This work identifies distinct contribution of direct (D1+) and indirect (Adora+, D2+) amygdalostriatal medium spiny cells in fear learning and plasticity. The authors combined freely moving calcium imaging with auditory fear learning assay to reveal tone, foot-shock and behavior (movement)-evoked activity of the two MSN population. While D1+ cells show plastic changes driven by fear learning and reaching their maximum tone responsiveness (PSTH) at fear retrieval, Adore+ cells activation remained constant. Furthermore, using optogenetic silencing they showed that the two MSN groups differently contribute to retrieval of fear memory. Both cells receive topographically organized insular cortical inputs which go through learning-induced long-term synaptic changes with opposite direction: postsynaptic LTP at D1 cells, while presynaptic LTD at Adora+ cells. These synaptic changes provide some level of explanation for distinct behavioral contribution of the two cell types in fear learning.

This study focuses on a so far neglected member of the 'extended' amygdalar circuitry, the amygdalostratal transition zone. The data is well-presented, the experiments are in logical order, built on each other and the paper is easy to read and follow.

However, some information regarding the connectivity (and function) of Astr have been presented in recent and earlier papers are missing from, or contradicting with, the present work. One reason to explain these is that the targeted striatal regions vary between experiments, and so, it is difficult to judge when the Astr and when the other part of the caudal (tail) striatum is examined. As these striatal regions are involved in different neuronal networks, their functional consequences could also be distinct. Without precisely clarifying and consistently targeting the aimed striatal region, it is difficult to interpret the findings of the present study (though those are relevant and important).

Main claim

Reviewer #1 (Public Review):

In this manuscript, Zhang and colleagues created a transgenic mouse strain that expresses SYT-1-tdt in all neurons. They showed that the labelled SYT-1 colocalizes with multiple synaptic markers and label synapses in different regions. More importantly, they showed that the transgenic expression does not alter synaptic function using ephys assays. This is a straightforward paper that generated a useful reagent that will be used broadly.

Reviewer #1 (Public Review):

This is an interesting and timely paper investigating the impact on participation in cancer screening programs across Italy during the COVID-19 pandemic where there was massive disruptions to health services. What is of particular interest in this analysis was the investigation of social, educational and cultural factors that might have impacted access and participation to screening.

- In the present study, the authors analyzed data collected by PASSI between 2017 and 2021, from interviews of more than 106,000 people, a representative sample of the Italian population aged 25-69 was selected but its not clear what was the representativeness by region, gender and age educational attainment? Also what is the total population (so I don't have to look it up). I am wondering if participation differed by characteristics and what approach to achieving the representative sample was made (e.g. replacement of individuals or oversampling certain strata where participation was lower).

- For figures 5-8 what is the N for the different groups not just the %?

- Table 2 to me is a key piece of information and very interesting can the authors formally test if there are signficant differences between the time periods?

Reviewer #1 (Public Review):

The authors introduce an online tool, CausalCell, to explore causal links in single-cell datasets. The authors investigate the process through examples based on existing data, offer comparisons of different algorithms, and suggest tips about the requirements and limitations of this approach. In my opinion, the main shortcoming is that the authors do not adequately justify whether the methods included in their tool are the most suitable methods for their intended analyses. The lack of a definite "ground truth" or "gold standard" also comes in the way clearly deciding which algorithms perform the best, especially when there are considerable differences between the results of different algorithms.

Reviewer #1 (Public Review):

The authors conducted a thorough analysis of the correlation between height and measures of cognitive abilities (what are essentially IQ test components) across four cohorts of children and adolescents in the UK measured between 1957 and 2018. The authors find the strength of the association between height and cognitive measures declined over this time frame--for example, among 10- and 11-year-olds born in 1958, height explained roughly 3% of the variation in verbal reasoning scores; this dropped to approximately 0.6% among those born in 2001. These associations were further attenuated after accounting for proxy measures of social class.

The authors' analyses were performed carefully and their observations regarding declining height / cognitive measure associations are likely to be robust if we interpret their results with an important caveat: these results reflect measurements aimed at assessing cognition rather than cognition itself. The importance of this distinction is evidenced by the changing correlation structure of the cognitive measures over time. For example, age 11 verbal / math scores were correlated at >= 0.75 at the first two time points but dropped to 0.33 at the most recent time point. Similar patterns are present for the other cognitive measures and time points. The authors' conclude that such changes are unlikely to impact their primary findings, but I'm less certain. For example, one interpretation of this finding is that older cognitive measures were simply worse at indexing distinct cognitive domains and instead reflected a combination of cognitive ability together with non-specific factors relating to opportunity, health, class, etc. Further, height was historically a stronger proxy for class and economic status than it is today (e.g., by capturing adequate nutritional intake, risk for childhood disease, etc.). Together, then, previously high height / cognitive measure correlations might reflect the fact that both phenotypes previously indexed socio-economic factors to a greater extent than they might today (which is still non-negligible).

Additionally, their findings add an interesting data point to a collection of recent results suggesting that the relationship between cognitive and anthropometric measures is complex and difficult to interpret. For example, studies using genetic markers to examine shared genetic bases have virtually all relied on methods assuming mating is random, which is not the case empirically. Howe et al. (doi.org/10.1038/s41588-022-01062-7) recently reported that the ostensible genetic correlation of -.32 between years of education and BMI attenuates to -.05 when using direct-effect estimates, which should theoretically be immune to the effects of non-random mating and other confounding variables. Likewise, Keller et al. (doi.org/10.1371/journal.pgen.1003451) and Border et al. (doi.org/10.1101/2022.03.21.485215) used very different approaches to arrive at the same conclusion that ~50% of the nominal genetic correlation between IQ and height could be attributed to bivariate assortative mating rather than shared causal biological factors. Given that assortative mating on both IQ measures and height involves many other traits (not just two as assumed in such bivariate models), the true extent to which height / IQ correlations reflect causal factors is plausibly even lower than these estimates suggest. For these reasons, I do not entirely agree with the authors' review of previous findings in the introduction, where they write "recent studies have suggested that links between higher cognition and taller height can be largely explained by genetic factors", though it is certainly true that this claim has been made.

Reviewer #1 (Public Review):

Li et al. have designed a study that examines specific mechanisms for how different DNA sequence variants in the common cancer gene p53 (also known as TP53) influence the sensitivity of tumors to a variety of common cancer treatments. Specifically, they examine a handful of p53 variants with respect to glioblastoma and its response to platinum-based chemotherapy and to radiation therapy. The authors begin by mentioning that looking at DNA variants in cancer is useful but also incomplete: methylation, PTMs, and non-DNA sequence variants can also be critical. They then mention that they have created a model showing that nearly all cancers with p53 mutations have loss-of-function variants and that many cancers with "normal" wildtype p53 in fact have variants causing LOF. These p53 LOF tumors lead to worse patient outcomes, but the authors here show that these tumors appear to be more susceptible to radiation and platinum-based chemotherapy, which they say they have validated in glioblastoma xenografts. This potentially opens up a new avenue for precision medicine for many different sources of cancer that share common p53 LOF variants.

The authors have taken a modern approach towards cancer diagnosis and shown how this can improve targeted treatments across a large array of cancer types. They have provided a reasonably convincing proof of concept of this approach for n = 35 PDXs in one cancer type. By and large, the approach and results are reasonable, although many of the exact results concerning the genes and pathways identified that covary with the various treatments and p53 variants are unclear. For instance, the feature selection seems to be somewhat ad hoc, e.g. the method used to determine p53 LOF from p53 WT in the TCGA data was not the same method used for determining p53 LOF from p53 WT in the PDX data. The TCGA AUROCs were incredibly good - over 99% - versus more like 75% for the actual proof of concept. While any significant p-value is fine for basic research, it would be nice to know how this could be improved and bring the results in Figure 4 from ~75% to the >99% that would be necessary for use as a medical diagnostic or for treatment selection for precision medicine. However, there are significant questions regarding the specific findings uncovered: do the gene pathways identified through bioinformatic analysis fit in with the many highly-studied mechanistic roles of p53? Do the cohort selections - which vary by an order of magnitude in sample size, and come from different locations and different tissues - make statistical sense for cross-validation?

Reviewer #1 (Public Review):

NADPH oxidases are a family of membrane enzymes that produce reactive oxygen species (ROS). NOX2 is the most well-studied member of the NADPH oxidase family, and the proper function of NOX2 is critical for innate immunity against pathogens in mammals.

The study by Dr. Chen and colleagues used antibodies to facilitate the structural determination of the high-resolution structure of the NOX2-p22 complex, which is otherwise challenging for single-particle analysis due to its flexibility and relatively small molecular weight. The work uncovered the high-resolution information between NOX2-p22 interaction and conformational flexibility between the DH domain and the transmembrane domain of NOX2. This structural study provides valuable information for a mechanistic understanding of NOX2 activation at the molecular level.

The weakness of the paper is the lack of in-depth analyses regarding structural discoveries. In addition, a study by Noreng S et al on the structure of the NOX2-p22 complex is now available.

Reviewer #1 (Public Review):

Chondrosarcoma is a rare and aggressive cancer type with a poor prognosis and lacks effective treatment options. Developing an effective strategy for targeting chondrosarcoma is therefore considered an unmet clinical need. The goal of this study is to provide the molecular basis for chondrosarcoma progression and identify a potential strategy/agent for targeting chondrosarcoma. The study reveals that EZH2/hSULF1/c-Met axis is a critical signaling pathway for chondrosarcoma and provides proof of principle evidence that targeting c-MET by pharmacological approaches is an effective strategy to suppress tumor growth in chondrosarcoma mouse models. The aims to be explored for the study are novel and have been well accomplished. The conclusions from this current study are well supported by the compelling and robust datasets using diverse approaches. The study not only reveals a novel insight into how chondrosarcoma progression occurs but also offers the potential strategy for targeting chondrosarcoma, hence significantly advancing the field.

Reviewer #1 (Public Review):

It has been shown that selenium protects against the development of epilepsy, and behavioral comorbidities, as pointed out by the authors. This paper attempts to show it does if administered later after chronic seizures start. While clinically relevant, as noted by the authors, the paper seems not to be a major advance beyond the prior study. The antiseizure effect is also not very convincing because the effect size is so small and the variance so high. The data about behavior is more convincing but similar data were in the previous paper, so it is not very novel.

The data showing changes in PP2A are interesting and while logical that it contributes to the effects of sel, one would like to see proof that this is the basis of sel effects. Same for hyperphosphorylated tau, telomere length, etc. The doubt is because these are indices that change after many types of experiments and they change many aspects of brain and peripheral physiology. Regarding molecular data, how these provide insight and comparison to other data sets of this kind would be valuable.

Reviewer #1 (Public Review):

Estimating the effects of mutations on the thermal stability of proteins is fundamentally important and also has practical importance, e.g, for engineering of stable proteins. Changes can be measured using calorimetric methods and values are reported as differences in free energy (dG) of the mutant compared to wt proteins, i.e., ddG. Values typically range between -1 kcal/mol through +7 kcal/mol. However, measurements are highly demanding. The manuscript introduces a novel deep learning approach to this end, which is similar in accuracy to ROSETTA-based estimates, but much faster, enabling proteome-wide studies. To demonstrate this the authors apply it to over 1000 human proteins.

The main strength here is the novelty of the approach and the high speed of the computation. The main weakness is that the results are not compared to existing machine learning alternatives.

Reviewer #1 (Public Review):

This manuscript uses two OR molecues as a model to understand the mechanism behind their ligand specificity. It combines a series of targeted mutations and domain swapping followed by functional analysis in Xenopus oocyte expression system to analyse functional aspects of the modified ORs. It also models the various OR structures. The authors find that a single amino acid residue is critical for ligand specificity and that this is mediated by space constraints generated in the ligand docking region. The manuscript is generally well written and the data are clear and well represented.

Reviewer #1 (Public Review):

The authors investigate the relative importance of the bee host and bacterial microbiome in processing the nectar secondary metabolite amygdalin, with a focus on understanding the contributions of the different members of the microbiome, and the enzymatic basis for metabolic transformations. The manuscript clearly describes the experimental procedures, presents the results in graphically appealing figures and clear text, and puts the work into a broader context in the discussion. The conclusions are backed by sophisticated in vitro and in vivo experimental data. A particular strength of the manuscript is the combined use of genomic, gene expression, proteomic, and small metabolite analyses to pin down the mechanistic basis of the degradation of amygdalin. While at this stage the authors cannot infer the importance of their findings for bee health, their insights and methods should stimulate additional experiments into the role of microbial conversion of dietary metabolites for bee health.

Reviewer #1 (Public Review):

Building upon the previous evidence of activation of auditory cortex VIP interneurons in response to non-classical stimuli like reward and punishment, Szadai et al., extended the investigation to multiple cortical regions. Use of three-dimensional acousto-optical two-photon microscopy along with the 3D chessboard scanning method allowed high-speed signal acquisition from numerous VIP interneurons in a large brain volume. Additionally, activity of VIP interneurons in deep cortical regions was obtained using fiber photometry. With the help of these two imaging methods authors were able to extract and analyze the VIP cell signal from different cortical regions. Study of VIP interneuron activity during an auditory go-no-go task revealed that more than half of recorded cortical VIP interneurons were responding to both reward and punishment with high reliability. Fiber photometry data revealed similar observations; however, the temporal dynamics of reinforcement stimuli-related response in mPFC was slower than in the auditory cortex. The authors performed detailed analysis of individual cell activity dynamics, which revealed five categories of VIP cells based on their temporal profiles. Further, animals with higher performance on the discrimination task showed stronger VIP responses to 'go trials' possibly suggesting the role of VIP interneurons in discrimination learning. Authors found that reinforcement related response of VIP interneurons in visual cortex was not correlated with their sensory tuning, unveiling an interesting idea that VIP interneurons take part in both local as well as global processing. These observations bring attention to the possible involvement of VIP interneurons in reinforcement stimuli-associated global signaling that would regulate local connectivity and information processing leading to learning.

The state-of-the-art imaging technique allowed authors to succeed in imaging VIP interneurons from several cortical regions. Advanced analyses revealed the nuances, similarities and differences in the VIP activity trend in various regions. The conclusions about reinforcement stimuli related activity of VIP interneurons made by the authors are well supported by the results obtained, however some claims and interpretations require more attention and clarification.

Reviewer #1 (Public Review):

In this work the authors study the effects of the accumulation of endogenously produced Advanced Glycation End-products (AGEs) on feeding behaviors in C. elegans. AGEs are produced during the metabolism of all organisms, and also, they are produced by the food industry through Mainard reactions. In this sense, the objectives of this study are not only to provide basic information relevant to phenomena that are likely to be conserved throughout the animal kingdom but also to provide information that could be important in human health for the understanding of disorders caused by the consumption of processed foods.

The methodology includes as read out very robust and supercharacterized assays of food intake in C. elegans, such as pharyngeal pumping and food depletion.<br /> As a general evaluation of the manuscript, I think the authors could provide more detailed mechanistic information about how MGH-1 acts on the tyraminergic pathway to potentiate food intake. While they find important players, they do not quite find how these players interact with each other, nor which cells or neural circuits are governing the processes described.

In summary, I consider the initial objective of the manuscript to be extremely significant, but I believe it falls short in the mechanistic explanation of the observations described.

Reviewer #1 (Public Review):

This report describes an exhaustive analysis of behavior in a complex associative learning paradigm that blends aversive Pavlovian and appetitive instrumental elements. The hand-scoring technique is rigorous and documented to a greater degree than what is typically reported in papers using human raters to quantify animal behavior. Near-complete ethograms offer a novel, high-resolution look at how aversive cues exert distinct effects on appetitive and aversive behavior.

From the perspective of the rodent subject, there is quite a lot going on in the experimental chamber in this study. It's an environment in which appetitive instrumental action is set against multiple predictive cues signaling differing degrees of danger and safety. The test is fully on-baseline, occurring in the same place as training. The rich web of associations formed has a predictably complex influence on behavior. The authors contrast this complexity with much of the rest of the literature, in which freezing is reported to predominate when an aversive CS is presented. Indeed, most conventional studies of aversive associative learning train subjects on a single tone-shock association and test in a neutral context. The contrast between the common approach and the one taken by the authors suggests questions central to understanding the current report. Does being tested in an associatively complex context promote the pattern of behaviors that the authors observe? Or is it a question of learning history - would, following this kind of complex training, an off-baseline test in a neutral environment, produce the same suite of outcomes in response to the danger cue? Answers to these questions would go some distance toward nesting this paper in a wider body of knowledge about defensive reactions to aversive conditioned stimuli. Data speaking to these issues would also increase the work's impact by demonstrating the way in which a given response can be modulated by other learning.

Reviewer #1 (Public Review):

The paper, fundamentally, is a description of the accuracy of individual model and ensemble model short-term forecasts of COVID-19. This has been done before in both weather and infectious disease. So what are the contributions of this manuscript? I see the following:

1. The authors show that ensemble prediction (a straight average) generally outperforms individual component models. This is not new and has been shown, as the authors cite, for weather, climate, and infectious disease.<br /> 2. Use of the median estimate across models, rather than the mean, buffers against outliers. This is a well-recognized workaround for right-skewed distributions, though the specific finding in this study is of some importance, as this hasn't always been the case (noted by the authors in their discussion).<br /> 3. Deaths are better forecasted than cases. This is not new, either, as the authors note, as deaths are a lagged function of cases/infections.<br /> 4. It presents the archive of European COVID-19 forecasts.

Although I don't see a lot of novelty in these findings, this COVID-19 forecasting work is important and represents a considerable effort on part of the individual modelers. The paper is well written, but it doesn't show much that is novel methodologically. For instance, it doesn't propose and validate an approach for improving forecasting or projection accuracy. Are there new ways to handle or predict behavioral, vaccination uptake, or viral changes? Are there novel post-processing approaches, other than 'ensembling' that could improve forecast accuracy?

Reviewer #1 (Public Review):

Earlier this year Skolnick and colleagues managed to tweak AlphaFold to predict protein complexes (reference 23 in the current manuscript). They also added a score that allows the detection of true protein-protein interactions among arbitrary protein pairs. Thus, their methodology allows reliable prediction of homo- and hetero-meric protein-protein interactions, and predicting the structure of the corresponding protein complexes. Leveraging this methodology, the current manuscript describes a very interesting application to a set of about 1,500 E. coli proteins of the outer membrane, the periplasm and the inner membrane of this Gram negative bacteria. They explore protein-protein interactions among this protein set, which they refer to as 'envelome'. Their results reproduce known protein complexes, such as the translocon, and suggest many yet unknown interactions that make biological sense.

A main strength here is the generation of ample hypotheses to be tested in experiment, i.e., all protein-protein interactions of high predicted accuracy. Another strength is that the methodology is readily applicable to other systems. However, a few outstanding issues need to be clarified.

1. Even though the methodology was already introduced, it should be described in some detail. Most importantly, AlphAfold's measures of accuracy have been part of the loss function during training/testing. What about the measure of protein-protein interaction accuracy? Was it also in the loss function?<br /> 2. Figure 1a (upper panel, PpiD) includes quite a few promising hits but only the first, third, and 12th were considered. How were these chosen? For example, why not consider the second? The lower panel (YfgM) also shows many promising hits but only the first was chosen. Why not more?<br /> 3. Likewise, only two of the top hits in Figure 4 are considered. What about the rest? For example, why taking into account the second best hit while skipping the first?<br /> 4. Authors argue that the unstructured part of OmpA, which wraps around SurA, is to be trusted, which may be the case. But a more likely explanation is that it is an artefact, in agreement with the very low confidence assigned by AlphaFold.<br /> 5. Figure 5. How is this predicted structure compare with the known structure of the complex? In particular, how similar are the predicted and known structures of the individual subunits, and how similar are the predicted docking poses to the known ones?<br /> 6. Authors should make the results easily accessible to all. Maybe as Cytoscape and CyToStruct sessions for easy visualization.<br /> 7. Finally, AlphaFold was trained and tested mostly with water-soluble protein. Thus, application to outer membrane proteins is a bit risky. Maybe authors can comment on this.