yellow journalism in this manner did not have good effects. It was spreading misinformation to audiences.

This is what yellow journalism was- getting the information they needed at whatever cost, eve if it meant making up some facts in order to attract readers.

Reviewer #3 (Public Review):

This manuscript aims to address whether age-related iron status influences differential effects of estrogen replacement on atherogenesis in postmenopausal females. Specifically, whether age-related iron accumulation reduces estrogen signaling through ERa receptor in relevant cell types (endothelial cells and macrophages). They test this fairly rigorously using in vitro, preclinical and clinical data, and the data is presented logically.

First, the authors demonstrate that in postmenopausal women, there is an inverse correlation between age-related increase in iron levels and age-related decrease in ERa levels in atherosclerotic plaques. This is consistent with the fact that, in ovariectomized (OVX) pro-atherogenic (ApoE KO) mice, there is differential effect of estrogen (E2) on ERa, atherosclerosis, lipid profiles and key biomarkers (ABCA1, eNOS, etc) based on age (early: 16 weeks, versus late: 40 weeks), with young mice responding favorably while older mice responding negatively. Consistent with this, in the older OVX ApoE KO mice, E2 treatment worsened atherosclerosis. Importantly, they point this differential effect of E2 to iron overload in macrophages using ApoE KO; LysM-macrophage-specific Fpn1 KO as E2 now has deleterious effects regardless of age. Next, using ferric ammonium citrate (FAC) to supplement iron and deferiprone (DFP) to chelate iron, they show that iron manipulation impacts the E2 response appropriately in the relevant cell types (endothelial cells and macrophage cells). Then they provide evidence for the MdM2-mediated post-translational regulation of ERa as a mechanism by which iron status impacts differential E2 has differential effects on ERa. Finally, they test the impact of systemic iron chelation on the OVX ApoE KO mice model of atherosclerosis and show that iron chelation attenuates the deleterious effects of E2 in late postmenopausal mice.

Overall the evidence is solid and logically laid out. Given that serum iron levels do not correlate with the rest of the story, inclusion of LysM-macrophage-specific Fpn1 KO provides the key evidence that iron loading in macrophages drives differential effects to E2 in postmenopausal mice. The paper provides evidence that iron levels influence how the relevant cells respond to E2 with clinical implications to hormonal replacement therapy in younger and older postmenopausal women. While this study is limited by a small clinical sample size, it provides an important framework for future studies on the impact of age-related iron status on the response to hormonal replacement therapy.

Reviewer #3 (Public Review):

This paper demonstrates neural mechanisms important for the representation of moving stimuli. Specifically, using EEG, the authors investigated the temporal profiles of visual activities that correspond to changes in positions of moving stimuli.

Strengths:<br /> The authors examined an interesting question of how moving stimuli can be smoothly represented and perceived by using a neural recording modality with high temporal resolution. To my knowledge, the temporal dynamics of the neural correlates of successful motion perception are not well understood, and the study provides evidence for a plausible mechanism for this process. Additionally, the paper is well-written where the results are clearly communicated, and the figures are clearly presented.

Weaknesses:<br /> The findings reported are derived from a specific case of motion perception which may not reflect the general mechanisms optimized for motion perception. The limitations related to task designs and the neural readouts should be discussed as they affect the way that the reported results will be interpreted.

Reviewer #3 (Public Review):

This study provides interesting new information on the cellular roles of different Arp2/3 complex isoforms. Previous studies have shown that T cell receptor signaling induces both cytoplasmic and nuclear actin filament assembly, which are dependent on the Arp2/3 complex. Moreover, nuclear actin filament assembly can be induced in response to DNA damage by the Arp2/3 complex. However, the possible roles of different Arp2/3 isoforms in these processes have not been reported. Here, Sadhu et al. demonstrate that the two isoforms of the ARPC5 subunit (ARPC5 and ARPC5L) have specific functions in these processes. By using knockdown and knockout cells, they provide evidence that cytoplasmic actin polymerization induced by T cell receptor activation is dependent on the ARPC5 isoform, whereas consequent nuclear actin filament assembly relies on the ARPC5L isoform. Interestingly, the nuclear actin polymerization induced by DNA replication stress is dependent on ARPC5. The authors also examined the upstream signaling pathways, and provide evidence that nuclear calcium-calmodulin signaling and N-WASP are specific activators of ARPC5L containing complexes.

Majority of the data presented in the manuscript appear of good technical quality, and the study provides interesting new insights into specific cellular roles of different Arp2/3 isoforms in T lymphocytes.

Reviewer #3 (Public Review):

The article reports the functional analysis of one of the critical genetic determinants for bacterial infection, the RHIZOBIUM-DIRECTED POLAR GROWTH protein (RPG). The evolutionary pattern linking RPG to the Transcription Factor and the LysM receptor-like kinase, and the ability to form root nodule symbiosis makes it one of the prime candidates for engineering symbiotic nitrogen fixation in cereals. Therefore this analysis is timely and of huge importance. In a previous study, an EMS-induced mutant of M. truncatula (rpg-1 allele) was reported to have aberrant infection threads and poorly colonized nodules, the RPG expression was strongly associated with rhizobial infection, and the RPG protein showed nuclear localization when heterologously overexpressed in N. benthamiana (Arrighi et al., 2008), but its cellular and molecular function in M. truncatula have not been understood in detail.

In the present manuscript, the authors showed that RPG is a crucial component of the infectosome machinery. They conclude that RPG sustains polar growth of intracellular infection threads, being necessary to recruit via protein-protein interactions the VAPYRIN.<br /> In absence of rpg, the authors reported : (i) the absence of membrane polarization at the advancing tip of the infection thread using phosphoinositide reporters, (ii) the lack of connectivity between the infection thread tip and the nucleus via the microtubules cytoskeleton, (iii) the loss of polar secretion of the cell wall modifying enzyme NODULE PECTATE LYASE (NPL).

These results confirmed that RPG is part of the core host machinery required to support symbiont accommodation. Furthermore, this work shows that multimeric host factors work as a module committed to endosymbiosis to sustain the infection thread-mediated bacterial infection.

This paper is well written, with extremely beautiful cell biology and the conclusions are clearly connected to the data presented. However, in my view, some critical points need to be further addressed:

1. The author showed that RPG co-purified with EXO70H4 and VPY indicating that these proteins indeed belong to the same complex. Moreover, they showed that the major molecular determinant conferring functionality to RPG resides in its coiled-coil domain with structure-function analysis. Does the CC domain important for RPG interaction with "the multimeric host factors" described here? What is the localization of the truncated form of RPG (reported in this paper for rpg1 complementation analysis) in the WT plant?

2. It is not clear to me how RPG acts on the membrane polarity: Is there a direct role of RPG in the PIP2 polarization, and cytoskeleton arrangement in the exocytosis of cell wall modifying enzyme NODULE PECTATE LYASE (NPL)? Did the authors observe an increase in membrane polarity when RPG is overexpressed? The authors might consider adding a model for the different molecular components they studied in the context of this biotic interaction.

Reviewer #3 (Public Review):

In this manuscript, the authors propose that there is a special, previously unrecognized, high-frequency population of a/b TCRs that are shared between people, have high generation probabilities, and react to many unrelated viral epitopes. Here is the main flow of the results, with comments on the strengths of the conclusions:

"Thymopoiesis selects a large and diverse set of clustered CDR3s with high generation probabilities" – this seems correct and has been noted in earlier work by Mora and Walczak and others. Selection leads to a focusing of the CDR3 length which likely increases the degree of clustering and increases Pgen.

"Clustered CDR3s are enriched for publicness" This also seems correct and again it makes sense: publicness is equivalent to having been independently rearranged (and sequenced) in another individual, which is determined by Pgen, and clustering is also determined to a large extent by Pgen (the factors that contribute to Pgen, shorter CDR3s for example, are largely shared between neighbor TCRs).

"Clustered public CDR3s are enriched in viral specificities" – This claim is not justified by the data, which comes from sequence matching against literature-derived databases. Rather, what is true is that "Clustered public CDR3s are enriched in public viral specificities". But this might be a simple consequence of the previous observation, that "clustered CDR3s are enriched for publicness". One would need experimental specificity data on the very same datasets to make a conclusion about viral specificities in general.

"Identification of polyspecific TCRs" – In this section, the authors report that some of the CDR3 clusters contain CDR3 sequences from literature-derived TCRs with multiple specificities. They conclude that these must represent polyspecific TCRs. The problem with this conclusion is that even having the same CDR3beta, let alone similar CDR3beta sequences, does not imply the same specificity. One can see the problem if one imagines a very deeply sequenced dataset, and focuses on a short CDR3 length with high frequency. WIth sufficient sampling, one will be able to navigate from nearly any single CDR3beta to any other CDR3beta of the same or similar length by jumping between single-mismatch variants. But this doesn't imply that all the TCRs from which these CDR3s were sampled, which likely have many different Vbeta genes and completely different TCRalpha sequences, must all bind the same thing.

"Binding properties of polyspecific TCRs" – Here the authors look to validate these results with paired TCR sequences. They analyze a public dataset made available by 10X genomics, featuring single-cell gene expression, TCR sequencing, and dextramer UMI counts for ~150,000 T cells. This is an amazing dataset with lots of interesting features, but, like any large high-throughput dataset, it needs to be analyzed with care. The authors claim to see evidence for large-scale cross-reactivity. This comes mainly from a set of dextramers for A*03 and A*11-restricted peptides. But these dextramers appear to be binding in a uniquely non-specific manner (by comparison with the other dextramers) and non-TCR-dependent manner in this experiment. One can see this, for example, by comparing the consistency of binding within expanded clonotypes: for a specific dextramer like A*02-GIL(Flu), positive binding for one cell in a clonotype greatly increases the likelihood of binding for other cells in the clonotype, suggesting that the binding is mediated by the TCR. This is not true for the A*03 and A*11 dextramers (except for a few expanded clonotypes in an A*11 donor). TCR sequence doesn't appear to be the determining factor for binding to these dextramers; rather it may be expression of KIR genes or other surface proteins that can interact with MHC.

"Polyspecific T cells are activated in vitro by multiple viral peptides" Here the authors explore polyspecificity experimentally. First they report that polyclonal populations of T cells, sorted for binding to one dextramer, can also produce IFNgamma upon stimulation with a distinct peptide, albeit more weakly than for the cognate peptide. But it's not clear that the concentrations of the peptides are appropriate for stringently detecting cross-reactivity. Then the authors actually synthesize and characterize individual TCRs. Here what is seen is consistent with expectation and does not seem to support the idea of substantial fuzzy cross-reactivity: binding to the cognate peptide is 3-4 orders of magnitude stronger than to the alternative peptides. The only exception is the GAD 114-122 TCR, where the different peptides appear to be closer in binding strength. But in this case, the authors state that they "analyzed their response to a set of peptides comprising their cognate peptide and peptides with no significant structural commonalities, selected by testing combinatorial peptide libraries". If the competitor peptides came from peptide library screening then the observation of strong binding to alternative peptides does not seem as surprising as a TCR that binds well to a Flu peptide, say, and also a CMV peptide, selected from a smallish set of possibilities.

It is pretty well established that TCRs are cross-reactive, both for nearby peptides and also for sequence-dissimilar peptides. The question is whether widespread, functionally relevant (not just dextramer binding at some concentration) poly-reactivity to diverse viral peptides is a defining feature of a large fraction of the TCR repertoire. The paper does not appear to present sufficiently strong evidence to support this claim.

Reviewer #3 (Public Review):

This manuscript explores the concept of TCR convergence, defined here as the presence of TCRs with the same amino acid sequence but distinct nucleotide sequences. The central premise is that TCR convergence is a sign of antigen-driven selection. TCR convergence as a biomarker for immune checkpoint blockade (ICB) response is also investigated. Although both these ideas have been put forward in the literature, this manuscript provides some new analyses and a new perspective on these topics.

Main results:

- TCR convergence is different from publicity: The authors look at CDR3 sequence features of convergent TCRs in the large Emerson CMV cohort. Amino usage does not perfectly correlate with codon degeneracy, for example, arginine (which has 6 codons) is less common in convergent TCRs, whereas leucine and serine are elevated. It's argued that there's more to convergence than just recombination biases, which makes sense. (I wonder if the trends for charged amino acids could be explained by the enrichment of convergent TCRs in CD8 T cells, which tend to have more acidic CDR3 loops). There's also a claim that the overlap between convergent and public TCRs is lower in tumors with a high mutational burden (TMB), but this part is sketchy: the definition of public TCRs is murky and hard to interpret, and the correlation between TMB and convergence-publicity overlap is modest (two cohorts with low TMB have higher overlap, and the other three have lower, but there is no association over those three, if anything the trend is in the other direction). It's also not clear why the overlap between COVID19 cohort convergent TCRs and public TCRs defined by the pre-2019 Emerson cohort should be high. A confounder here is the potential association between convergence and clonal expansion since expanded clonotypes can spawn apparently convergent TCRs due to sequencing errors. The paper "TCR Convergence in Individuals Treated With Immune Checkpoint Inhibition for Cancer" (Ref#5 here) gives evidence that sequencing errors may be inflating convergence in this specific dataset.

- Convergent TCRs are more likely to be antigen-specific: This is nicely shown on two datasets: the large dextramer dataset from 10x genomics, and the COVID19 datasets from Adaptive biotech. But given previous work on TCR convergence, for example, the Pogorelyy ALICE paper, and many others, this is also not super-surprising.

- Convergent T cells exhibit a CD8+ cytotoxic gene signature: This is based on a nice analysis of mouse and human single-cell datasets. One striking finding is that convergent TCRs are WAY more common in CD8+ T cells than in CD4+ T cells. It would be interesting to know how much of this could be explained by greater clonal expansion of CD8+ T cells, together with sequencing errors. A subtle point here is that some of the P values are probably inflated by the presence of expanded clonotypes: a group of cells belonging to the same expanded clonotype will tend to have similar gene expression (and therefore similar cluster membership), and will necessarily all be either convergent or not convergent collectively since they share the same TCR. So it's probably not quite right to treat them as independent for the purposes of assessing associations between gene expression clusters and convergence (or any other TCR-defined feature). You can see evidence for clonal expansion in Figure 3C, where TRAV genes are among the most enriched, suggesting that Cluster 04 may contain expanded clones.

- TCR convergence is associated with the clinical outcome of ICB treatment: The associations for the first analysis are described as significant in the text, and they are, but just barely (0.045 and 0.047, but you have to check the figure to see that).

- Introduction/Discussion: Overall, the authors could do a better job citing previous work on convergence, for example, papers from Venturi on convergent recombination and the work from Mora and Walczak (ALICE, another recombination modeling). They also present the use of convergence as an ICB biomarker as a novel finding, but Ref 5 introduces this concept and validates it in another cohort. Ref 5 also has a careful analysis of the link between sequencing errors and convergence, which could have been more carefully considered here.

Reviewer #3 (Public Review):

Gupte and colleagues develop an individual-based model to examine how the introduction of a novel pathogen influences the evolution of social cue use in a population of agents for which social cues can both facilitate more efficient foraging, but also expose individuals to infection. In their simulations, individuals move across a landscape in search of food, and their movements are guided by a combination of cues related to food patches, individuals that are currently handling food items, and individuals that are not actively handling food. The latter two cues can provide indirect information about the likely presence of food due to the patchiness of food across the landscape.

The authors find that prior to introducing the novel pathogen, selection favors strategies that home in on agents, regardless of whether those agents are currently handling food items. The overall contribution of these social cues to movement decisions, however, tends to be relatively small. After pathogen introduction, agents evolve to rely more heavily on social information and to either be more selective in their use of it (attending to other agents that are currently handling food and avoiding non-handlers) or avoiding other agents altogether. Gupte and colleagues further examine the ecological consequences of these shifts in social decision-making in terms of individuals' overall movement, food consumption, and infection risk. Relative to pre-introduction conditions, individuals move more, consume less food, and are less likely to be infected due to reduced contact with others. Epidemiological models on emergent social networks confirm that evolved behavioral changes generate networks that impede the spread of disease.

The introduction of novel pathogens into wild populations is expected to be increasingly common due to climate change and increasing global connectedness. The approach taken here by the authors is a potentially worthwhile avenue to explore the potential eco-evolutionary consequences of such introductions. A major strength of this study is how it couples ecological and evolutionary timescales. Dominant behavioral strategies evolve over time in response to changing environmental conditions and impact social, foraging, and epidemiological dynamics within generations. I imagine there are many further questions that could be fruitfully explored using the authors' framework. There are, however, important caveats that impact the interpretation of the authors' findings.

First, reproduction bears no cost in this model. Individuals produce offspring in proportion to their lifetime net energy intake, which is increased by consuming food and decreased by a set amount per turn once infected. However, prior to reproduction, net energy intake is normalized (0-1) according to the lowest individual value within the generation. This means that individuals need not maintain a positive energy balance nor even consume food at all to successfully reproduce, so long as they perform reasonably well relative to other members of the population. Since consuming food is not necessary to reproduce, declining per capita intake due to evolved social avoidance (Fig. 1d) likely decreases the importance of food to an individual's reproductive success relative to simply avoiding infection. This dynamic could explain the delayed emergence of the 'agent avoiding' strategy (Fig. 1a), as this strategy potentially is only viable once per capita intake reaches a sufficiently low level across the population (Fig. 1d). I am curious to know what the results would be if reproduction required some minimal positive net energy, such that individuals must risk food patches in order to reproduce. It would also be useful for the authors to provide information on how net energy intake changes across generations, as well as whether (and if so, how) attraction to the food itself may change over time.

A second important caveat is that the evolutionary responses observed in the model only appear when novel pathogen introductions are extremely frequent. The model assumes no pathogen co-evolution, but rather that the same (or a functionally identical) pathogen is re-introduced every generation (spillover rate = 1.0). When the authors considered whether evolutionary responses were robust to less frequent introductions, however, they found that even with a per-generation spillover rate of 0.5, there was no impact on social movement strategies. The authors do discuss this caveat, but it is worth highlighting here as it bears on how general the study's conclusions may be.

Reviewer #3 (Public Review):

The authors have accomplished large amounts of work to prove the role of VPS9D1-AS1 in promoting immune escape from cytotoxic T cells, and the mechanistic exploration is valid enough to support the conclusions, as well as the translational significance of this target through in vivo experiments. However, the logicality of the diagram requires improvement, and several revisions are warranted.

Reviewer #3 (Public Review):

The authors do an excellent job of producing relatively simple model neurons to characterize the key properties of two different cell classes (feedforward Somatostanin, SOM, interneurons and serotonergic, 5-HT, output neurons) in the dorsal raphe nucleus. They make a strong case for the role of an A-type potassium current and strong spike-threshold adaptation in the 5-HT neurons in reproducing the measured single-cell responses to stimuli and characterize well the interplay of these two currents in producing a transient measure of the derivative of inputs under some circumstances. While the demonstration of rapid adaptation leading to an output that resembles a derivative of the input under some circumstances is far from novel, the authors here have succeeded in matching such a computation to known properties of neurons in known circuitry. However, a little more care is needed in how the authors describe the output of serotonergic neurons being linear in the derivative of the inputs, because in general that is not the case. For example, the sustained response of the neurons depends on the net input current when the derivative of the input is zero in all cases. A neuron that really follows a derivative would, for example, respond in a manner independent of baseline current and produce a cosine output to a sinusoidal input. Rather, it is a transient output response that in some limited ranges of constant baseline input and ramping rate of the input, has a peak that is linearly dependent on the ramping rate. Also, only positive slopes were considered. Thus, it seems unlikely that the serotonergic output in the model is very close to the derivative of a general input signal, nor does it appear likely to operate on the long temporal timescale needed for reinforcement learning, as suggested.

Reviewer #3 (Public Review):

In this work, the authors have assessed the bone phenotype of a mouse with targeted ablation of the vacuolar ATPase accessory protein ATP6AP2 in the osteoblast lineage. They observe a clear increase in cortical thickness, but the cortex is highly porous and contains remnant cartilage as well as extensive woven bone. They then follow this by suggesting that one cause of this phenotype may be a change in the surface expression of the protein MMP14, a matrix metalloproteinase, known to be involved in bone matrix degradation, at least in osteoclasts. They provide evidence that this protein may also regulate matrix degradation surrounding osteocytes and an increase in this protein in osteocytes lacking ATP6AP2 may be a cause of the initial phenotype described.

While the phenotype described is very dramatic, the interpretation that it reflects a defect in osteoblast to osteocyte transition is questioned by this reviewer. The phenotype appears to be an osteopetrosis, including a lack of remodelling of the cortex. Cartilage and woven bone are not replaced effectively by lamellar bone. The bone contains ample osteocytes, but they are the osteocytes typical of woven bone, with rounded cell bodies, disordered organisation, low sclerostin expression, and short dendritic processes. The defect in the ATP6AP2 mice is a lack of cortical remodelling during cortical consolidation (for review see PMID: 34196732). Cartilage and woven bone remnants, which are normally remodelled as cortical bone matures, remain in the cortex until adulthood. It is not clear whether this results from reduced or increased remodelling of the cortex, but it is not because the osteoblasts cannot form osteocytes.

Some of the data is very challenging to interpret because of low sample numbers (n=4 for much of the analysis), and lack of detail as to the sex of the animals. Regions used for imaging, histomorphometry, and dynamic histomorphometry all need to be defined throughout the work. Since the cortex differs dramatically by site, and by distance from the growth plate (due to the different stages of maturation) this is critical. Some methods are not defined, although they could be of great use to the field (e.g. the method for assessing bone degradation by MMP14).

Reviewer #3 (Public Review):

The aim of Weber and colleagues' study was to generate arthropod environmental DNA extracted from a unique 30-year time series of deep-frozen leaf material sampled at 24 German sites, that represent four different land use types. Using this dataset, they explore how the arthropod community has changed through time in these sites, using both conventional metabarcoding to reconstruct the OTUs present, and a new qPCR assay developed to estimate the overall arthropod diversity on the collected material. Overall their results show that while no clear changes in alpha diversity are found, the β-diversity dropped significantly over time in many sites, most notable in the beech forests. Overall I believe their data supports these findings, and thus their conclusion that diversity is becoming homogenized through time is valid.

While overall I do not doubt the general findings, I have a number of comments. Firstly while I agree this is a very nice study on a unique dataset - other temporal datasets of insects that were used for eDNA studies do exist, and perhaps it would be relevant to put the findings into context (or even the study design) of other work that has been done on such datasets. One example that jumps to my mind is Thomsen et al. 2015 https://besjournals.onlinelibrary.wiley.com/doi/full/10.1111/1365-2656.12452 but I am sure there are others.

From a technical point of view, the conclusions of course rely on several assumptions, including (1) that the biomass assay is effective and (2) that the reconstructed levels of OTU diversity are accurate,

With regards to biomass although it is stated in the manuscript that "Relative eDNA copy number should be a predictor for relative biomass ", this is in fact only true if one assumes a number of things, e.g. there is a similar copy number of 18s rDNA per species, similar numbers of mtDNA per cell, a similar number of cells per individual species etc. In this regard, on the positive side, it is gratifying to see that the authors perform a validation assay on 7 mock controls, and these seem to indicate the assay works well. Given how critical this is, I recommend discussing the details of this a bit more, and why the authors are convinced the assay is effective in the main text so that the reader is able to fully decide if they are in agreement. However perhaps on the negative side, I am concerned about the strategy taken to perform the qPCR may have not been ideal. Specifically, the assay is based on nested PCR, where the authors first perform a 15cycle amplification, this product is purified, then put into a subsequent qPCR. Given how both PCR is notorious for introducing amplification biases in general (especially when performed on low levels of DNA), and the fact that nested PCRs are notoriously contamination prone - this approach seems to be asking for trouble. This raises the question - why not just do the qPCR directly on the extracts (one can still dilute the plant DNA 100x prior to qPCR if needed). Further, given the qPCRs were run in triplicate I think the full data (Ct values) for this should be released (as opposed to just stating in the paper that the average values were used). In this way, the readers will be able to judge how replicable the assay was - something I think is critical given how noisy the patterns in Fig S10 seem to be.

Next, with regards to the observation that the results reveal an overall decrease in arthropod biomass over time: The authors suggest one alternate to their theory, that the dropping DNA copy number may reflect taxonomic turnover of species with different eDNA shedding rates. Could there be another potential explanation - simply be that leaves are getting denser/larger? Can this be ruled out in some way, e.g. via data on leaf mass through time for these trees? (From this dataset or indeed any other place).

With regards to estimates of OTU/zOTU diversity. The authors state in the manuscript that zOTUs represent individual haplotypes, thus genetic variation within species. This is only true if they do not represent PCR and/or sequencing errors. Perhaps therefore they would be able to elaborate (for the non-computational/eDNA specialist reader) on why their sequence processing methods rule out this possibility? One very good bit of evidence would be that identical haplotypes for the individual species are found in the replicate PCRs. Or even between different extractions at single locations/timepoints.

With regards to the bigger picture, one thing I found very interesting from a technical point of view is that the authors explored how modifying the mass of plant material used in the extraction affects the overall results, and basically find that using more than 200mg provides no real advantage. In this regard, I draw the authors and readers attention to an excellent paper by Mata et al. (https://onlinelibrary.wiley.com/doi/full/10.1111/mec.14779) - where these authors compare the effect of increasing the amount of bat faeces used in a bat diet metabarcoding study, on the OTUs generated. Essentially Mata and colleagues report that as the amount of faeces increases, the rare taxa (e.g. those found at a low level in a single faeces) get lost - they are simply diluted out by the common taxa (e.g those in all faeces). In contrast, increasing biological replicates (in their case more individual faecal samples) increased diversity. I think these results are relevant in the context of the experiment described in this new manuscript, as they seem to show similar results - there is no benefit of considerably increasing the amount of leaf tissue used. And if so, this seems to point to a general principal of relevance to the design of metabarcoding studies, thus of likely wide interest.

Reviewer #3 (Public Review):

Park and Bafna et al. applied a genetics-based epidemiological approach, the Mendelian randomization analysis (MR), to evaluate the potential causal roles of triglycerides across 2,600 disease traits (i.e., the phenome). In a typical two-sample MR framework, they utilized existing genome-wide association study (GWAS) summary statistics from two separate studies. They are Global Lipids Genetics Consortium (GLGC) and UK Biobank in the discovery analysis, and UK Biobank and FinnGen in the replication analysis. This replication design is a great strength of the study, enhancing the robustness and reproducibility of the results. For the candidate pairs of causal associations, the authors further perform multiple sensitivity analyses to evaluate the robustness of the results to possible violations of assumptions in MR. To disentangle the independent effects of triglycerides from other lipid fractions (i.e., LDL-cholesterol and HDL-cholesterol), the authors performed multivariable MR analysis. In the end, possible causal associations were revealed in three tiers, based on statistical significance in the two-stage analysis. The results support the causal effects of triglycerides in increasing the risk of atherosclerotic cardiovascular disease. They also reveal novel conditions, which are either new treatable conditions (e.g., leiomyoma, hypertension, calculus of kidney and ureter) for repurposing of triglycerides-lowering drug, or possible side effects (e.g., alcoholic liver disease) the triglyceride-lowering treatment should pay special attention to.

The analysis approaches in the paper are standard and solid. The discovery-replication study design is a great strength. Correction for multiple testing was implemented in a conservative way. The sensitivity analyses and MVMR strengthen the robustness of the results. The manuscript is very clearly written and pleasant to read. The limitations were well-presented. The conclusions and interpretations are mostly supported by the data, with one major concern as explained below. But overall, in addition to the specific findings, this study could be an exemplar study for the use of phenome-wide MR in identifying treatable conditions and side effects for most existing drugs.

1. My major concern is about reverse causation. For example, having atherosclerotic cardiovascular disease increases circulating triglycerides. Reverse causation can induce false positives in MR analysis. With the existing data in this study, the authors can perform a reverse MR to evaluate the effect of the 19 disease traits on triglycerides. Ruling out the presence of reserve causation is important to make sure that the current findings are not false positives.

Reviewer #3 (Public Review):

This study examines behavior of humans and monkeys in a standard two-player game theory game called Bach or Stravinsky (also known as Battle of the Sexes) and, more technically, the iterated version of this game. This game is less well studied than the Prisoner's Dilemma or the Stag Hunt, but has an interesting twist relative to these - the optimal strategy in an iterated version is one of two options that provide a better reward to one player. For this reason, humans will typically show alternating behavior. This game then lets us ask whether and how well monkeys will also come to the same alternating behavioral pattern.

The study is unique in that it uses a novel format for interaction - the "transparent game" in which subjects press their fingers on a clear glass screen. Among other benefits, this feature allows the experimenters to study dynamics of choice, so that the decision takes on properties of continuousness. That in turn allows for reaction time biases.

In summary, this is an excellent and fascinating study. The authors have asked important and interesting questions, and have done a careful study that provides answers. I anticipate that their "transparent game" technique will become more popular due to its utility.

Another strength of this paper is the combination of human and macaque players (and the return of the macaque to the macaque-macaque group play). The result is exciting and surprising. This is a novel and remarkable element of the study and a source of great strength.

Limitation - lots of conceptual differences, including primary vs secondary reward, expectations, that may be due to learning/socialization. Having said that, these need to be acknowledged, but the study is good anyway. I will also note the authors already include a good and healthy limitation section in the Discussion.

Reviewer #3 (Public review):

This papers builds on a previous publication from the same group that showed compartmentalisation model of beta-cell fuel metabolism in which plasma membrane-localized pyruvate kinase is sufficient to close KATP channels required for insulin secretion. In this current manuscript the authors identified the PK isoforms involved in this process using tissue specific KO mouse models. Using excised patch-clamp experiments, they demonstrated that although redundant in their function both the constitutively active PKm1 and allosterically PKm2 are associated with the PM and locally regulate KATP channel closure. Further, the authors showed that the mitochondrial PEP carboxylase (PCK2) is essential for amino acids to promote an increase in cytosolic ATP/ADP and closure of KATP channels. Therefore, this study very nicely demonstrates that he distinct response of PK isoforms to the mitochondrial and glycolytic sources of PEP impacts beta cell nutrient preference and affects the oscillatory cycle regulating secretion. These findings do provide new mechanistic information about the control of the regulated secretory pathway and will be of interest to broader audience.

Strength<br /> The major strength of the study is the use of tissue/isoform specific KO mouse models. Although limited by constitutive KOs with compensatory increase in other isoforms, the authors have achieved what they were set out to do i.e identify the PK isoform involved in the regulation of PM ATP generation and regulation of KATP channel closure. Their experimental rigorosity including the ability to perform the excised patch clamp experiments and use of PKa to show the specific effect of the allosterically regulated PKM2 are also strength.

Weakness<br /> It is not clear from the manuscript what the "littermate controls" are used in all the experiments. Given the limitations of the cre lox system, it is really important to clearly show what controls have been used and their phenotypes (and the rationale for pooling the different controls if that is what is done here).

The data adds to our understanding of the role of PM localised PK on the regulated exocytosis pathway however the claim that these findings question the canonical mitochondrial ATP coupled to KATP channel closure is not fully supported by the data especially given glucose induced insulin secretion is not affected by any of the KO models.

Reviewer #3 (Public Review):

This manuscript presents a new tool, SIMMER, to predict bacterial enzyme-mediated transformations of compounds, an important and incompletely understood aspect of microbiome drug metabolism. The authors compare their resource to existing resources that allow users to generate hypotheses related to compound toxicity and putative routes of compound metabolism. The authors identify the key innovations of their resource as including full chemical representations of reactions and a novel method to predict an enzyme's EC number (a description of function) from its reaction.

Strengths:

• Generating user-friendly tools to explore existing knowledge of bacterial enzymes and their reactions is important.

• SIMMER is a novel resource where the user provides the substrates and products as input and receives a list of potential microbiome enzymes as output.

• SIMMER includes a novel EC predictor based on reaction rather than based on sequence.

Weaknesses:

• Validation claims are not well supported by the results.

• Need for the user to know both the substrate and the product for a reaction of interest limits the utility of the resource.

• Reliance on homology transfer annotation to predict enzyme function; this approach has important, microbiome-relevant, limitations.

Reviewer #3 (Public Review):

The manuscript by Voufo et al. aims to advance our understanding of the mechanisms responsible for the earliest pattern of spontaneous activity in the mouse retina, stage I retinal waves. These waves occur during embryonic development (E16-18) and are the least known form of activity in the immature retina.

The authors show that stage I waves have broad spatiotemporal features and are mediated by circuitry involving subtypes of nicotinic acetylcholine receptors (nAChRs) and gap junctions. The authors also found that the developmental decrease of intrinsic photoreceptor retinal ganglion cells (ipRGCs) density is preserved between control and ß2-nAChR-KO mice, indicating that processes regulating ipRGC distribution are not influenced by early spontaneous activity.<br /> The quality of the data is excellent, and the conclusions of this paper are mostly well supported by data, but the presentation of the data and the analysis need to be clarified and extended.

Strengths:<br /> The earliest patterns of spontaneous activity are crucial for the correct development of sensory circuits. In the visual system, most studies focus on postnatal activity (stage 2 and 3 retinal waves) overlooking embryonic stages, likely due to challenges related to methods and animal handling. Therefore, in this manuscript, the authors from a laboratory pioneer in studying retinal waves in the mouse, tackle a very relevant subject that has not been explored in detail. The bibliography that encompasses most of the current knowledge about stage 1 retinal waves in mammals is compressed into three fairly dated publications: Galli and Maffei 1988, Bansal et al 2000, and Syed et al 2004. These publications were pioneering attempts to describe early spontaneous activity; however, much work remained to be done regarding the molecular and cellular mechanisms involved. Here, Voufo and colleagues provide additional fundamental details about the properties and components of stage 1 waves. The dataset has excellent quality and plenty of information could be extracted from it. The authors used a macroscope that allows the acquisition of images from the entire retina while preserving a good spatial resolution.

Weakness:<br /> The authors distinguish different subtypes of activity during embryonic stages in the retina of mice. However, they do not provide a detailed characterization that allows a clear definition of these subtypes (and specifically stage 1 waves). Moreover, throughout the manuscript, there are many technical details of the analysis that are missing and preclude a complete understanding of the robustness of the data. The authors have an excellent dataset that needs more analysis and an improvement in the presentation of the results.

Reviewer #3 (Public Review):

This work provides a detailed single-cell transcriptomic analysis of endothelial cell (EC) differentiation from induced pluripotent stem cells in a suspension culture. The data demonstrates that the protocol produces a large number of both endothelial cells and mural cells, which is comparable to a 2D monoculture, with differences observed mainly in the expression of ECM genes. This first part of the study shows that EC differentiation works well both in 2D monolayer and in suspension cultures, with some key differences in their gene expression patterns, which has been shown before. Here, a detailed transcriptomic landscape of single cells during differentiation is provided. The second part of the paper examines how the transcriptome of ECs and pericytes (PC) that are differentiated in a suspension culture is remodeled in a 3D hydrogel environment, when cells sprout and form tubular structures. Based on the single-cell transcriptomes, the present study allows detection of different EC and PC populations during tube formation and cell maturation, and the identification of the genes and transcription factors, which regulate cellular behavior and phenotype, such as coalescing and sprouting ECs.

Strengths:<br /> The study is an extensive description of remodeling of the transcriptomic landscape in endothelial cells and mural cells during differentiation from human induced pluripotent stem cells. It demonstrates the potential of endothelial cell and pericyte differentiation in suspension culture, which allows larger yields compared to 2D monolayer cultures.<br /> The novelty in the study is the detailed transcriptomic characterization of the different cell clusters formed during tube formation and maturation in 3D hydrogel. This provides important information for future experiments studying the mechanisms of vasculogenesis and angiogenesis in vitro and for designing vascularization for organ-on-chip approaches.<br /> The data gathered here provides a great possibility to identify unknown interaction mechanisms between PC and EC during vascular development and maturation. Especially identification of PC subpopulations, which seem to produce both common and individual ligands for ECs is intriguing and creates several new research questions.

Weaknesses:<br /> The paper has a lot of transcriptomic data from different cell culture conditions and time points, making it difficult to identify the key observations and novel findings of the study. The paper provides a resource for future studies, but does not answer a clear biological question or test a hypothesis.<br /> One of the concerns is the large number of mural cells in maturing 3D hydrogel cultures (66-85%), indicating that the protocol directs the cells more towards mesenchymal than endothelial cells or ECs lose their identity over time. It would be important to show the localization and organization of these two cell types in the 3D cultures and to demonstrate if the different EC and pericyte subclusters localize to certain parts of the network.

Reviewer #3 (Public Review):

Using a robust transient transgenic approach in the zebrafish embryonal rhabdomyosarcoma (ERM) model, Chen et. al. identified diverse activities of several disease-relevant TP53 variants in ERM pathogenesis. The useful tools established in this study would allow rapid in vivo assessment of the effect of newly identified TP53 mutations on ERM tumorigenesis.

Strengths:<br /> • It's the first time to dissect the activities of several rare patient-specific TP53 mutations in ERM tumor initiation and progression in vivo.

• This study demonstrates the robustness of transient co-injection transgenic approach for rapid structure function analyses of disease-relevant variants in vivo.

• This study also suggests distinct activities of different TP53 structure variants, such as their potential functions as a hypomorphic allele, a gain-of-function mutation, or a predisposition mutant for the head musculature ERMs.

Weaknesses:<br /> • The role of tp53 loss in promoting the initiation of kRASG12D-driven ERM has been demonstrated previously using a similar strategy by coauthors (Ignatius, M. S., eLife, 2018; Langenau, D. M., Genes Dev, 2007).

• The data from TP53-null SaOS2 osteosarcoma cell line did not consistently support the findings from in vivo zebrafish studies, which is confusing and would need to be addressed.

• It is not clear how overexpression of the TP53P153△ or TP53Y220C mutant induced different effects on the tumor initiation and cell survival of kRASG12D-driven ERM but led to similarly enhanced head ERM development.

• This study mainly applied the overexpression approach to understand the function of TP53 mutants in ERM pathogenesis and demonstrated the distinct effects of their overexpression on kRASG12D-driven tumor initiation, cell survival and proliferation. However, these mutations are not gained or amplified in human ERMs. Hence, overexpression approach could provide some insights of their function, but cannot faithfully mimic the ERM disease situation to uncover the real function of these mutants in ERM pathogenesis.

Reviewer #3 (Public Review):

In their manuscript, McKitterick and Bernhardt perform a screen to determine host factors, such as receptors, which are important for bacterial viruses (phages) to infect Corynebacterium glutamicum., an organism that shares the unique membrane of mycobacteria (mycomembrane), with M. tuberculosis. To do so, they challenge a previously described Tn-seq library with a high MOI of 2 phages - Cgl and Cog. The surviving strains are those in which genes important for phage infection (such as receptors) are disrupted. The authors' screen is successful, and the authors identify and validate several factors important for the infection of each phage, providing the first such screen in Corynebacterium. Moreover, the authors perform a suppressor screen to identify additional factors and experimentally follow up several genes of interest. Finally, the authors use the newly determined host specificity of te phages to implicate new genes in mycolic acid synthesis. As a whole, this is a strong work that paves the way to a deeper understanding of Corynebacterial and (by extension) Mycobacterial phages and should be of broad interest.

Below, we suggest additional analyses, context, and elaboration that will help the ms. elaboration to fully realize its impact.

Major points:

1. Although the authors' experimental design is fundamentally sound, I am worried about the possibility of "jackpotting" in shaping their results, particularly in the uninfected control experiment. If the authors' Tn-seq library is ~200,000 strains, and they don't plate at least 10-100x times that many colonies then any given strain (regardless of its phenotype) may or may not be represented in the output of the experiment, causing false phenotypes to be ascribed to genes based on chance. This is particularly a problem for the uninfected control, where the authors choose to dilute the culture 1000-fold to mimic the number of colonies that survive infection. They may be better served by plating the whole culture on the plates, to ensure adequate representation of the library. Part of the reason for this concern is that an overwhelming majority of statistically significant hits (something like 80-90%) appear to confer susceptibility rather than resistance (source data Fig 2) - something the authors' experimental design should not be able to measure. The lack of accurate representation of distributions of strains in the starting culture also calls into question the quantitative differences they present in the results

a. L138. Where the authors describe their initial experimental design it would be helpful to add more details. What is the size of the Tn library? What is the coverage in their experiment? Approximately how many colonies are recovered on the plates after phage infection and in the uninfected control?

b. it is important to know how the number of colonies on the plates compares to the number of reads in the experiment. In the analysis of most HT screens, one implicitly assumes that each read corresponds to 1 cell, hence each read can be treated as statistically independent. This assumption is critical to the statistical methods used to analyze this data. By scraping a plate of colonies (which may be required for efficient phage infection), the authors potentially violate this assumption (since the number of cells → number of colonies, which are the actual statistically independent entities in the experiment). Does this assumption hold (or approximately hold) for the screen? If not, a different statistical method should be used to determine p-values.

2. The authors' Tn-seq methodology is different from previously published HT-phage screens (e.g. Mutalik et al., 2020 and Rousset et al., 2018). Based on my knowledge of classical phage biology, I agree that plating the infected cells has advantages. However, the rationale will not be clear for most people performing such experiments. Please explain the rationale for the experimental protocol.

a. Why did the authors plate the cultures after initial phage absorption instead of remaining in liquid?

b. How reproducible are the authors' Tn-seq results? The SRA ascension shows multiple replicates but this is not described in the manuscript nor reflected in the supplementary data. Given the potential for bottleneck and jackpotting effects in this assay, some measure of reproducibility is important for interpreting the results (see point 1).

c. L587 "Significant hits with fewer than 10 insertions on each strand were manually removed." Why did the authors choose this criterion? Almost all of the genes they removed have very asymmetric distributions (e.g. in the Cog experiment, cgp3051 has 47853 fwd reads and 6 rev reads. Asymmetric distribution of insertions suggests that overexpression of downstream genes has an important (positive or negative) effect. This is a worthwhile pursuit, and many automated analysis pipelines can disambiguate these effects, including those developed in the Walker Lab (e.g. doi: 10.1038/s41589-018-0041-4). These genes shouldn't be thrown away when they are arguably some of the most informative hits!

3. There is a somewhat extensive phylogeny of M. smegmatis phages (phagesdb.org). Are the phages that the authors work on related to any of these phages? If so, what cluster do they map to? What is the host range of other phages in that cluster? If not, may be worthwhile to mention that these are quite distinct from other studied phages.

4. Given that cgp_0475 was a strong hit in the Tn-seq, why was it not identified in the previous chemical genomics experiments from the lab (https://doi.org/10.7554/eLife.54761) ?

5. Is there any relationship between the growth-rate of the mutants and their phage susceptibility? This can be analyzed using the authors' previous studies of this library.

Reviewer #3 (Public Review):

In this manuscript, the authors Castro, Shortill, Dziurdzik, Cadou and colleagues perform an extensive systematic analysis of six membrane contacts sites (MCSs) to uncover novel proteins required for organelle tethering and modulation of membrane contacts. This work is critical as few if any proteins have been identified to regulate the formation and/or function of these contact sites. The authors identify over 100 new potential contact site proteins and effectors, including identification of proteins associated with the recently discovered plasma membrane-LD (pClip) and Golgi-peroxisome (GoPo) contact sites. This data set alone represents a huge contribution to the MCS field. The authors go on to identify and characterize novel proteins associated with the pClip homologous to known lipid binding proteins which may facilitate the transfer of lipids at membrane contact sites. Finally, the authors investigate the lipid droplet-ER (LiDER) contact site associated protein Lec1, which contains a novel putative lipid binding domain and may facilitate ergosterol transport between the plasma membrane and lipid droplets. The screening approach and experiments characterizing the function of Lec1 are for the most part straight forward, well-controlled, and easy to interpret. However, the authors' conclusions regarding the identified family of VPS13 like proteins role at the pCLIP is not strongly supported. Overall, these findings greatly expand our knowledge of proteins that regulate MCSs and will serve as the foundation for the identification of MCS tethers.

Reviewer #3 (Public Review):

Strege et al. addressed the mechanism underlying the well-known mechanosensitivity of voltage-gated sodium channels. They cleverly bypassed the complexities of working with the mammalian NaV channels using a non-inactivation version of the bacterial homologue NaChBac T220, validating its use as a model for studying mechanical modulation of voltage-gated Na channel gating.

By performing a high-quality single channel recording the authors demonstrated that mechanosensitivity affected the channel Po nor single-channel conductance, and importantly, that the effects of pressure on channel gating were reversible. This is particularly appreciated due to the exceptionally low unitary conductance of the channel makes it exceedingly difficult to obtain this type of result.

The authors performed kinetic modelling over the single channel recording at different voltages and pressures, using linear gating schemes that contrasted two extreme situations: mechanosensitivity was a property of the voltage sensing transitions - mechanosensitive activation (MSA) - or, alternatively, it was a feature of the voltage-independent step that opens the channel pore - mechanosensitive opening (MSO). The MSO performed much better than MSA model, suggesting that mechanosensitivity arises from conformational changes at channel regions different than the VSD during the pore opening. The latter gains further support with the experiments showing that pressure modulates the D93A - a mutation that stabilizes the VSD in its resting state - resembles wt channels, but is ineffective on the I228G channel, a channel with altered closure.

Overall, the manuscript presents a nice experimental approach, making use of kinetic analysis in terms that are very accessible to the reader to shed light on a physiologically relevant question.

Reviewer #3 (Public Review):

In this manuscript from Wang et al., the authors use cryo-EM and complementary functional assays to examine nucleosome recruitment and histone H3 engagement by the S. cerevisiae Yta7 chromatin remodeler. The authors investigate H3 tail engagement by determining two high-resolution structures of Yta7 in different conformations - an ADP-bound conformer absent of H3, and another bound to ATPgS, where the H3 tail is bound in the central channel of AAA1. The most striking finding is an unexpected organization of the N-terminal bromodomains (BRDs) in the ADP-bound conformer. This structure shows that the BRDs oligomerize via a novel interaction with a C-terminal bromo-interaction motif (BIM), which together assemble a spiraling assembly above the AAA1 ring. This structural feature is only observed in conformers that are absent of bound H3 peptide, and since this BRD/BIM organization appears to block entry to the central channel of the AAA1 ring, the authors propose that nucleosome binding by these domains induces a rearrangement that exposes the AAA1 pore loops for H3 tail engagement. The authors provide biochemical evidence that the BRD interacts with the H3 tail, and the role of these domains in nucleosome recruitment is further supported by a low-resolution cryo-EM structure showing that the nucleosomes bind to Yta7 via the BRDs. The cryo-EM studies are performed with expertise, which includes advanced processing methodologies to improve the resolution of the flexible regions such as the BRDs. However, further clarification of the mechanistic details would increase the study's impact in the field, particularly regarding the relationship between nucleotide state and Yta7 conformation, since two conformations are observed in the presence of ATPgS. Also, while BRD & BIM binding to the H3 tail is shown in isolation, it remains to be seen how these domains impact H3 tail binding in the context of the assembled hexamer. Lastly, the low resolution of the nucleosome-bound complex complicates concise mechanistic interpretation of nucleosome binding, which is an important aspect of the study. Overall, the work provides a generalized mechanism of nucleosome recruitment and the rearrangements associated with positioning of a histone tail for subsequent nucleosome disassembly, which will be of broad relevance to the chromatin remodeling field, although there remain ambiguities regarding many of the mechanistic details.

While he previously recommended using note cards of the same size, the examples in Goutor (1980) have 3x5" cards for bibliographic notes and 5x7" or larger cards for content notes. (p19, 21)

Is there a reason stated anywhere here for this discrepancy or change? One would ostensibly keep them in different places/sections of one's card index, but does the size difference help to differentiate the two to aid in sorting? Is the larger card intended to hold more long form writing?

Goutor is in Canada, so were 5x7" cards more common or standardized there in the late 1970s and early 80s?

A5 measures 148 × 210 millimeters or 5.83 × 8.27 inches, so is a bit larger than 5x7".

5x7" is a more standard photo size, so was this chosen as the result of storage options from the photography space?

5x7" is scantly available in America in 2022, but only from Hamilco. A few others make cardstock in that size but not specifically as index cards.

Reviewer #3 (Public Review):

Using experimental evolution with a nematode model system in a novel salt environment, Mallard et al. present a very nice experiment testing whether plasticity aligns with genetic variance and whether phenotypic divergence can be predicted from patterns of genetic variance. They find that although plasticity is not in the direction of genetic variance, estimates of selection that predict divergence are concordant with observed divergence. However, direct selection on a trait not included in the analysis is expected to be the underlying cause of phenotypic evolution. I commend the authors on their experiment and the framing of such a conceptually difficult topic.

Strengths:

Comparing a common ancestor to evolved populations to predict evolution has rarely been achieved, and the authors provide a strong test for predicting evolution in a novel environment.

Weaknesses:

Although a valuable dataset, the framing of the paper needs to be more focused on the question of predicting phenotypic evolution and comparing direct versus indirect selection. There are many details that are missing in the methods, which include the biological importance of the traits that are being studied, and how adaptation to the novel environment has occurred. In the discussion, they reveal that a measured trait that hasn't been included in the analyses is likely responsible for the observed patterns and it is unclear why this trait wasn't included in the analyses. In addition, direct versus indirect selection is not formally compared, which makes it difficult to interpret their results.

Reviewer #3 (Public Review):

The flowering heads of species in the Asteracaeae comprise large number of flowers, and this phenotype is thought to contribute to their reproductive success. The Harmer lab has developed sunflower as an experimental model to investigate the contribution of circadian regulation to the processes of reproduction in the Asteraceae, and this paper presents a new addition to this line of research.

The novelty of the article is that it resolves unanswered questions around the processes that underlie coordinated flowering within the disc structure of the floral capitulum. The authors demonstrate a role for circadian clock in the temporal structuring of this process. They identify a free running rhythm in constant darkness of floral anthesis, and this rhythm has several key characteristics of circadian rhythms. The data collected also indicate that the circadian clock might gate the response of anthesis to darkness.

I like the presentation of an external coincidence model for the interaction of light and circadian cues in the floral developmental program of the capitulum. However, I wonder whether this is the only potential explanation. The data in Fig. 4C look like classical entrainment responses. Are the authors sure that they are not just seeing an entrainment process within the capitulum, combined with a masking effect of continuous light upon the rhythmic phenotype? I encourage the authors to retain speculation about the coincidence model within the discussion – it's so important for future work – but perhaps consider alternative interpretations of the data also.

Reviewer #3 (Public Review):

The manuscript by Jelen and colleagues aims at investigating the neuronal circuits underlying aversive and appetitive gustatory Pavlovian conditioning in Drosophila. To this end Jelen and colleagues employ an intuitive novel training device that allows for automatic optogenetic activation of neuron populations of choice upon physical contact of freely walking flies with a food source. Whereas olfactory appetitive or aversive conditioning is well established for Drosophila, learning paradigms using other sensory modalities like acoustic or gustatory stimulation are either not yet well established or cumbersome for the broad community. In this concern the advancement existing optogenetic gustatory learning setups to the automated optogenetic learning device "STROBE" finds a remedy to allow for high throughput experiments on gustatory learning in freely walking and behaving animals.

In the first part of their study Jelen and colleagues employ the experimental setup to induce an aversive memory in the fly to low concentrated sugar in combination with activation of bitter neurons optogenetically recapitulating earlier studies where low concentrated sugar was presented in combination with quinine. However, in contrast to those earlier studies where sugar was presented to the tarsi of the fly and quinine to the proboscis, allowing for differentiated detection of taste modalities in the study by Jelen and colleagues the presentation of both taste modalities are synchronous. Sugar and bitter appear to be sensed simultaneously and bitter neurons are globally activated once the fly gets in contact with the sugar solution. In this scenario it is difficult to understand how the bitter neuron activation does not directly interfere with the sensation of sugar changing the perception of the sugar itself instead of being sensed as a punitive stimulus. The strong aversion of the flies towards the sugar during the training phase may reflect indeed such a change in perception mixed with the learning. Further, during the first 5 - 10 min of the test phase for short-term memory the flies appear to show a stronger preference to the aversively conditioned sugar when compared the control gustatory stimulus. According to the theory during this phase short-term memory should be displayed the strongest and decline over time reaching a nearly complete attenuation after one hour. However, the displayed cumulative average preference indices let assume that for the aversive conditioning the memory recall takes place after about 30 minutes when middle-term memory starts to emerge. In this regard it is further worth noting that after an initial increase of the sugar preference to about 0.25 the preference index of the trained flies remain stable whereas the control flies only reach the same level of attraction to the sugar after 12-15 min and only start increasing their sugar preference after about 20 - 25 min. Compared to the dynamics of the cumulative average preference indices of the appetitive gustatory neuron activation and the artificial activation of dopamine neuron subsets the dynamics of the cumulative average preference after the aversive reinforcement through bitter gustatory neuron activation appears drastically different. This may further indicate competing pathways between sensing and conditioning the bitter taste stimulus as well as a delayed memory recall according to the metabolic need of the animal, as again the strongly delayed dynamics of the cumulative preference index indicates.

In the subsequent part Jelen and colleagues investigate the role of different subsets of dopaminergic neurons in the formation of aversive or appetitive gustatory short- and long-term memory. Similar to olfactory memory, gustatory memory relies on mainly two major sets of dopaminergic neurons that drive aversive and appetitive memory, namely the PPL1 and the PAM cluster that innervate different compartments of the mushroom body where they provide aversive or appetitive input to the conditioned stimuli encoded through the sparse activity of mushroom body Kenyon cells. As a consequence, in the following experiments Jelen and colleagues interfere with the hereinafter layer of memory and silence the mushroom body during conditioning while opto-genetically activating the PAM dopaminergic neurons, conceptionally recapitulating earlier studies that demonstrated the role of the mushroom body in gustatory memory earlier. Analogous to earlier findings for olfactory learning Jelen and colleagues use their intuitive setup to functionally subdivide the dopaminergic neurons in functional subunits with different roles in memory formation. These results strongly demonstrate how conserved the value-giving neuronal circuits are independent from their stimulus modality.

Consequently, extrapolating questions on olfactory memory formation on gustatory learning the authors use the STROBE essay to investigate how different nutrients may affect the formation of a long-term memory. In accordance with the findings on olfactory memory formation Jelen and colleagues find that long-term memory formation depends on readily accessible energy sources.

The study is interesting and rigorously conducted and reveals striking similarities between olfactory and gustatory memory formation. However, it appears that the authors have put large focus on the recapitulation of an already demonstrated mode of action of learning circuits using their new technique and many of the parallels between olfactory and gustatory memory formation appear pertinent as e.g., the need of readily accessible energy sources for long-term memory formation. The need for energy to form a long-term memory should not depend on the stimulus modality you learn but on the cellular mechanisms underlying learning itself. The innovative technique Jelen and colleagues present in their manuscript has such a strong potential that to me as a reader it appears a pity that the study did not exploit the possibilities of their technique to investigate virgin soil instead of walking on beaten tracks.

Reviewer #3 (Public Review):

In this article, Juette et al employed single-molecule FRET, cryo-EM, and Hpg incorporation (in cell translation assays) to compare the mechanisms by which Didemnin B and Ternatin-4 inhibit translation elongation. They found that, while binding to the same pocket of eEF1A and blocking accommodation after GTP hydrolysis, Didemnin B had an irreversible effect on protein synthesis, but Ternatin-4, while still a potent inhibitor, allowed more flexibility in complexes (increased disorder of regions in cryo-EM structures) that allowed increased sampling of on-pathway accommodated states (observed by smFRET), and reversibility of effects on protein synthesis in cultured cells (by Hpg incorporation). This is a straightforward study and the conclusions are well-supported by the data using appropriate techniques. The work will be of impact to the ribosome field, which may use these drugs in other mechanistic studies, and researchers wanting to employ the drugs to combat cancer and other diseases.

Reviewer #3 (Public Review):

The manuscript describes a novel and well-designed psychopharmacological investigation of the mechanisms underpinning reward-induced pain relief in healthy humans. In a within-subjects design, ~28 healthy volunteers participated in three sessions (placebo, dopamine enhancement, opioid blockade) assessing subjective and behavioural effects of winning a temporary pain decrease (compared to no change or losing, i.e. a temporary pain increase). The task combines a simple two-choice gambling task with the capsaicin-heat model of pain to uncover the mechanisms involved in endogenous processing of pain reduction. A number of outcomes are presented. Pain ratings are decreased following heat decrease (from winning in the decision-making task) in all conditions, but more strongly so in conditions where the winning cue has the highest informational value, i.e. after active vs passive choice, under unpredictable vs predictable conditions, and after pharmacological enhancement of dopamine. These and several other interesting findings represent a substantial increase in the field's mechanistic understanding of learning and motivation related to pain and pain relief. The manuscript is very well written, the analyses well-reasoned and the results instructive and intriguing.

Reviewer #3 (Public Review):

Wild et al examined several syngeneic breast cancer cell lines using the novel cell tracing technology they have developed. This identified subpopulations of cells that were differentially represented in the in vitro pool and when grown in vivo. This difference extended to immunodeficient as compared to immunocompetent hosts. Treatment altered the dynamics of clonal persistence, allowing the authors to interrogate pathways expressed in cells in response to treatment with a BET inhibitor or docetaxel, revealing the exciting finding that taxane resistant subclones were dependent on NRF2 and emerged with a collateral sensitivity to l-aspariginase, which was demonstrated in vivo, and further observed in human tumor samples.

Overall, this is an exciting, elegant, and rigorously developed strategy, which gives a new level of insight into the tumor biology. The authors make an important discovery regarding dynamic intratumoral response to systemic therapy.

Reviewer #3 (Public Review):

Nuclear shape and size have long been characterized and can indicate and influence cell fates. The present study starts with a knockdown screen of size and shape, adds some information on lamins known to influence size and shape, proceeds to focus on 'subtle' modulators that are often epigenetic factors, then provides in vitro pulldown and array studies that support histone-lamin interactions, and concludes with further such evidence from one small final cell study. Some concerns temper enthusiasm. I found it important that they restricted analyses "To eliminate hits due to cell death or altered cell-cycle behavior, we excluded any hits with a cell number z-score of less than -2." Some mention of this in the abstract seems important. Secondly, The histone-H3 mutation effects on nuclear morphology in Fig.6 are especially important, but it is unclear whether the histone intensities are uniform or enriched in places with LaminA, nor what happens to LaminA levels or localization.

Reviewer #3 (Public Review):

The authors proposed an antibody catenation strategy by fusing a homodimeric protein (catenator) to the C-terminus of IgG heavy chain and hypothesized that the catenated IgGs would enhance their overall antigen-binding strength (avidity) compared to individual IgGs. The thermodynamic simulations supported the hypothesis and indicated that the fold enhancement in antibody-antigen binding depended on the density of the antigen. The authors tested a catenator candidate, stromal cell-derived factor 1α (SDF-1α), on two purposely weakened antibodies, Trastuzumab(N30A/H91A), a weakened variant of the clinically used anti-HER2 antibody Trastuzumab, and glCV30, the germline version of a neutralizing antibody CV30 against SARS-CoV-2. Measured by a binding assay, the catenator-fused antibodies enhanced the two weak antibody-antigen binding by hundreds and thousands of folds, largely through slowing down the dissociation of the antibody-antigen interaction. Thus, the experimental data supported the catenation strategy and provided proof-of-concept for the enhanced overall antibody-antigen binding strength. Depending on specific applications, an enhanced antibody-antigen binding strength may improve an antibody's diagnostic sensitivity or therapeutic efficacy, thus holding clinical potential.

Reviewer #3 (Public Review):

This paper is of interest to scientists within the field of iNKT cells. The authors conducted scRNA-seq to longitudinally profile activated iNKT cells and generated a transcriptomic atlas of iNKT cells at the activation states. The study suggests that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations and that the adipose iNKT cells undergo blunted activation and display constitutive enrichment of memory like population, plus identifying a conserved cMAF- associated network in NKT10 cells. This study provides some new insights into the NKT biology.

Reviewer #3 (Public Review):

This is a computational modeling study to evaluate the merits (likely success) of different 'suppression' gene drive systems. Gene drives offer a possible simple and low-effort means of suppressing or even extinguishing pest populations. Using CRISPR technology, several gene drive systems have been developed in the last decade for key mosquito vector species. As no gene drive has been approved for release in the wild, efforts to evaluate their likely success are limited to cage trials and modeling, the latter as done here. In contrast to some modeling studies, the effort here is to develop and analyze models that match the gene drive and mosquito biology closely. The models are thus parameterized with values representative of what is known about mosquito biology and of the various gene drive constructs that have been developed for lab studies.

In these models, gene drive success or failure in population suppression largely depends on (i) how well the drive spreads throughout the population, and (ii) whether the population persists because of a type of ongoing spatial 'group selection' in which local pockets invaded by the drive die out and are then repopulated by migrants lacking the drive. Formal evolution of functional resistance is not allowed. The numerical results show striking differences in suppression success with different gene drive constructions, and these differences are likely to be of use when designing drives for actual releases.

The basic group selection outcome that allows population persistence amid a suppression gene drive has been shown before, as cited in the ms. The novelty provided by the present study is to tie the models to the biology of known gene drive constructions. Given the high specificity of the models, the audience for this work is likely to be somewhat narrow, confined to those involved in gene drive design. The work is nonetheless significant in view of the strong potential of gene drives in global public health efforts.

The software used to generate the trials is freely available from one of the authors for anyone wishing to repeat the simulations. There is an extensive supplement of results referenced (but not otherwise included) in the main text.

Reviewer #3 (Public Review):

Zhang et al. examined a novel articular cartilage progenitors NFATc1 expressing cells. Through multiple pulse-chase experiments, they found that NFATc1 expressing cells generated most of the articular chondrocytes, but not chondrocytes in the growth plate primordium. In vitro and in vivo transplantation of NFATc1 expressing progenitors demonstrated that these cells exhibit pluripotency to chondrocytes, osteoblasts, and adipocytes. RNA-seq analysis of NFATc1 expressing progenitors demonstrated that these cells are enriched with articular cartilage stem cell markers such as Prg4. Interestingly, NFATc1 expression in chondrocytes diminished as mice aged, suggesting that NFATc1 expressing progenitors are no longer expressing NFATc1. Through CRISPR-mediated knockdown and conditional deletion in Prrx1-cre cells, authors found that NFATc1 negatively regulated chondrocyte differentiation with its putative binding sites on Col2a1 promoter and intron 1. These data support authors' conclusion that NFATc1 negatively regulates chondrocyte differentiation but it also marks chondrocyte progenitors.

The major strengths of the manuscript are the rigorous approach to examine NFATc1 expressing progenitors, including in vivo pulse-chase experiments, in vitro differentiation and in vivo transplantation studies, and transcriptomic profiling. Authors also use multiple approaches to demonstrate functional role of NFATc1, which is negatively regulating chondrocyte differentiation. All these findings generally support authors conclusions. There are some minor weaknesses, such as discordance between NFATc1 expression and NFATc1 expressing cells on articular cartilage, comparison of NFATc1 to the well-known articular chondrocyte progenitors such as GDF5 expressing progenitors, and lack of analyses of in vivo multipotency of NFATc1 expressing progenitors. Nevertheless, authors' findings will substantially advance the field that have long sought to examine the mechanism of joint development by revealing novel population of progenitors and chondrocyte differentiation mechanism. This could ultimately lead to novel treatment strategies for articular cartilage diseases.

Reviewer #3 (Public Review):

Gajwani et al present an intriguing study which concludes that a key aspect of endothelial TNFa-induced inflammation involves mitochondrial clearance (mitophagy) and secretion of formylated peptides that activate neutrophils. TNFa is shown to not only increase mitophagy but also increase the secretion of mitochondrial contents, which are expected to promote enhanced inflammation. A severe mouse lung inflammation model shows that mice with reduced endothelial PINK1, a major driver of mitochondrial damage-induced mitophagy, demonstrated enhanced survival and reduced neutrophil recruitment.

This manuscript is of interest as it proposes a novel mechanism of cell-cell signaling in inflammation, involving the surprising release of mitochondrial proteins via mitophagy. This is potentially an important advance. However, the results are far from conclusive. In addition to specific technical problems, they provide no evidence that this mechanism operates in vivo.

Conceptual issues.

Fig 2. In general, inflammation in endothelial cells is associated with high glycolysis, not high mitochondrial metabolism. Thus, it is important to address the question, how does TNFa trigger increased mitophagy? Is it preceded by elevated mitochondrial oxidative phosphorylation, reactive oxygen production and mitochondrial damage? Or simply mitochondrial depolarization? Is it a consequence of general upregulation of ROS? General upregulation of autophagy? Figure 3 is relevant to this question but does not answer it.

It is not clear what figures 2C,D add to the paper. Why is the occasional contact of mitochondria and mitolysosomes relevant? The absence of controls or quantification further detract from this figure.

Fig 5. What about other leukocyte populations? Is the effect of PINK1 ECKO specific to neutrophils or were they the only cells examined?

Fig 6. These experiments appear to be compromised by the presence of TNFa or FCCP in the EC conditioned medium, which could act directly on the neutrophils. Additionally, the authors provide no evidence that the effect requires PINK1.

Figure 6 also raises an important question of specificity. If the consequence of mitophagy is the release of mitochondrial content and the activation of neutrophils, wouldn't other cell types that have more mitochondrial content and more mito-phagosomal flux contribute more to neutrophil activation? Perhaps the authors could compare to other cell types and test if endothelial cells are more prone to secrete their mitochondrial content. Time courses would also improve this panel.

Reviewer #3 (Public Review):

Li et al. present cryo-EM structures of the insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R), exploring the functional roles of the disulfide-linked alphaCT regions in ligand binding and receptor activation.

Cryo-EM structures of mutants of IGF1R and IR designed to increase the flexibility between disulfide-linked alphaCT regions revealed conformational states that were distinct from those of the wild-type (WT) receptors. Mutant (P673G4) IGF1R displayed conformations in which two IGF1 molecules were bound, rather than the 1:1 ligand:receptor state observed previously for WT IGF1R. Mutant (3CS) IR displayed asymmetric conformations with four insulin molecules bound, as well as the symmetric T conformation with four insulin molecules bound observed previously for WT IR. In each case, the mutant receptor was shown in cells to be poorly activated by its respective ligand.

This study demonstrates the importance of the disulfide-coupled alphaCT regions in the IR and IGF1R for ligand binding and receptor activation. What is not resolved in this study is whether differences in the alphaCT regions of these two highly related receptors contribute to their disparate active states - asymmetric for IGF1R (and 1:1 IGF1:IGF1R) vs. symmetric (T) for IR (and 4:1 insulin:IR).

Reviewer #3 (Public Review):

Zuber et al. investigated various KOW domains and found that the thermodynamically less stable EcRfaH and VcRfaH domains can switch between an all-alpha and all-beta state. This property has been known so far only for the KOW domain of the E. coli orthologue. For the latter, a very detailed thermodynamic and structural biology study could be performed at residue resolution by combining DSC and CD spectroscopy with very sophisticated NMR methods. The latter revealed the role of hydrogen bonds of the switching KOW domains by analyzing long range scalar couplings along hydrogen bonds. The second elegant approach was to use 15N and 13C CEST experiments to characterize the ensemble of conformations forming the unfolded state. This is a very difficult task to do experimentally. The authors can show experimentally at residue resolution that residues forming the alpha helices in the all-alpha switched form have already some alpha helical content in the U ensemble. Requirements for fold switching proteins from published theoretical approaches could be all experimentally confirmed and were convincingly discussed together with the Gibbs free energy landscape of all relevant conformational states. There is only one minor weakness concerning the interpretation of chemical shift changes of U with increasing urea concentrations (Figure 6B -Figure supplement 2).

Reviewer #3 (Public Review):

Shen et al. investigated the relationship between the diagnosis of cardiovascular disease (CVD) and subsequent diagnosis of psychiatric disorders using national databases and health records over a 30-year period in Sweden. They also investigated the association between the diagnosis of psychiatric disorder and subsequent CVD-related mortality. Comparisons were made between participants diagnosed with CVD and siblings without CVD, and between the CVD participants and random age- and sex-matched controls from the general population.

They show that diagnosis of all types of CVD investigated was associated with increased risk of all types of psychiatric disorders considered, both in comparison to non-CVD siblings and general population controls. They also showed that diagnosis of psychiatric diagnosis subsequent to CVD diagnosis was associated with greater CVD-related mortality.

A key strength of this study is the use of national databases and populations, as it has allowed for sufficiently large numbers for important subgroup analyses investigating specific types of CVD and psychiatric disorders. In addition to disease and disorder subtypes, the authors have investigated many other factors that may be important for understanding these relationships, including time of diagnosis during follow-up, year of diagnosis, age of participant, and various comorbidities. The duration of follow-up is another important strength of this study, as is the use of sibling controls to mitigate the potential confounding effect of genetic and early-life environment.

However, while it is acknowledged as a limitation by authors, the lack of lifestyle data is a notable weakness of the study. The authors allude to causal inference in the abstract and discuss controlling for important confounding factors, but this is somewhat undermined by not being able to account for lifestyle factors, particularly since there are shared biological pathways such as inflammation linked to both CVD and many psychiatric disorders. As such, the associations reported in this study are potentially influenced substantially by unmeasured confounding related to lifestyle factors.

Overall, this is important data, and the conclusions around these findings supporting surveillance of psychiatric disorders in individuals diagnosed with CVD due to its association with increased risk of mortality may be of interest to clinical settings.

Reviewer #3 (Public Review):

This work aims at testing hypotheses derived from the field of behavioral economics (Kahneman's theories), related to subjective value perception in ants foraging for food. The work was conceived to test how ants react to a specific feature which is the segregation or the bundling of food resources. Behavioral economics posits that individuals value more segregated resources than the same amount of resources presented in a bundled way. At the same time, if accessing the segregated resources implies an increase in energetic costs to access them (i.e. longer displacements), then costs would be also perceived as higher in the segregated-resource case than in the bundled-resource case.

Whether ants conform or not to this model is an interesting question, and irrespective of the results obtained, the experiments presented by the authors have been conceived to address this model as the experimental parameters varied refer to resource separation (drops of sucrose solution with different degrees of spacing between them) and to walking distances.

Yet, the manuscript suffers from various serious deficits that preclude being enthusiastic with respect to its present form. Various problems are listed below, which reduce the quality of this work. Hopefully, the authors can amend some of these problems to reach a more consistent version.

1) The inconsistent and unjustified "wrapping" with a "wanting vs liking" framework<br /> While it is unquestionable that the question raised by the authors revolves around behavioral-economic hypotheses on value perception and is fully addressed by the experiments performed, the "extra wrapping" of the "wanting/liking" framework added, probably to make the manuscript more attractive, is unjustified and excessively speculative. The use of a "wanting vs liking" interpretation framework is inappropriate as neither the experiments were conceived to address this topic, nor the results allow any robust conclusion on this point. These concepts originate in neuroscience analyses of neural-circuit activation in the mammalian brain upon situations that allow distinguishing several components related to reward: 1) the hedonic effect of pleasure itself (liking); 2) motivation to obtain the reward (wanting or incentive salience); and 3) and reward-related learning(1-3). These components refer to different identified neural circuits and brain areas as wanting for reward is generated by a large and distributed dopaminergic brain system including the frontal cortex, while liking is generated by a smaller set of hedonic hot spots within limbic circuitry and which are not dopamine-dependent.

Clearly, the use of the wanting vs liking terminology requires accuracy and appropriate studies to support it. This is not the case in the present manuscript which was not conceived to tackle this issue. Moreover, inconsistent testing procedures (see below point 3) undermine the use and interpretation of choice data as wanting. The authors have no proof of the involvement of wanting vs. liking systems in their design and even more, cannot disentangle between these components based on their behavioral data. Considering that pheromone deposits after food experience express "liking" can be questioned as it does not dissociate between individual liking and social information transfer (the liking and wanting systems are individually based systems). Moreover, the assignment of a choice in a binary-choice test to a wanting system is also questionable as the experiments cannot disentangle between the eventual individual wanting and the reward-related learning as animals are making choices based on odorant cues they have learned during their previous foraging bouts. In the absence of neurobiological data, the hypotheses of wanting vs. liking remain on a shaky, highly speculative ground.

Thus, the whole "wanting vs liking interpretation" (which attains alarming speculative levels in the Discussion section) should be omitted entirely from the manuscript if the authors want to provide a solid convincing framework articulated exclusively around the bundling vs. the segregation effects, which is precisely what their experiments tested. The rest is speculation in the absence of analyses supporting the wanting vs liking dissociation. An example of the kind of analysis necessary to go in this direction is provided by a recent work in which a dopamine-based wanting system was shown in honey bees(4), a work that the authors did not consider. We are clearly far from this kind of analysis in the present manuscript. As the authors wrote, "the present study is the first to examine bundling vs. segregation in an animal (line 99)", yet not liking vs. wanting.

2) Some experimental assumptions are not substantiated by data<br /> The experimental procedure relies on separating or aggregating reward (drops of sucrose solution) and determining the impact of this variation on pheromone deposition while returning to the nest and subsequent choice in a dual test situation in which two of the three treatments designed - distinguished by the odorant experienced en route to reward - were presented. While the "Segregated All Treatment" (Fig. 2A) managed to space the 0.2 µl reward drops by significant 25-cm segments, thus enhancing potentially both reward appreciation (segregated food drops) and cost appreciation (successive segments to be negotiated), the "Segregate Reward Treatment" (Fig. 2B) raises doubts about its validity.<br /> In this case, three drops were offered at the end of three consecutive 25-cm segments, with the assumption that drops spaced by 5 mm should be perceived as being segregated (two of 0.2 µl and 1 ad libitum). Yet, there is no proof - at least in the manuscript - that spacing two food drops by 0.5 mm induces a segregated perception in ants. The first experience with the first drop may induce both sensitization and a local search that may last until the very close next drop is detected so that for the ant, these drops would be perceived as belonging to the same resource rather than being perceived as segregated resources. The same applies to the vicinity between the 0.2 µl drop and the ad libitum drop.<br /> This raises the question of the real volume of the ad libitum drop, which is not mentioned (it is just described as beings "large"; line 205). One could argue that if drops separated by 5 mm were bound together, the results would be similar to those of the "Bundled Treatment" (Fig. 2C). Strictly speaking, this is not necessarily true if the volume of the large drop was known. If this were the case, the Bundled Treatment offered a volume that was 0.4 µl smaller than the total food provided in the "Segregate Reward Treatment".<br /> Overall, further controls are needed to support the assumptions of the different treatments chosen.

3) Unclear design in the testing procedures<br /> The authors did not specify in the methods if a reward was provided in the tests in which a Y maze was presented to the ants having experienced a succession of short and long segments. This information was provided later, in the Results section (line 309) and, as expected, no reward was provided during the tests, thus raising the question of the necessity of the three consecutive tests, with no refreshment trials in between. This procedure is puzzling because it induces extinction of the odor-length association - as verified by the authors (see lines 306-309) - and makes the design questionable. Only the results of the very first test should be kept and analyzed in the manuscript.<br /> The same remark applies to the three tests performed after comparing the experimental treatments, which - one discovers only in the Results Section - were also performed in the absence of refreshment trials. In fact, the absence of coherence in the results of these tests (e.g. lines 328-332) could be precisely due to a change of strategy between the tests following the absence of reward in the first test. This underlines the necessity of focusing exclusively on the first test and dismissing the data of the 2nd and 3rd tests in which performance may have been affected by extinction and strategy change. This again shows why speaking about "wanting" in this inconsistent framework makes no sense at all.

1 Berridge, K. C. & Robinson, T. E. Am Psychol 71, 670-679. (2016).<br /> 2 Berridge, K. C. & Kringelbach, M. L. Neuron 86, 646-664. (2015).<br /> 3 Berridge, K. C. & Kringelbach, M. L. Curr Opin Neurobiol 23, 294-303. (2013).<br /> 4 Huang, J. et al. Science 376, 508-512. (2022).

Reviewer #3 (Public Review):

This paper provides a novel framework for understanding prediction-based learning rules that are potentially employed by the hippocampus to optimize behavior. Specifically, the authors examined how a cognitive map containing predictive information (termed the successor representation) is computed in the hippocampus with spike-timing-dependent synaptic plasticity (STDP).

Strengths:<br /> By using an ecologically plausible computational model that is embedded with important biological characteristics, the authors propose a novel framework that demonstrates a set of computational principles employed by the hippocampus to achieve successful predictive learning. The paper clearly and thoroughly explains different components of the model with concrete examples and illustrations. Analytical solutions are also provided in addition to narratives to help readers understand the model setup as well as its relevance and connection to biological studies. Among the set of biologically realistic computational dynamics achieved by the modeling framework, the proposed model can elegantly account for both exponential and hyperbolic discounting by demonstrating that exponential discounting is utilized while the animals travel through space, whereas hyperbolic discounting is capitalized while the animals travel through time. Additionally, this paper discusses the model findings in the context of experimental and theoretical work which help readers understand how the proposed framework can be utilized in future work to guide investigations on predictive learning both empirically and computationally.

The proposed model makes connections to other theoretical frameworks

Weaknesses:<br /> While the framework proposed in this paper is potentially powerful in capturing different aspects of hippocampus-based predictive learning, the links between the model results and experimental findings are not sufficiently demonstrated. There are several biological concepts that are discussed in the context of the model. It is, however, unclear if the implementations of these concepts within the model capture the same underlying principles that happen in nature. For example, there is rich literature on hippocampal replays including their heterogeneity across contexts and species. The paper does not provide sufficient information regarding the specific types of replays or the specific aspects of replay dynamics that are observed in the model.

Reviewer #3 (Public Review):

The Nkx3.2 transcription factor is both necessary and sufficient for jaw joint development. In this paper the authors use comparative genomics to identify a conserved Nkx3.2 enhancer that they call a jaw regulatory sequence (JRS). With transgenic zebrafish they show that JRSs from multiple species of gnathostomes, including humans, can drive reporter expression in the larval jaw joint. Transgene expression in each case includes the joint interzone as well as adjacent cartilage and perichondrium. With CRISPR-targeted deletion they show requirements for the zebrafish JRS in larval jaw joint development. Analyses of additional gnathostome genomic sequence near Nkx3.2 suggest that jawless hagfish lack JRS-like sequences, consistent with an important role for this enhancer and Nkx3.2 in the evolutionary origin of jaws. Despite a growing recognition of the importance of enhancers in development and disease, very few have been shown to have essential functions in vivo.

The first part of the paper is straightforward, with clear evidence that the JRSs tested act as functional enhancers, since they drive reporter expression in very specific and similar subsets of skeletal cells. The images are high quality and the cellular resolution is impressive. There are also several moderate/major weaknesses that should be addressed. The experiments showing larval joint defects following JRS deletion in zebrafish are less clear, since the phenotypes are subtle in mutants and later recover to resemble wild type siblings. One major weakness is that there is no evidence that JRS deletion alters Nkx3.2 expression at any stage. In the absence of these data, it is possible that the JRS acts on other nearby genes. Another weakness is a lack of quantification of variation in this phenotype in the JRS mutants. Though the images of larval joint defects in the JRS mutants are clear, they are single examples. Finally, the apparent absence of an obvious JRS in the region adjacent to Nkx3.2 in hagfish is used to argue that it arose together with the origin of jaws during vertebrate evolution. Alternatively, this may instead reflect a unique loss of this element in hagfish.

Reviewer #3 (Public Review):

In this manuscript, the authors present data supporting FOXP2 as an oncogene in PCa. They show that FOXP2 is overexpressed in PCa patient tissue and is necessary and sufficient for PCa transformation/tumorigenesis depending on the model system. Over-expression and knock-down of FOXP2 lead to an increase/decrease in MET/PI3K/AKT transcripts and signaling and sensitizes cells to PI3K/AKT inhibition.

Key strengths of the paper include multiple endpoints and model systems, an over-expression and knock-down approach to address sufficiency and necessity, a new mouse knock-in model, analysis of primary PCa patient tumors, and benchmarking finding against publicly available data. The central discovery that FOXP2 is an oncogene in PCa will be of interest to the field.

However, there are several critically unanswered questions.

• No data are presented for how FOXP2 regulates MET signaling. ChIP would easily address if it is direct regulation of MET and analysis of FOXP2 ChIP-seq could provide insights.<br /> • Beyond the 2 fusions in the 100 PCa patient cohort it is unclear how FOXP2 is overexpressed in PCa. In the discussion and in FS5 some data are presented indicating amplification and CNAs, however, these are not directly linked to FOXP2 expression.<br /> • There are some hints that full-length FOXP2 and the FOXP2-CPED1 function differently. In SF2E the size/number of colonies between full-length FOXP2 and fusion are different. If the assay was run for the same length of time, then it indicates different biologies of the over-expressed FOXP2 and FOXP2-CPED1 fusion. Additionally, in F3E the sensitization is different depending on the transgene.<br /> • The relationship between FOXP2 and AR is not explored, which is important given 1) the critical role of the AR in PCa; and 2) the existing relationship between the AR and FOXP2 and other FOX gene members.

Reviewer #3 (Public Review):

Helsell et al. uses atomistic molecular dynamics simulations to characterize the structural dynamics of the M2 protein together with continuum elastic models to evaluate the energetic cost of the protein-induced bilayer deformations. Using unbiased simulations (without constraints on the protein) they show that the M2 structure is dynamic and that the AH helices are mobile (though they tend to retain their secondary structure), in agreement with experimental observations. Then, using simulations in which the peptide backbone was restrained to the starting structure, they were able to quantitatively characterize the protein-induced bilayer deformations as well as the acyl chain dynamics.

Both the atomistic simulations and the continuum-based determinations of the bilayer deformation energies are of high quality. The authors are careful to note that their unbiased simulations do not reach equilibrium, and the authors' conclusions are well supported by their results, though some issues need to be clarified.

1. P. 7: Choice of lipid composition: POPC:POPG:Cholesterol 0.56:0.14:0.3. This lipid composition (or POPC:POPG 0.8:0.2) has been used in a number of experimental studies that the authors use as reference. It differs, however, substantially from the lipid composition of the influenza membrane (Gerl et al., J Cell Biol, 2012; Ivanova et al., ACS Infect Dis, 2015), which is enriched in cholesterol, has a 2:1 ratio of phosphatidylethanolamine to phosphatidylcholine, and almost no PG. The choice of lipid composition is unlikely to impact the authors' major conclusions, but it should be discussed briefly. As noted by Ivanova et al., the lipids of the influenza membrane are enriched in fusogenic lipids. How will that impact the authors results.

2. The definition of the lipid tilt needs to be revisited. On P. 13 (in the Pdf received for review, the authors do not provide page numbers), the tilt is defined/approximated as "the angle between the presumed membrane normal (aligned with the Z axis of the box) and the vector pointing from each phospholipid's phosphate to the midpoint between the last carbon atoms of the lipid tails." This (equating the normal to the interface with the Z axis of the simulation box) may be an acceptable approximation for the lower leaflet, which is approximately flat, but probably not for the upper leaflet where the interface is curved in the vicinity of the protein. The authors should, at least, discuss the implications of their approximation in terms of their conclusion that there is little lipid tilt in the upper leaflet.

3. P. 14, last paragraph, Figure 5 and 6: The snapshots in Figure 5 are too small to see what the authors refer to when they write "tilt their lipid tails to wrap around the helices." The authors should consider citing the work of H W. Huang, e.g., Huang et al. (PRL, 2004), who introduced the notion of curvature stress induced by antimicrobial peptides, a concept similar to what the present authors propose.

4. P. 17-18, Figure 7: The authors introduce the bilayer midplane, which becomes important for the determination of the deformation energy in the (unnumbered) equation on P. 17, but do not specify how it is determined. This is a non-trivial undertaking, but critical for the evaluation of the deformation energy; please add the necessary details.

5. P. 18-19, Figure 8: The comparison of the MD and continuum membrane deformations is very informative, but the authors should discuss the implications of the increased symmetry further in terms of the estimated deformation energies. (I do not believe the authors really mean that they predicted the energies, they estimated/approximated them.)

Reviewer #3 (Public Review):

This communication describes the molecular design of antinociceptive peptides with the aim to improve the peptide affinity and blocking activity towards Nav1.7. The authors performed in vivo experimental assays of such molecular designed peptides to validate them. The methods incorporate state-of-the-art techniques, and the results are clear and of great quality.

Strengths: Many synthetic variants were generated to accomplish the best antinociceptive peptides including its in vitro and in vivo assays.

Weaknesses: Some of the reasonings for creating some of the in-silico peptide variants were not clear at all even though they were designed based on ligand-receptor models.

Reviewer #3 (Public Review):

The manuscript by Pauli and Chen et al. is a beautiful and much-needed study that characterizes the cell types that make up these nuclei.

The major strength of this study is it combines a profiling approach to identify subtypes (single-cell sequencing) with microscopy that reveals where the cells reside within the PBN (multiplex FISH) and a genetic approach to visualize the projection pattern of these cell types throughout the brain (genetic labeling with Cre-lines). The result is important new insight about the cell types in this structure. Highlights include (1) the identification of two inhibitory neuron subtypes and 19 excitatory cell types, (2) the discovery of two output streams, (3) insight about the ontogeny of cell types and transcription factors that may be involved in fate specification/determination, (4) clarification of which neuropeptides are expressed/not expressed in which cell types, (5) and the identification of suitable genetic markers to target many of the cell types. Importantly, all the data are available for researchers to mine their genes of interest. This information is a major advance in the field.

Reviewer #3 (Public Review):

Grande and colleagues used high-resolution 7 Tesla fMRI to investigate the topographical distribution of functional connectivity, and sensitivity to scene and object stimuli, across subregions of the entorhinal-hippocampal circuitry. They report scene-specific activations in functionally connected voxels of the posterior-medial entorhinal cortex (EC) and the distal subiculum. In contrast, no specific preference for object stimuli was detected.

The authors managed to characterize functional connectivity patterns in the entorhinal-hippocampal circuitry to an impressive level of detail. The division of the subiculum and CA1 region in 5 and 3 segments, respectively, extends the very sparse body of literature on the organization of connectivity across the transversal axis of the hippocampal formation in humans. Notably, the authors replicate findings by Maass et al. 2015 of a dissociation of functional connectivity preference between voxels in anterior-lateral and posterior-medial EC with voxels in proximal and distal subiculum (informed by the well-described connectivity architecture of the hippocampal formation in rodents; see e.g. Witter et al. 2000). In addition, they report the novel finding of specific functional connectivity preference of voxels in anterior-medial and anterior-lateral EC seed regions with distal CA1 (again consistent with previous findings in rodents, as well as diffusion MRI findings in humans; Syversen et al. 2021).

After having established 4 different clusters of entorhinal voxels based on functional connectivity to 4 'source' regions, the authors report specific scene sensitivity in a posterior-medial cluster, partially replicating previous studies. In addition, they describe another novel finding of specific scene sensitivity in the two most distal segments of the subiculum.

I agree with the authors that understanding the connectivity of hippocampal subregions and their functional preferences is an important goal with relevance for many research disciplines, such as on episodic memory, spatial navigation, or Alzheimer's disease.

While the paper makes a number of important contributions to help understand entorhinal-hippocampal function and connectivity, I feel that the premise and research question is somewhat unclear and potentially misleading. Most notably, I don't think the conclusions pertaining to processing of object (item) information are supported by the results. An absence of a difference does not provide evidence for similar levels of processing of object and scene information.

Reviewer #3 (Public Review):

This manuscript, "Variation in Ubiquitin System Genes Creates Substrate-Specific Effects on Proteasomal Protein Degradation" studies the genetic basis of differences in protein degradation. The authors do so by screening natural genetic variation in two yeast strains, finding several genes and often several variants within each gene that can affect protein degradation efficiency by the Ubiquitin-Proteasome system (UPS). Many of these variants have "substrate-specific effects" meaning they only affect the degradation of specific proteins (those with specific degrons). Also, many variants located within the same genes have conflicting effects, some of which are larger than others and can mask others. Overall, this study reveals a complex genetic basis for protein degradation.

Strengths: Revealing the genetic basis for any complex trait, such as protein degradation, is a major goal of biology. The results of this paper make a significant step towards the goal of mapping the genes and variants involved in this specific trait. Fine mapping methods are used to home in on the specific variants involved and to measure their effects. This is very nicely done and provides a detailed view of the genetic basis of protein degradation. Further, the GFP/RFP system used to quantify the efficiency of the protein degradation system is a very elegant system. Also, the completeness of the analysis, meaning that all 20 N-degrons were studied, is impressive and leads to very detailed findings. It is interesting that some genetic variants have larger and opposite effects on the degradation of different N-degrons.

Weaknesses: Some of the results discussed in this paper are not surprising. For example, the finding that both large effect and small effect genetic variants contribute to this complex trait is not at all surprising. This is true of many complex traits. The discussion of human disease is also a bit extensive given this study was performed on yeast. It might be more productive to use these findings to understand the UPS better on a mechanistic level. Why does the same genetic variant have opposite effects on the degradation of different degrons, even in cases where those degrons are of the same type?

Overall, this manuscript excels at mapping the genetic basis of variation in the UPS system. It demonstrates a very complex mapping from genotype to phenotype that begs for further mechanistic explanation. These results are important to the UPS field because they may help researchers interrogate this highly conserved essential system. The manuscript is weaker when it comes to the broader conclusions drawn about the relative importance of large vs. small effects variants on complex traits, the amount of heritability explained, and the effects of genetic variation on protein abundance vs transcript abundance. Though in the case of protein vs transcript, I feel the cursory examination of the trends is perhaps at an appropriate level for the study, as it is mainly meant to show these things differ rather than to show exactly how and why they differ.

Reviewer #3 (Public Review):

This paper reports the humoral (neutralizing antibody concentrations from serum) and cellular (cytopathic effect on Vero cells) immune responses of volunteers enrolled in a randomized clinical trial for the CoronaVac® SARS-CoV-2 vaccine. The findings are useful and, through solid reporting, discussion, and statistical analyses, provide context for the efficaciousness of the 0-14 day and 0-28 day dosing schedules of CoronaVac®. The results show that these two dosing schedules are similar across most metrics. Furthermore, the findings pave the way for key future work, including reporting and understanding the clinically relevant protective effects, and how long they last, of CoronaVac® against the emerging variants/subvariants.

Reviewer #3 (Public Review):

Meissner et al. employ stochastic Gal4 labeling with MCFO to ease the identification candidate lines for split-Gal4 line generation to genetically target neurons of interest identified in EM traces. Data basis for the approach is a novel resource of 74k MCFO images aligned to the JRC18 template allowing the matching between EM and LM traces of single neurons. The resource is released in combination with data processing and query tools. In addition, an open web-based data portal to the released data collection and data mining tools is made available. This will allow broad access to this novel resource with the potential to create high impact in the community.

Strength:

The possibility to bridge between EM neuron traces and expression patterns in LM images is a key method to achieve and accelerate genetic access to individual neurons. The proposed resource and tools contribute to this effort and provide open and easy access to it. This also includes the possibility to upload and analyze own data using the provided infrastructure, which is a great asset.

Weaknesses:

While the generation and analysis of the MCFO data is described in great detail and the overall technical approach seems feasible, the description of the technical part and its evaluation are lacking important implementation details and scientific rigor. Although this is primarily a life science paper introducing a new data resource it's the mining capability making this resource really valuable. The provided evaluation of the image mining capabilities however is currently insufficient to support the very general claims on effectivity and speed of the method.

Reviewer #3 (Public Review):

Noel et al provide a neural representational account of three brain areas in a virtual, visual navigation task paradigm especially designed to achieve a closed action-perception loop closely resembling natural behaviour. The authors recorded hundreds of neurons from three monkeys while the animals were engaged in the task where latent cognitive variables like distance travelled and distance to target continuously changed. The authors build on their previous work where they robustly characterized animal behaviour on this task paradigm. Here, they aim to find neural codes of dynamic, latent variables and report a mixed and heterogeneous profile of task variable coding distributed across the two brain areas in the parietal cortex (MSTd and area 7a) and one in the prefrontal cortex (dlPFC).

Major strength: Multi-area recording and the close-loop behavioural paradigm are major strengths of this study. The robust model-based analysis of neural data strengthens the paper even more. The correlation of coupling between MSTd and dlPFC and behaviour, albeit in a coarse time scale (of sessions), is particularly interesting and makes the paper strong by quantitatively relating behaviour to neural activity.

Major weakness: The paper mainly gives a long list of what task variables the three brain areas code for along with measures of connectivity between areas. Although this is a valuable contribution to the field, the study is not designed to test predictions of specific computational hypotheses. Towards the end of the paper, the authors bring up the two alternate mechanisms: vector-coding vs distance-coding, but only as a speculation. These two hypotheses could have been developed further at the outset to make specific predictions for neural dynamics and subsequently be tested in their data. This will likely lead to richer findings going beyond representations of task variables. Nevertheless, the findings presented in the paper are surely novel and exciting.

Impact: The main impact of the paper is neurophysiology under a novel, naturalistic behavioral paradigm. The data, both behavioral and neurophysiological, is rich and has potential to test predictions of more fine-grained computational hypotheses. However, the observation that MSTd codes for latent variables is not as surprising as the authors claim. Given the recent observations of heterogeneous variables represented in brain areas traditionally thought to be highly specific (e.g. locomotion variables in V1, mixed coding in EC etc.), it is not surprising to find latent variables in a 'traditionally' sensory area, especially in a continual behavioral paradigm where many variables are changing and are correlated.

Based on their previous work and this work, the authors mention multiple times the task strategy and its embodied nature. While the authors conclusively show the involvement of eye movement in solving the task, it is difficult to imagine a concrete definition of an embodied task strategy without clear alternate hypotheses. How would the animals behave if their eye movements were prevented? Worse performance (like humans did in their previous paper) or unable to perform (akin to a bird unable to fly without wings) or a different strategy? What should we predict based on the neural observation reported here? The impact of this paper would be greater if the authors bring up these questions and provide some speculations rooted in neurophysiological observations.

Reviewer #3 (Public Review):

This manuscript should be of broad interest to readers not only in the field of gap junction (GJ) mediated cell-to-cell communication but also to scientists and clinicians working on the function of mitochondria and metabolism. Their data elucidates a new function of Cx43 in regulating the energy (ATP) generation of mitochondria, e.g., under oxidative stress.

The canonical function of gap junctions is in direct cell-to-cell communication by forming plasma membrane traversing channels that electrically and chemically connect the cytoplasms of adjacent cells. These channels are assembled from connexin proteins, connexin 43 (Cx43). However, more recently new, non-canonical cellular locations and functions of Cx43 have been discovered, e.g. mitochondrial Cx43 (mtCx43). However, very little is known about where Cx43 transported into mitochondria is derived from, how Cx43 is transported into mitochondria, where it is located in mitochondria, in which form Cx43 is present in mitochondria, (polypeptides, hemi-channels (HCs), complete GJ channels), and what the function of mtCx43 is. The authors addressed the latter question. The authors provide convincing evidence that mtCx43 modulates mitochondrial homeostasis and function in bone osteocytes under oxidative stress. Together, their study suggests that mtCx43 hemi-channels regulate mitochondrial ATP generation by mediating K+, H+, and ATP transfer across the mitochondrial inner membrane by directly interacting with mitochondrial ATP synthase (ATP5J2), leading to an enhanced protection of osteocytes against oxidative insult. These findings provide important information of a role of Cx43 functioning directly in mitochondria and not at the canonical location in the plasma membrane. While most of the functional assays presented in Figures 2-8 appear solid, the mitochondrial localization of Cx43, its translocation into mitochondria under oxidative stress, and its configuration as hemi-channels (Figure 1) is less convincing. I have five general comments that should be addressed:

1) This study was performed in MLO-Y4 osteocyte cells. Is the H2O2 induced increase of mitochondrial Cx43 MLO-Y4 cell type or osteocyte specific, or is Cx43 playing a more general role in mitochondrial function, e.g. under oxidative stress? Osteoblasts such as MC3T3-E1 and MG63, and many other cell types endogenously express Cx43, and oxidative stress is a general physiological stressor, not only for osteocytes and bone cells. Attending to this question would address the generality of the findings for mitochondrial function.

2) The images of MLO-Y4 cells (Figure 1A) and the primary osteocytes isolated from Csf-1+/- and control mice (Figure 8) do not show visible gap junctions. I guess this is due to the fact that slides were stained with the Cx43(E2) antibody. I feel, staining of these cells in addition with the Cx43(CT) antibody would be helpful to get a better understanding on the distribution of Cx43 in gap junctions and undocked/un-oligomerized Cx43 in these cells.

3) The images of cells presented in Figure 1A are quite fussy. No mitochondria are visible, and the Cx43 staining is hazy and does not localize to any subcellular structures. Also, it is not clear if the higher resolution image presented in Figure 1C actually represents a mitochondrion. A good DIC image, or co-staining with another mitochondrial marker such as MitoTracker (as shown in Figure 4-S1) would make the localization and translocation of Cx43 into mitochondria upon oxidative stress more convincing. This is especially important as the translocation, although statistically significant, increases only by about 10% or less (Figure 1B). Such a small difference (also represented in the Western analyses presented in Figure 1D) could easily be artefactual, depending on how the correlation coefficient was generated. Of note in this respect is that control cells in Figure 1A appear larger (compare the size of the nuclei) and are spread out more than the H2O2 treated cells. Better, more clear images would make the mitochondrial localization/translocation more convincing.

4) How pure are the mitochondria that were probed for Cx43 by Western shown in Figure 1D? The preparation method described is relatively simple, collecting the 10,000xg supernatant (here 9,000xg supernatant) as mitochondrial fraction. Is it possible that the Cx43 signal, at least in part, is derived from other, contaminating membranes, such as PM, Golgi, or ER? Testing the mitochondrial preparation by Western with marker proteins specific for these compartments would strengthen the author's results.

5) The authors rely on previous studies to postulate that Cx43 in mitochondria forms hemichannels in their system, is localized in the inner membrane, and is oriented with the Cx43 C-termini facing the inter-membrane space (as schemed in Figure 8C). The authors use lucifer yellow (LY) dye transfer and carbenoxolone, but both are not hemi-channel specific probes. They are transferred by, and block GJ channels as well. Experiments, using hemi-channel specific probes would be more convincing. This is important, as the information cited is based on only two references (Boengler et al., 2009; Miro-Casas et al., 2009), and it still is highly unclear how a membrane protein that is co-translationally inserted into the ER membrane, then traffics through the Golgi to be inserted into the plasma membrane is actually imported into mitochondria and in which state (monomeric, hexameric). Why the Cx43(CT) specific antibody traverses the outer mitochondrial membrane and reaches the Cx43CT while the Cx43(E2) specific antibody is not described and clear either. Where are these mitochondria permeabilized with Triton X-100 as described in M&M?

Reviewer #3 (Public Review):

With resting-state fMRI data, recent work has mapped the organisation of the cortex along a continuous gradient, and regions that share similar patterns of functional connectivity are located at similar points on the gradient (Margulies et al., 2016). In the current study, the authors investigate how this dimension of connectivity changes during conceptual retrieval with different levels of semantic association strength. Specifically, they perform gradient analysis on task-fMRI informational connectivity data and reveal a similar principal gradient to the previous study, which captures the separation of heteromodal memory regions from the unimodal cortex. More importantly, by comparing the gradient generated with data from different experimental conditions (i.e., strong vs. weak association), the authors find the separation of the regions at the two ends of the gradient can be regulated by the association strength, with more separation for stronger association. They also examine the relationships between the gradient values and dimensionality and brain-semantic alignment measures, to explore the nature of this shifting gradient as well as the corresponding brain areas.

Strengths:<br /> 1. The aim of this study is clear and the relevant background literature is covered at an appropriate level of detail. With the cortical gradient analysis approach, this study has the potential to make a contribution to the understanding of the topographical neural basis of semantics in a fine-grained manner.<br /> 2. The methodology in the current study is novel. This study validates the feasibility of performing gradient analysis on task-fMRI data, which is enlightening for future research. Using the number of PCs generated by PCA as a measure of dimensionality is also an interesting approach.<br /> 3. The authors have conducted multiple control analyses, which tested the validity of their results. Specifically, a control task without engaging semantic processing was built in the experimental design (i.e., the chevron task), and the authors conducted multiple parallel control analyses with the data from this control task as a comparison with their main results. Other control analyses were also performed to validate the robustness of their methodological choices. For example, varied thresholds were used during the calculation of dimensionality and similar results were obtained.

Weaknesses:<br /> 1. As a major manipulation in the experiment, it is not very clear how the authors split/define their stimuli into strong and weak semantic association conditions. If I understood correctly, word2vec was used to measure the association strength in each pair of words. Then the authors grouped the top 1/3 association strength trials as a "strong association" condition and the bottom 1/3 as "weak association" (Line 689), and all analyses comparing the effect of "strong vs. weak association" were conducted with data from these two subsets of stimuli. However, in multiple places, the authors indicate the association strength of their stimuli ranges from completely unrelated to weakly related to highly related (Line 612, Line 147, Line 690, and the examples in Figure 1B). This makes me wonder if the trials with bottom 1/3 association strength (i.e., those were used in the current study) are actually "unrelated/no association" trials (more like a baseline condition), instead of "weak association" trials as the authors claimed. These two situations could be different regarding how they engage semantic knowledge and control processing. Besides, I am very interested in what will the authors find if they compare all three conditions (i.e., unrelated vs. weak association vs. strong association).<br /> 2. Following the previous point, because the comparison between weak vs. strong association conditions is the key of the current study, I feel it might be better to introduce more about the stimuli in these two conditions. Specifically, the authors only suggested the word pairs fell in these two conditions varied in their association strength, but how about other psycholinguistic properties that could potentially confound their manipulation? For example, words with higher frequency and concreteness may engage more automatic/richer long-term semantic information and words with lower frequency and concreteness need more semantic control. I feel there may be a possibility that the effect of semantic association was partly driven by the differences in these measures in different conditions.<br /> 3. The dimensionality analysis in the current study is novel and interesting. In this section, the authors linked decreasing dimensionality with more abstract and less variable representations. However, most results here were built based on the comparison between the dimensionality effects for strong and weak association conditions. I wonder if these conclusions can be generalised to results within each condition and across different regions (i.e., regions having lower dimensionality are doing more abstract and cross-modal processing). If so, I am curious why the ATL (a semantic "hub") in Figure 3A has higher dimensionality than the sensory-motor cortices (quite experiences related) and AG (another semantic "hub").<br /> 4. I am not sure about the meaning/representational content underlying the semantic similarity matrix in the semantic-brain alignment analysis. According to the authors, this matrix was built based on the correlation of participants' ratings of associative strength (0, no link; 1~4, weak to strong) across trials. The authors indicate that this matrix reflects the global similarity of semantic knowledge between participants (Line 403). However, even though two participants share very similar ratings of association strength across trials, they could still interpret the meaning/knowledge underlying the associations very differently. For example, one participant may interpret the link between "man" and "car" as a man owns a car but another participant may interpret it as a man is hit by a car, although both associations could be rated as strong for this trial. This situation may be even more obvious for those pairs with weak association. Therefore, I am not confident this is a measure of similarity of semantic knowledge.

Reviewer #3 (Public Review):

Higher BMI in childhood is correlated with behavioral problems (e.g. depression and ADHD) and some studies have shown that this relationship may be causal using Mendelian Randomization (MR). However, traditional MR is susceptible to bias due to population stratification, assortative mating, and indirect effects (dynastic effects). To address this issue, Hughes et al. use within-family MR, which should be immune to the above-listed problems. They were unable to find a causal relationship between children's BMI and depression, anxiety, or ADHD. They do, however, report a causal effect of mother's BMI on depression in their children. They conclude that the causal effect of children's BMI on behavioral phenotypes such as depression and anxiety, if present, is very small, and may have been overestimated in previous studies. The analyses have been carried out carefully in a large sample and the paper is presented clearly. Overall, their assertions are justified but given that the conclusions mostly rest on an absence of an effect, I would like to see more discussion on statistical power.

1) The authors show that the estimates of within-family MR are imprecise. It would be helpful to know how much power they have for estimating effect sizes reported previously given their sample size.

2) They used the correlation between PGS and BMI to support the assertion that the former is a strong instrument. Were the reported correlations calculated across all individuals? Since we know that stratification, assortative mating, and indirect effects can inflate these correlations, perhaps a more unbiased estimate would be the proportion of childrens' BMI variance explained by their PGS conditioned on the parents' PGS. This should also be the estimate used in power calculations.

3) In testing the association of mothers' and fathers' BMI with children's symptoms, the authors used a multivariable linear regression conditioning on the child's own BMI. Was the other parent's BMI (either by itself or using the polygenic score) included as a covariate in the multivariable and MR models? This was not entirely clear from the text or from Fig. 2. I suspect that if there were assortative mating on BMI in the parent's generation, the effect of any one parent's BMI on the child's symptoms might be inflated unless the other parent's BMI was included as a covariate (assuming both mother's and father's BMI affect the child's symptoms).

4) They report no evidence of cross-trait assortative mating in the parents generation. The power to detect cross-trait assortative mating in the parents' generation using PGS would depend on the actual strength of assortative mating and the respective proportions of trait variance explained by PGS. Could the authors provide an estimate of the power for this test in their sample?

5) Are the actual phenotypes (BMI, depression or ADHD) correlated between the parents? If so, would this not suffice as evidence of cross-trait assortative mating? It is known that the genetic correlation between parents as a result of assortative mating is a function of the correlation in their phenotypes and the heritabilities underlying the two traits (e.g., see Yengo and Visscher 2018). An alternative way to estimate the genetic correlation between parents without using PGS (which is noisy and therefore underpowered) would be to use the phenotypic correlation and heritability estimated using GREML or LDSC. Perhaps this is outside the scope of the paper but I would like to hear the author's thoughts on this.

6) It would be helpful to include power calculations for the MR-Egger intercept estimates.

7) Finally, what is the correlation between PGS and genetic PCs/geography in their sample? A correlation might provide evidence to support the point that classic MR effects are inflated due to stratification.

Reviewer #3 (Public Review):

This study outlines the role of osteoclast-mediated resorption in integrating the skeletal elements during limb regeneration, using axolotls that can regenerate the entire limb upon amputation. Using calcium-binding vital dyes (calcein and alizarin red), the authors first demonstrated that a large portion of amputated skeletal elements is resorbed prior to blastema formation. They further show that 1) inhibiting bone resorption by zoledronic acid impairs proper integration of the pre-existing and regenerating skeletal elements, 2) removing the wound epithelium using the full skin flap surgery inhibits bone resorption, and 3) bone resorption and blastema formation are correlated. The authors reached the major conclusion that bone resorption is essential for successful skeletal regeneration. Notably, this study applies a well-established and elegant axolotl limb regeneration model and transgenic reporter strains to reveal the potential roles of resorption in limb regeneration.

Strengths:<br /> 1. The authors utilized a well-established axolotl limb regeneration model and applied elegant vital mineral dyes and transgenic reporter lines for sequential in vivo imaging. The authors also provided quantitative assessment by examining multiple animals, particularly in the early sections, ensuring the rigor and the reproducibility of the study.<br /> 2. The authors further performed important interventions that can impinge upon successful limb regeneration, including inhibition of bone resorption by zoledronic acid and impairment of the wound epithelium by full skin flap surgery. These procedures gave rise to useful insights into the relationship between bone resorption and successful limb regeneration.<br /> 3. The imaging presented in this manuscript is of exceptionally high quality.

Weaknesses:<br /> 1. Despite the high quality of the work, many analyses in this study are incomplete, making it insufficient to support the major conclusion. For example, in Figure 4, the authors did not provide any quantitative assessment to show how zol affects the integration of the skeletal elements (angulation?), which seems to be essential for supporting the conclusion. Likewise in Figure 7, the analyses of EdU+ cells and Sox9 reporter expression were not included in zol-treated animals. Similarly in Figure 5, quantification of osteoclasts was not performed with the full skin flap surgery group. Analyses of only normally regenerated animals are not sufficient to support many of the conclusions.<br /> 2. The phenotype of zol-treated animals in limb regeneration is somewhat disappointing. Although zol-treated animals show decreased blastema formation and unresorbed pre-existing skeletal elements, limb regeneration still occurs and the only phenotype is a relatively minor defect in skeletal integration. It is possible that zol-induced defect in blastema formation is not directly linked to the failure of integration at a later stage.<br /> 3. As an integration failure of the newly formed skeleton still occurs in untreated animals, it is not entirely clear how the authors can attribute this defect to a lack of bone resorption. More quantitative analyses would be necessary to demonstrate the correlation between zol treatment and lack of integration.

Reviewer #3 (Public Review):

In this work, Dumrongprechachan et al. impressively expanded their earlier work on the identification of cell type-specific subcellular proteomes from mouse brain by APEX2 proximity labeling. Instead of using viral expression of APEX2, the authors now created a Cre-dependent APEX2 reporter mouse line using CRISPR knock-in, which can be combined with multiple Cre-driver lines for proteomic applications. Using this novel tool in combination with sophisticated mass spectrometry and elegant bioinformatics, they mapped the temporal dynamics of the axonal proteome in corticostriatal projections (instead of only identifying a static cell type- and compartment-specific proteome) together with its phosphorylation status (instead of only looking at protein abundance). The data will provide a valuable resource on developmental trajectories at the proteomic and phosphoproteomic level, and will allow for pathway- and phosphosite-centric systems-level analyses as exemplified by the identification of proline-directed protein kinases as major regulators of corticostriatal projection development.

Strengths:<br /> The key tool developed in this work is the APEX2 reporter mouse line as it enables capturing of early postnatal time points, which was not possible before due to the time window of 2-4 weeks required for viral APEX expression. Thus, this tool puts the authors into position to access the temporal dynamics of the developing axon at time points spanning from neonate (as early as P5) to young adult (P50). Within this complex experimental design, the authors even managed to introduce a crucial compartment control at least for the time point P18, in which APEX expression is restricted to nucleus and soma upon viral expression. The resulting resource will be of high value as the data are derived from advanced mass spectrometric methods and stringent data handling. Examples of this high level of scrutiny include the use of MS3 methodology for the acquisition of TMT data to address the ratio distortion issues typically seen with isobaric labeling and thereby increase the quantification accuracy and the limitation to proteins quantified in all biological replicates.

Weaknesses:<br /> As to sample preparation for mass spectrometry, the authors follow the interesting concept of first enriching the phosphopeptides from the pool of TMT-labeled tryptic peptides and then using the unbound fraction from that step for further peptide fractionation, followed by mass spectrometric protein quantification. While this strategy sounds very straightforward in principle, one would expect that the phosphopeptide enrichment comes with an unspecific loss of other peptides in general, and with a semi-specific loss of acidic peptides in particular. Was this potential issue investigated by comparison with samples that were fractionated directly without prior phosphopeptide enrichment? Or with other words: the rationale for this sequential procedure is compelling - quantification of both protein and phosphopeptide abundance from the same (limited) sample, but what is the price for it as to peptide loss?

The APEX2 reporter mouse line is a novel tool with broad applicability for proximity labeling approaches and, understandably, the authors advertise its advantages, mainly via the suitability for short temporal windows. However, the discussion on the limitations of the approach falls short. The authors should make clear that the APEX method in general is limited to ex vivo approaches such as the acute brain slices used here due to the limitation that potentially toxic reagents (i.e. low membrane-permeable biotin-phenol and H2O2) have to be delivered to the target tissue. Although treatment with H2O2 is rather short, undesired oxidative stress signaling may have to be taken into account, particularly when protein phosphorylation rather than protein abundance is assessed. It would also be interesting to discuss the pros and cons of perfusing the mice prior to preparation of brain slices; e.g., in the context of removal of catalases/endogenous peroxidases or potential for substrate delivery (like recently shown in heart, doi: 10.1038/s41586-020-1947-z). Another issue with the Discussion is that the authors do not properly reflect the involvement of proline-directed kinases in the development of corticostriatal projections, which stands in contrast to the fact that they sell this as one of their major findings throughout the manuscript, including the Abstract.

Reviewer #3 (Public Review):

This study investigates the neuronal correlates of low-frequency changes in respiration volume per unit time (RVT). The authors report distributed patterns of correlations between RVT and fMRI that may represent a respiration-driven brain network. The ability to demonstrate that this pattern has neuronal origins would make an important contribution to the fMRI field, especially as physiological signals are typically treated as artifacts in fMRI analysis.

A major strength of this paper is the use of concurrent fMRI, physiological monitoring, and invasive electrophysiology (electrode in the anterior cingulate cortex; ACC) in the anesthetized rat, which allows for directly measuring local neuronal activity associated with changes in respiration. A second strength is that the authors demonstrate coherence between respiration (the raw signal as well as RVT) and gamma-band power in the ACC, and furthermore replicate prior findings of a close link between gamma-band power and the BOLD fMRI signal. The authors also take care to ensure that the pattern of correlation between RVT and fMRI is distinct from artifacts resulting from breathing-induced static field changes as well as from CO2-related effects of breathing on the BOLD signal. The findings are clearly presented throughout the paper.

I believe that additional information would help to more strongly support the main claim, i.e., that the reported RVT-fMRI correlation pattern is of neuronal origin. One analysis that supports this claim is that in the lightly anesthetized state, regressing out the gamma-band power signal considerably reduced correlations between RVT and fMRI. However, the more direct test of this possibility involves the experiment in which neural activity across the brain is silenced (isoelectric state) while respiration is artificially maintained. The resulting disappearance of correlation between RVT and fMRI data points to the neuronal nature of RVT-fMRI correlation. Yet, since the amount of temporal variation in RVT during the iso-electric state was not reported, it was not clear whether RVT itself also exhibited less temporal variation in the isoelectric state. Since respiration was maintained by a ventilator in the isoelectric state, I wondered if the respiration depth and volume was more constant compared to in the lightly anesthetized state, in which it is mentioned that spontaneous respiration occurred. Importantly, the authors do mention that the respiration patterns were visually similar between these conditions (Fig. 5C and line 219), which is very promising, but quantification of RVT properties would be important to provide as well.

Reviewer #3 (Public Review):

The study presents interesting new data on the role of the PTPRK D2 pseudophosphatase domain recruiting determining substrate specificity. The paper also demonstrates the utility of predicted structural models, an aspect that has been nicely integrated into this study. However, many open questions remain and additional experimental data should be provided to experimentally confirm the proposed substrate recognition model.

In particular:<br /> 1) Validation of reagents: The authors generated a pY1230 Afadin antibody claiming that (page 6) "this new antibody is specific to tyrosine phosphorylated Afadin, and that pY1230 is targeted for dephosphorylation by PTPRK, in a D2-domain dependent manner". The WB in Fig 1B shows a lot of background, two main bands are visible which both diminish in intensity in ICT WT pervanadate-treated MCF10A cell lysates. The claim that the developed peptide antibody is selective for pY1230 in Afadin would need to be substantiated, for instance by pull down studies analysed by pY-MS to substantiate a claim of antibody specificity for this site. However, for the current study it would be sufficient to demonstrate that pY1230 is indeed the dephosphorylated site. I suggest therefore including a site directed mutant (Y1230F) that would confirm dephosphorylation at this site and the ability of the pY antibody recognizing the phosphorylation state at this position.<br /> 2) The authors claim that a short, 63-residue predicted coiled coil (CC) region, is both necessary and sufficient for binding to the PTPRK-ICD. The region is predicted to have alpha-helical structure and as a consequence, a helical structure has been used in the docking model. Considering that the authors recombinantly expressed this region in bacteria, it would be experimentally simple confirming the alpha-helical structure of the segment by CD or NMR spectroscopy.<br /> 3) Only two mutants have been introduced into PTPRK-ICD to map the Afadin interaction site. One of the mutations changes a possibly structurally important residues (glycine) into a histidine. Even though this residue is present in PTPRM, it does not exclude that the D2 domain no longer functionally folds. Also the second mutation represents a large change in chemical properties and the other 2 predicted residues have not been investigated.<br /> 4) The interface on the Afadin substrate has not been investigated apart from deleting the entire CC or a central charge cluster. Based on the docking model the authors must have identified key positions of this interaction that could be mutated to confirm the proposed interaction site.<br /> 5) A minor point is that ITC experiments have not been run long enough to determine the baseline of interaction heats. In addition, as large and polar proteins were used in this experiment, a blank titration would be required to rule out that dilution heats effect the determined affinities.

Reviewer #3 (Public Review):

This paper uses transient dark exposure to induce plasticity in the adult visual cortex. It shows that transient dark exposure in the adult mice has opposing effects at the single neuronal level versus the population level. At the population level, the stimulus representation is degraded following dark exposure but rebounds back to normal within 8 days of light re-introduction. Thus, dark exposure does not have a lasting negative impact on the visual cortex. Unexpectedly, at the single neuronal level, following dark exposure a fraction of neurons show more stable responses and higher correlations among pairs of neurons. It is inspiring to hypothesize that this fraction of neurons may form a plastic substrate for representation of complex natural scenes.

Strengths:

The paper uses a combination of single neuron and population analyses to identify the effects of transient dark exposure on visual responses in the adult mouse visual cortex. It succeeds in identifying degradation of stimulus representation at the population level following dark exposure, and stabilization of visual stimulus preference at the single neuron level as well as stabilization of stimulus correlations among pairs of neurons. This success is in part due to an impressively large set of simple visual stimuli used (180 different stimuli). This large set allows the authors to identify even small changes in stimulus preferences at the single neuronal level.<br /> This paper uses transient dark exposure to induce plasticity. An alternative and commonly used method to induce plasticity is monocular deprivation. This paper shows that at the single neuron level, the effects of transient dark exposure are different from the previously reported effects of monocular deprivation. This is an important finding for the field.

Weaknesses:

The analysis methods used are thoughtful and complementary. The statistical tests are mostly performed on visual responses pooled across 6 mice. These statistical tests support the claims of the paper. However, we are left wondering whether the effects identified would also be significant for visual responses of each individual mouse.

Reviewer #3 (Public Review):

In a previous study, the authors screened a genome-wide CRISPRi library for sensitivity to a panel of antibiotics. One of the hits on this screen was found to be an essential adenylate cyclase, Rv3645. Rv3645 is a multidomain adenylate cyclase (AC), membrane-associated, and carries a HAMP domain (often associated with two-component signal transduction pathways). Surprisingly, Rv3645 was the only AC exhibiting this broad sensitivity to antibiotics. These observations were validated using a knock-down strategy and were also shown to be complemented by expressing a CRISPRi-resistant allele. To confirm that the sensitivity is not due to weakened cell walls or increased permeability of the cell to antibiotics, they measured the uptake of vancomycin using fluorescently conjugated vancomycin and by mass-spec. Interestingly, the essentiality and drug sensitivity of rv3645KD was found to be dependent on long-chain fatty acids. When Mtb was cultured in absence of fatty acids, rv3645 was no longer essential which allowed them to construct an rv3645 deletion strain. To determine the role of AC in lipid metabolism, the authors carried out a suppressor screen to identify mutants that reversed the fatty-acid phenotype. Mutants were identified in fatty acid transporter genes and in a cAMP phosphodiesterase gene, rv1339. The role of cAMP levels in mediating fatty acid metabolism and antibiotic resistance was further confirmed through the measurement of cAMP levels using mass spectrometry and expression of an enzymatically inactive mutant of rv3645.

Overall, this is a very elegant study that uses cutting-edge bacterial genetics to address the role of cAMP in mycobacterial pathogenesis. All the experiments have been well designed with the necessary controls and rigor. The studies clearly establish the role of cAMP, especially mediated through rv3645 in fatty acid metabolism and resistance against different classes of antibiotics. While the upstream signal is still unknown this can be for future follow-up studies.

While it may be slightly more difficult to find larger metal/wood cases for the 4x6 or 5x8 cards, it's a minor nuisance and anyone who wants them will eventually find the right thing for them. Beyond this, choose the card size that feels right to you.

If you don't have an idea of what you need or like, try things out for 10-20 cards and see how it works for you, your handwriting size, and general needs. People have been using 3x5, 4x6, and even larger for hundreds of years without complaining about any major issues. If Carl Linnaeus managed to be okay with 3x5, which he hand cut by the way, I suspect you'll manage too.

Of course I won't mention to the Americans the cleverness of the A6, A5, A4 paper standards which allows you to fold the larger sizes in half to get the exact next smaller size down. Then you might get the benefit of the smaller size as well as the larger which could be folded into your collection of smaller cards, you just have to watch out for accidentally wrapping ("taco-ing") a smaller card inside of a larger one and losing it. I suppose you could hand cut your own 5" x 6" larger cards to do this if you found that you occasionally needed them.

For the pocketbook conscious, 3x5 does have the benefit of lower cost as well as many more options and flexibility than larger sizes.

At least commercial card sizes are now largely standardized, so you don't have deal with changing sizes the way Roland Barthes did over his lifetime.

My personal experience and a long history of so many manuals on the topic saying "cards of the same size" indicates that you assuredly won't have fun mixing different sized slips together. I personally use 3x5" cards in a waste book sense, but my main/permanent collection is in 4x6" format. Sometimes I think I should have done 3 x 5, but it's more like jealousy than regret, particularly when it comes to the potential of a restored fine furniture card catalog. But then again...

Reviewer #3 (Public Review):

This paper presents a rigorously performed series of studies to improve the ability of the PhIP-seq method to discover autoantibodies against peptide antigens that span the whole peptidome at scale, and increase the ease of validation and definition of disease specificity. The paper is an extension of a recent paper from the DeRisi and Anderson groups done on APS1 patients, which defined and validated a novel series of tissue-specific autoantigens in APS1. The current studies show that the authors can find the antibodies they previously defined, and using larger numbers of disease and control samples, can expand some what they detect. They then use the new method to look at multiple additional processes in which autoimmunity has been demonstrated/postulated.

The dataset may be of use to others interested in defining novel autoantibodies. The findings really did not share significant new insights into the processes they studied,. As the authors note, they were unable to detect the antibodies (~10% of patients) recognizing type I IFNs in severe COVID-19, where these had been demonstrated effectively using ELISA previously. Unlike APS1, where their findings about uncommon tissue specific autoantibody responses across a population with known genetic deficiency and heterogeneous phenotypes could really illustrate the power of the method and approach, that elegance and powerful and novel conclusion is not as evident here.

Reviewer #3 (Public Review):

The authors have determined a range of conformations of the high-affinity prokaryotic K+ uptake system KdpFABC, and demonstrate at least two novel states that shed further light on the structure and function of these elusive protein complexes.

The manuscript is well-written and easy to follow. The introduction puts the work in a proper context and highlights gaps in the field. I am however missing an overview of the currently available structures/states of KdpFABC. This could also be implemented in Fig. 6 (highlighting new vs available data). This is also connected to one of my main remarks - the lack of comparisons and RMSD estimates to available structures. Similarity/resemblance to available structures is indicated several times throughout the manuscript, but this is not quantified or shown in detail, and hence it is difficult for the reader to grasp how unique or alike the structures are. Linked to this, I am somewhat surprised by the lack of considerable changes within the TM domain and the overlapping connectivity of the K indicated in Table 1 - Figure Supplement 1. According to Fig. 6 the uptake pathway should be open in early E1 states, but not in E2 states, contrasting to the Table 1 - Figure Supplement 1, which show connectivity in all structures? Furthermore, the release pathway (to the inside) should be open in the E2-P conformation, but no release pathway is shown as K ions in any of the structures in Table 1 - Figure Supplement 1. Overall, it seems as if rather small shifts in-between the shown structures (are the structures changing from closed to inward-open)? Or is it only KdpA that is shown?

My second key remark concerns the "E1-P tight is the consequence of an impaired E1-P/E2-P transition" section, and the associated discussion, which is very interesting. I am not convinced though that the nucleotide and phosphate mimic-stabilized states (such as E1-P:ADP) represent the high-energy E1P state, as I believe is indicated in the text. Supportive of this, in SERCA, the shifts from the E1:ATP to the E1P:ADP structures are modest, while the following high-energy Ca-bound E1P and E2P states remain elusive (see Fig. 1 in PMID: 32219166, from 3N8G to 3BA6). Or maybe this is not what the authors claim, or the situation is different for KdpFABC? Associated, while I agree with the statement in rows 234-237 (that the authors likely have caught an off-cycle state), I wonder if the tight E1-P configuration could relate to the elusive high-energy states (although initially counter-intuitive as it has been caught in the structure)? The claims on rows 358-360 and 420-422 are not in conflict with such an idea, and the authors touch on this subject on rows 436-450. Can it be excluded that it is the proper elusive E1P state? If the state is related to the E1P conformation it may well have bearing also on other P-type ATPases and this could be expanded upon.

Reviewer #3 (Public Review):

The authors introduce an integrative platform for identifying small molecule ligands that can disrupt RNA-protein interactions (RPIs) in vitro and in cells. The screening assay is based on prior work establishing the MT bench assay (Boca et al. 2015) for evaluating protein-protein interactions in cells by utilizing microtubules as a platform to recruit and detect PPIs in cells. In the current manuscript, the authors adapted this methodology to evaluate small molecules targeting RNA-binding protein (RBPs) interactions with mRNA in cells. By combining the MT bench assay with computational docking/screening and ligand-binding evaluations by NMR, the authors discover inhibitors of the RBP YB-1, which included FDA-approved PARP-1 inhibitors. The impact of this work could be high given the critical roles of RNA-binding proteins in regulating the function and fate of coding and non-coding RNA. While the presented data are promising, the ability to generally apply this method beyond YB-1 and to RBPs in general remains to be addressed.

Reviewer #3 (Public Review):

Gicking et al. analyze the relation between DDB (retrograde motor complex) and different members of kinesin family. The authors directly linked DDB and kinesin-1, -2 and -3 using DNA linker. Consistent with previous force measurement, kinesin-1 can dominate DDB. On the other hand, it is an unexpected and interesting observation that kinesin-2 and -3 can withstand loads by DDB because these motors are sensitive to load and easily detach from microtubules under loaded conditions in optical tweezers experiments. The authors performed computer simulations and suggested that fast detachment in kinesin-2 and -3 can be antagonized by fast reattachment. The work will impact thinking about physical properties of kinesins under loaded conditions.

Weaknesses:<br /> (1) To show DDB-kinesin-2 relation, the authors analyzed KIF3A/KIF3A homodimers. This reviewer does not think KIF3A/KIF3A homodimers represent kinesin-2. The motor domain of kinesin-2 is a heterodimer composed of KIF3A and KIF3B in the cell. The authors have previously shown that properties of KIF3A/KIF3A homodimer are different from those of KIF3A/KIF3B (Andreasson et al., Curr Biol., 2015).

(2) While these in vitro results are interesting, physiological meaning of these findings is not very clear.

Reviewer #3 (Public Review):

The manuscript provides evidence that larvae are capable of operant, as opposed to classical, conditioning: optogenetic activation of serotonergic neurons after a larva bends in one direction increases the likelihood that it will bend in that direction again.

Furthermore, the manuscript shows that serotonergic neurons located in the ventral nerve cord are capable of inducing this associative conditioning. While dopaminergic and serotonergic neurons, notably the dopaminergic PAM cluster in the Mushroom Bodies, have previously been implicated in classical conditioning, where different subsets apply positive or negative valence, these data suggest that specific serotonergic neurons may also contribute to learned behaviors. Although the cellular and circuit mechanisms remain unclear, and the consequences of silencing these neurons in contexts where the larva might more naturally employ operant learning are not tested, this research suggests new areas for exploring the adaptive capacity of a powerful model organism.

https://web.archive.org/web/20080412071219/http://eastgate.com/catalog/Briefcase.html

Eastgate systems used to make a "3x5 Card Briefcase" to capture short notes on the go which could later "be scanned or transcribed to Tinderbox."

Tinderbox was one of the first digital tools to be used in a way very similar to zettelkasten of old, particularly by academics, who are a large portion of their power user base.

https://web.archive.org/web/20140707053048/http://www.getfrictionless.com/products/capture-cards-red

Simple index cards, but sold with a purpose in mind: capturing notes!

One is reminded here of waste books and fleeting notes.

Image:<br />

The documentary Casting By (2012) shows casting director Marion Dougherty's 3 x 5" index card collection which she used for casting notes. In particular they show an example card for Dustin Hoffman with his details. In Hoffman's case, his card included the older telephone numbers with exchanges (EN2-6933 or Endicott2 6933), so these cards may have also served a contact purpose similar to more modern rolodexes. Different from them however, Dougherty's also included heights, credits, and other observations relevant to the casting process.

Screen capture from the movie

Reviewer #3 (Public Review):

The authors demonstrate in mice that the amount of sleep is related to stress resilience, and specifically that increased sleep after stress exposure supports resilient behavior. The aims are achieved through an array of methodologies, which highly strengthens the conclusions of the work. The question of whether sleep is related to stress resilience is highly significant and in the current research, the authors tackle the questions by evaluating differences in sleep homeostasis in stress-resilient compared to stress-susceptible mice. To induce more stress-susceptibility, the authors challenge the mice with sleep restriction, and to induce more stress-resilience, the authors chemogenetically induce increased NREM sleep. Mechanistically the authors demonstrate that cortically mediated NREM sleep is sufficient to promote resilience. Despite the challenging nature of the technological approaches at hand, particularly in mice, the experiments are well-designed and the authors are commended for the execution of these studies.

It is very difficult to separate sleep loss and stress responses, as losing sleep is inevitably a stressful experience. The authors attempt to quell the notion that sleep restriction was also stressful to the animals by measuring fecal corticosterone, however, the measurements were from fecal pellets collected during an entire 24 hr period raises concerns that acute changes in HPA response may not be evident through this measurement. This is a challenging notion to tackle and deserves a bit more consideration.

Chemogenetic experiments induce a beautiful increase in NREM sleep at the expense of REM loss, yet all the animals treated with chemogenetic agents are resilient to social defeat stress. The authors conclude that because sleep restriction also reduced REM, yet opposite effects occur on social interaction, REM sleep is unlikely to be related to resilience. In this context, it would be beneficial to discuss the theories of sleeping to forget and sleeping to remember, and supporting literature that REM sleep is critical to the consolidation of memories, particularly upon stressful experiences.

Reviewer #3 (Public Review):

This study addresses fundamental aspects of the eco-evolutionary dynamics of highly fecund organisms experiencing huge mortality rates during early life stages. In such species, a mechanism called "sweepstakes reproductive success" (Hedgecock, 1994) has been proposed to understand the dynamics of recruitment, in which individual reproductive success shows high variance and skewed distribution. Sweepstakes reproductive success can be either neutral due to random environmental variation influencing the recruitment of reproducing offspring, or selective because genetic variation at particular loci influences the likelihood of successful recruitment. Unfortunately, empirical tests of sweepstakes reproduction remain scarce due to the difficulty of studying individual reproductive success directly, particularly in highly fecund marine organisms.

By analysing genome-wide genetic diversity data under different coalescent models representing alternative recruitment dynamics, this pioneering study specifically tests whether random or selective sweepstakes reproduction occurs in the highly fecund Atlantic cod. Using the classical Kingman coalescent and two multiple-merger coalescent models approximating random sweepstakes (the Xi-Beta-coalescent model) and selective sweepstakes (the Durrett-Schweinsberg model), the authors show that genetic diversity in the Atlantic cod genome is most likely shaped by pervasive selective sweeps of new beneficial mutations. The best-fit selective sweepstakes model is able to reproduce the main characteristics of the allele frequency spectrum of each Atlantic cod population, while alternative models include either random sweepstakes or other biologically plausible scenarios (i.e. historical demographic changes, cryptic breeding structure, and background selection) show a much poorer fit.

These findings have a broad impact on evolutionary genomics since they provide a new and exciting perspective on the choice of appropriate coalescent models for the study of highly fecund organisms that may experience high rates of selective mortality during early life stages. The low-fecundity low-variance reproductive success model classically used in evolutionary genetics may simply not apply in highly fecund organisms with skewed offspring distribution.

By confronting different alternative models of coalescence with genome-scale genetic diversity data, this work provides a roadmap for exploring fundamental processes at the crossroads between ecology and evolution. It highlights the importance of (i) understanding the potential impact of species-specific biological characteristics when inferring demography and selection from molecular data, and (ii) being aware of the potentially significant effects of unaccounted aspects of the data (e.g. variant misorientation, past admixture) on the interpretation of results.

Reviewer #3 (Public Review):

The manuscript by Ronzano et al presents a rigorous neuroanatomical study to convincingly demonstrate that there is no difference in the medio-lateral organization of flexor and extensor premotor interneurons. The study uses monosynaptic restricted transsynaptic tracing from ankle flexor and extensor muscles with several (4) strategies for delivery of the G protein complement and delta G Rabies virus, and additional (2) variations that consider titer and cre line. The authors went to great lengths here in attempt to replicate prior studies for which they had initial conflicting findings. Further, the experiments are performed in laboratories in four different locations. The variations on the Rabies and complement delivery, regardless of lab performing the experiment and analysis, all converge on the same conclusion. Aside from the primary conclusion, the paper can be used as a manual for anyone considering transsynaptic tracing as it details the benefits and caveats of each strategy with examples.

The initial conflicting results put the onus on the authors to demonstrate where the divergence occurred. The authors took a highly comprehensive approach, which is a clear strength of the paper. All of the data is fully and transparently presented. Standardizations and differences between experiments run or analyzed in each lab are well laid out. Figure 1 and Table 2 provide a great summary of the techniques and their limitations. These are also well thought out and discussed within each section of results.

The only thing missing is a likely explanation for the differences seen. Although the authors made several attempts to provide such explanation, the question remains - how did two groups who published independent studies using different strategies demonstrate flexor and extensor separation in the dorsal horn, when this study, using several strategies in multiple labs, show that the premotor neurons are in complete overlap? Additional small differences in methodologies could be identified which are not discussed and may provide potential explanations, but only for discrepancies in results of single techniques, not for all of the strategies used. The lack of reason for the discrepancy with prior studies despite the extensive efforts is unsatisfying, but, most importantly, the experiments were rigorously performed and the data support the conclusions presented.

Reviewer #3 (Public Review):

Zhao and Sharpe identified telocytes in the periodontium. To address their contribution to periodontal diseases, they conducted scRNA-seq analysis and lineage tracing in mice. They demonstrated that telocytes are activated in periodontitis. The activated telocytes send HGF signals to surrounding macrophages, converting M2 to M1/M2 hybrid status. The study implies that targeting telocytes and HGF signal for the potential treatment of periodontitis.

The significance of the study could be improved by authors testing if targeting telocytes or HGF signals could ameliorate periodontitis in the mouse model. The current form of the manuscript lacks the data that demonstrate the actual contribution of telocytes in the homeostasis of periodontium or progression of periodontitis.

Major comments:

1) I see the genetic validation of the role of telocytes or HGF signals are crucial to assure the significance of this manuscript. I recommend either of two experiments. a. testing the role of HGF signals by deleting the Hgf gene in telocytes. Using Wnt11-Cre; Hgf f/f mice, the authors could address the role of HGF signals in periodontitis. CX3CR1-Cre; cMet f/f mice will delete HGF signals in monocyte-derived macrophages. This will be another verification, but not sure if the PDL macrophages are derived from yolk sac or monocytes. b. measuring the contribution of telocytes in the homeostasis or disease progression. The mouse model could be challenging though, the system if achieved will be very informative. The authors could first check the expression of telocyte enriched genes, such as Lgr5 or Foxl1 reported previously in other tissue telocytes. Delete those genes under the Wnt1-Cre driver and check if telocyte lineage is removed. The system would be very useful for next-level study. DTA model could be an alternative, but Wnt1-Cre is vastly expressed in neural crest lienage.

2) This paper points out that the M1/M2 hybrid state of macrophages appears upon periodontitis. The authors could further characterize the hybrid macrophages by the expression of more markers, production of cytokines, and morphology. Need to clarify if this means some macrophages are in M1 state and others are in M2 state, or one macrophage possesses both M1 and M2 phenotype. Please conduct either FACS or immunofluorescence to demonstrate if one macrophage expresses both markers. Please introduce more information about the M1/M2 hybrid state of macrophage based on other present literature.

3) In the introduction part, the author lists several markers that can be used for telocyte identification, such as CD34+CD31-, CD34+c-Kit+, CD34+Vim+, CD34+PDGFRα+. Could authors explain why they chose CD34 CD31, but not other markers?

4) In figure 5g, I don't think the yellow color cell shows the reduction trend in the Tivantinib treatment group compared with a control group. Please validate the observation by gene expression analysis, WB, etc. In addition, please show c-Met+ cells level in the Tivantinib treatment group and control group.

Reviewer #3 (Public Review):

In this MS by Sefton et al., the authors investigate the role of HGF/MET pathway, as well as the cellular source of these molecules, during diaphragm development. In particular, the authors address the function of this pathway on muscle progenitors and phrenic nerve. They further provide evidence for the expression of HGF in pleuroperitoneal folds and for its requirement for muscle progenitor recruitment and maintenance during diaphragm muscle formation. This study is interesting and in general the results support the conclusions. The work could be improved by (1) providing appropriate controls for the role of HGF in the connective tissue and (2) linking the muscleless diaphragms and HGF to the hernia phenotype.

Reviewer #3 (Public Review):

This manuscript aimed to reveal the difference and similarity of sharp-wave ripples in awake vs. sleeping mice. To do this, the authors used wide-view voltage and glutamate activity imaging in awake head-fixed mice. The two-photon Ca imaging was applied to examine the spiking activity of the retrosplenial cortex.

They showed that the mean membrane potential and glutamatergic transmission of the neocortex's superficial layers were suppressed and enhanced, respectively, just after the sharp-wave ripples in awake mice, contrary to the same authors' previous findings in urethane-anesthetized and naturally sleeping mice. The retrosplenial cortex was most strongly modulated in membrane potential and glutamatergic transmission by awake sharp-wave ripples. The authors also found two groups of retrosplenial cortical neurons, whose spiking activity, measured by Ca dynamics, was suppressed and enhanced by awake sharp-wave ripples. These findings revealed the critical difference between sharp-wave ripples during waking vs. sleep, which would impact the field of memory research.

This manuscript's strength is that it compares the dynamics of membrane potential and glutamate transmission using wide view imaging. Both experimental and analytical methods were appropriate and supported their main conclusions.

Reviewer #3 (Public Review):

The authors attempted to, and succeeded at, testing the recent hypothesis that the large theropod dinosaurs Spinosaurus was a fast and capable swimmer and diver.

The strengths of the paper are the extent of the analyses which address and test numerous aspects of the 'aquatic hypothesis' with some depth, though in places this needs a better explanation of the details of the process and organisation. The results do support the conclusions, though these should be more specific and clear and relate to the recent literature on Spinosaurus habits to say what is, and is not, possible/plausible based on their analyses.

Overall this is likely to be a major step forwards in resolving the biology of this animal (and kin) and add considerable depth to the discussion by adding new data and results.

Reviewer #3 (Public Review):

In the manuscript by Bosada et al, the authors present work identifying and interrogating two cis-regulatory elements at TBX5 associated with atrial fibrillation. Mouse models lacking both copies of either element, but particularly one in the last intron of the Tbx5 gene, referred to as RE(int), results in increased Tbx5 expression, changes to cardiac electrophysiology, and downstream gene expression changes. Of interest, RE(int) induces expression of Prrx1, also associated with atrial fibrillation, and compound mutants partially rescues the RE(int) phenotype. Overall, this paper is of interest and advances our understanding of TBX5 in atrial fibrillation risk in humans. Critically, this study focuses on the impact of risk SNPs, which increase TBX5 expression in patients, while previous studies involving TBX5 in atrial fibrillation have focused on decreased expression.

The authors' work presents the following major claims:<br /> Figure 1. Identification of two, independently segregating risk regions at TBX5, which are conserved in humans and mice with predicted cis-regulatory functions termed RE(int) and RE(down).<br /> Figure 2. Homozygous RE(int) and RE(down) mutants, with a particular focus on RE(int) mutants, resulting in increased expression of Tbx5. The functional SNP appears to be rs7312625 A>G.<br /> Figure 3. Homozygous RE(int) mutants demonstrate several cardiac electrophysiological changes consistent with increased atrial fibrillation risk in humans.<br /> Figure 4. Cellular electrophysiology of RE(int) mutant cardiomyocytes demonstrates additional supportive changes to explain whole organ phenotypes presented in Figure 3.<br /> Figure 5. Transcriptional profiling of RE(int) and RE(down) homozygous mutants demonstrates many significant differences from control samples that are suggestive of specific mechanisms disrupting cardiac electrophysiology, including calcium and potassium regulators and gap junctions.<br /> Figure 6. Homozygous RE(int) and Prrx1(enh) mutant alleles genetically interact and result in partial rescue of phenotypes from RE(int) alone.

Reviewer #3 (Public Review):

Experimental and computational works have proposed that neurons in the hippocampus represent a predictive map of the environment called the successor representation. This theoretical study examines how plasticity in a model network of recurrently connected neurons can lead to such a representation. The main conclusion is that any plasticity rule that encodes transition probabilities in synaptic weights gives rise to the successor representation at the level of neural activity. This fundamental theoretical insight gives additional credibility to the idea that the hippocampus can implement the successor representation.

Strengths:<br /> - elegantly designed theoretical study<br /> - very clear writing that progressively introduces the main result and argues for its generality<br /> - comparison of the model with data in a random foraging task

Weaknesses:<br /> - certain technical choices need additional motivation

Reviewer #3 (Public Review):

The present study is a comparison of brain magnetic resonance imaging (MRI) of mice who developed in an enriched environment laboratory environment, in which some mice become habituated while other mice maintain active exploration of the environment over time. Between these groups, differences are shown in the pattern of correlations between brain regions of interindividual variability, which may indicate differences in brain connectivity or other shared maturational processes between regions. Because the mice are genetically inbred and have the same shared environment, these differences are attributed to individual-level differences in environment and behavior, which are extremely difficult to isolate in non-laboratory settings.

My comments are focused on aspects of the paper that overlap with my area of expertise which is human brain MRI methods. The strengths of the paper include the unique environmental paradigm that provides support for important hypotheses about individual-level variation. The imaging methods are rigorous and sound, and there is a nice convergence with human work. The application of structural covariance is interesting. The weaknesseses of the paper are that the writing could be clearer. Alternative explanations for structural covariance and alterations in "down roamers" should be more fully considered. The statistical approaches could be more rigorous in places. The areas of novelty relative to past work should be more explicitly articulated.

Reviewer #3 (Public Review):

Lillvis et al present a new method for quick targeted analysis of neural circuits through a combination of tissue expansion and (lattice) light sheet microscopy. Three color labeling is available which allows to label neurons of a molecularly specific type, presynaptic and/or post-synaptic sites.

Strengths:<br /> - The experimental technique can provide much higher throughput than EM<br /> - All source code has been made available<br /> - Manual correction of automatic segmentations has been implemented, allowing for an efficient semi-automatic workflow<br /> - Very different kinds of analyses have been demonstrated<br /> - Inclusion of electrical connections is really exciting, what a great complement to the existing EM volumes!

Weaknesses:<br /> - Limitations of the method are not really discussed. While the approach is simpler and cheaper than EM, it's still important to give the readers a clear picture of the use cases where it's not expected to work before they embark on the journey of acquiring tens of terabytes of data. Here are just a few examples of the questions I would have if I wanted to implement the method myself - I am a computational person and can easily imagine my "wet lab" colleagues would have even more to ask about the experimental side:

-- It is not very clear to me if the resolution of the method is sufficient to disentangle individual neurons of the same type. It has been demonstrated for a few examples in the paper, but is it generally the case? Are there examples of brain regions/neuron types where it wouldn't be possible? If another column was added to the table in Figure 1, e.g. "individual neuron connectivity", EM would be "+", LM "-", what would ExLLSM be?<br /> -- Similarly, the procedures for filling gaps in the signal could result in falsely merged neurons. Does it ever happen in practice?<br /> -- How long does semi-manual analysis take in person-hours/days for a new biological question similar in scope to the ones demonstrated in the paper?<br /> -- How robust are the networks for synaptic "blob" detection? The authors have shown they work for different reporters, when are they expected to break? Would you recommend to retrain for every new dataset? How would you recommend to validate the results if no EM data is available?

Reviewer #3 (Public Review):

Almedia and Macklin sought to characterize oligodendrocyte behavior at the earliest onset of myelination in the central nervous system. By sparsely labeling oligodendrocytes with transgenic fluorescent reporters in zebrafish larvae, they show that oligodendrocytes in the dorsal and ventral spinal cord have characteristic numbers of sheaths and sheath lengths. With impressive and technically laborious time-lapse imaging, they demonstrate that oligodendrocytes repetitively sample axon segments before stabilizing a nascent sheath, with most (~90%) immature sheaths failing to stabilize. They quantify differences in dorsal and ventral oligodendrocyte sampling and convincingly show that dorsal oligodendrocytes form more sheaths due to increased sampling relative to ventral, with similar rates of sheath retraction. Finally, the authors conclude that Rab5 and Rab11 promote myelination locally and cell-autonomously in oligodendrocytes, showing specifically that Rab5 is critical for stabilization of nascent sheaths but is dispensable for sampling. Altogether, the authors provide novel and detailed visualization of early myelin sheath development by oligodendrocytes.

Strengths:<br /> This is one of the first studies to closely examine early oligodendrocyte behavior at high resolution and adds to a body of work showing how oligodendrocytes initiate and maintain myelin sheaths. The authors find that sampling is widespread while most immature sheaths destabilize. This is an intriguing finding, as sampling is likely an energetically intensive process, but its prevalence in wild-type animals suggests that it is an important part of development.

The authors' claims are substantiated by technically challenging mosaic labeling experiments that monitored individual oligodendrocyte development over the course of days. Importantly, the authors have measured the sheath accumulation and loss phase for a single oligodendrocyte over the course of several days, and can pinpoint when individual sheaths are maintained with high resolution.

The imaging acquisition and data analyses are thorough, and the labor-intensive nature of the experiments is commendable. The authors carefully use statistics to validate their conclusions, including power analyses to determine appropriate sample size.

Limitations:<br /> Prior studies suggested that the potential for each oligodendrocyte to produce myelin sheaths is at least partially dependent upon the diameter of axons enwrapped. While axons are labeled to demonstrate ensheathment, axonal diameter is not measured, and it is unclear whether dorsal and ventral oligodendrocytes behavior could be explained by regional or individual differences in axon caliber.

Other recent studies suggest that early myelination is driven by axonal factors as much as oligodendrocyte-intrinsic factors. For instance, neuronal activity stabilizes myelination for a subset of early-born neuronal types. Because of the sparse labeling techniques and focus on oligodendrocyte behavior, it is unknown how or whether axonal subtypes and activity influence early oligodendrocyte sheath sampling and stabilization.

While the authors provide a tantalizing suggestion that early oligodendrocyte sampling primes axonal segments for myelination, it is not tested directly here. Thus, the paper does not address why, or even if, repeated sampling is important in development.

Reviewer #3 (Public Review):

The authors have constructed a large-scale interaction perturbation network from 2,167 CRC tissues and 308 normal tissues, deciphering six GINS subtypes with particular clinical and molecular peculiarities. In addition, the GINS taxonomy was rigorously validated in 19 external datasets (n =3,420) with distinct conditions. From an interactome perspective, this study identified and diversely validated a high-resolution classification system, which could confidently serve as an ideal tool for optimizing decision-making for CRC patients. The multifariously biological and clinical peculiarities of GINS taxonomy improve the understanding of CRC heterogeneity and facilitate clinical stratification and individuation management. Additionally, candidate specific-subtype agents provide more targeted or combined interventions for six subtypes, which also need to be validated in clinical settings.

Reviewer #3 (Public Review):

In this report, the authors examined 3 mutations in the BRC-repeat region of BRCA2 in a series of functional assays. They found that two of the mutants showed severe defects in BRCA2 function, whereas the third mutant had no clear phenotype. The two mutants with functional defects are tested most thoroughly. The assays used here a numerous and have been validated and performed with appropriate controls and statistics. There are no concerns about the experiments themselves or the conclusions. So, the strength of the study is the number of assays performed in a rigorous manner.

However, the weakness of the study is that it is unclear why these results are impactful. Several reports over the years, including some recent studies mentioned at the end of the Discussion, have involved parallel functional analysis of hundreds of alleles of BRCA2, with a clear end goal of improving medical decision-making for carriers of these BRCA2 alleles. Certainly, these studies have usually focused on other domains of BRCA2, like the DNA binding domain, but nonetheless, since these studies have typically involved testing hundreds of BRCA2 alleles, it is unclear how this manuscript studying 3 alleles fits into a broader population science effort to categorize BRCA2 variants of unknown significance (VUS). Perhaps the authors would argue that their study involves a comprehensive analysis of the 3 alleles, whereas other studies typically involve one or two functional assays. However, if that is the case, then is the argument that multiple assays are needed for accurate characterization of VUS? If so, what is the evidence for that assertion? Are there particular assays that are more likely to be predictive of pathogenicity based on their analysis?

The mechanistic insight of the study is also unclear. These alleles are in conserved residues of the BRCA2 BRC repeats, which have been established as being important for BRCA2 function. Indeed, in the Discussion, it appears that the findings here are largely confirmatory for other mechanistic studies of the BRC repeats of BRCA2. What new information has been determined about the role of the BRC2 and BRC7 repeats from this study?

Reviewer #3 (Public Review):

This study was designed to test the hypothesis that output from a subpopulation of neurons (PKCδ neurons) in the central nucleus of the amygdala (CeA) inhibits ZI neurons in a neuropathic pain condition and this ZI inhibition results in pain-related behaviors (Fig. 5).

First, the targets of CeA-PKCδ neurons were identified using cre-dependent viral vector for anterograde labeling with red-shifted channelrhodopsin (CrimsonR-tdTomato) or mCherry, and cholera toxin B (CTB) in PKCδ-tdTomato mice for retrograde labeling. The ZI was identified as one of the targets with approximately 19% of CTB+ CeA neurons identified as PKCδ- tdTomato positive, which is significant and makes this pathway worth exploring.

Next, electrophysiological (patch-clamp) studies showed monosynaptic inhibitory transmission from CeA to both VGAT+ and VGAT- neurons of the ZI and found no significant difference between these projections (from CeA to GABAergic or non-GABAergic ZI neurons).

Finally, chemogenetics are used to activate or silence GABAergic ZI neurons and determine behavioral consequences. Inhibition of GABAergic ZI neurons induced hypersensitivity in naïve mice and activation of these neurons reversed hypersensitivity in a neuropathic pain model. Interestingly, these effects were modality specific.

The combination of tracing techniques, electrophysiology, chemogenetics and behavior is a strength of this study, and so this the impressive amount of high-quality data. The focus on CeA-PKCδ neurons in the modulation of ZI is an important novelty of the present study.

However, slice physiology and behavioral data presented here do not actually link CeA-PKCδ neurons to ZI. Electrophysiological data show inhibitory transmission from CeA to ZI, but not specifically from CeA-PKCδ neurons to ZI. Behavioral studies assess the effects of modulation of ZI neurons but not of CeA-PKCδ to ZI projections. Previous data already showed the effects of activation and inhibition of GABAergic ZI neurons on pain behaviors, including in a neuropathic pain model.

Therefore, although the proposed model of CeA-PKCδ to ZI interactions in pain (Fig. 5) is novel and significant, additional experiments focusing on CeA-PKCδ neurons and their ZI projections would be needed to fully support this concept and enhance impact of the work.

Reviewer #3 (Public Review):

The authors have used both overall and local genetic correlations to understand how genes associated with two traits relate to those same traits. Their work focuses on understanding why in some cases local genetic correlations may disagree with overall correlation in terms of the direction of effect and exploit known biology to understand why and when this arises.

Overall the work is solid methodologically as it relies on well-established statistical methods and known biology. I don't see particular weaknesses in this work limited to the presented examples. It remains unclear how these observations will generalise to other less well-known biology or traits, but this is a matter of future work.

The work is in my opinion highly impactable as it creates a framework to be used to investigate the pleiotropic effects of genes and could help understand their biological role.

Reviewer #3 (Public Review):

This study reports on the phenotypes of a CRISPR-engineered zebrafish mutants in kinesin light chain 4 (KLC4). KLC4 is expressed prominently in spinal cord sensory neurons, and mutants have defects in peripheral axon branching/stabilization and branch repulsion, as well as make occasional ectopic axon branches. Imaging also demonstrates that axonal microtubule growth dynamics are altered. These axonal phenotypes are nicely characterized with beautiful light sheet time-lapse microscopy and clever image analyses methods. Additionally, the growth of adult KLC4 mutants is stunted, and they exhibit a variety of behavioral defects.

The strengths of this paper are the creation of a new mutant for studying axonal transport, the impressive imaging methods, and the development of image analysis methods for characterizing axonal trajectories across a population.

The main weaknesses is the lack of a specific mechanistic explanation for how kinesin dysfunction leads to axonal defects-what kinesin cargoes play a role in branch stabilization and branch repulsion? How does kinesin-mediate transport affect microtubule growth?

Another weakness is the lack of a connection between the cellular defects characterized in larval sensory neurons, and the behavioral defects in adults. Since the adult behavioral defects likely do not involve sensory neurons, these two parts of the paper don't fit together. The authors may want to consider moving the behavior to a different paper. Additionally, the cellular basis of the adult behavioral defects is unknown, and likely involves a complex combination of defects in multiple cell types.

Reviewer #3 (Public Review):

In this manuscript, Zhou et al describe basal cell heterogeneity in the mouse trachea. They describe how dorsally vs ventrally located tracheal basal cells which are supported by different stromal cell populations show differential potential to undergo squamous metaplastic differentiation. Furthermore, they suggest that the differences in these basal cells might be epigenetically programmed as they are maintained after these basal cells have been isolated and cultured in vitro. However, it is not clear whether dorsal vs ventral supporting stromal cell populations made it into the culture medium.

Reviewer #3 (Public Review):

This work is important for understanding both how immune cells are regulated and how alterations in receptor signaling can affect the balance of health and development of autoimmune diseases. The work uses CRISPR-based genetic manipulation of the autoimmunity associated PTPN22 gene in single donor human cord-derived naïve T cells to analyze T-cell receptor functions. The authors conclude that the autoimmunity associated PTPN22 variant PTPN22(620W) is a loss-of-function mutant as T cells expressing PTPN22(620W) phenocopies PTPN22 deficient T cells. The use of a single donor minimizes potential other effects that would be observed when comparison cellular functions from multiple donors.

Reviewer #3 (Public Review):

Angueyra et al. tried to establish the method to identify key factors regulating fate decisions in the retinal visual photoreceptor cells by combining transcriptomic and fast genome editing approaches. First, they isolated and pooled five subtypes of photoreceptor cells from the transgenic lines in each of which a specific subtype of photoreceptor cells are labeled by fluorescence protein, and then subjected them to RNAseq analyses. Second, by comparing the transcriptome data, they extracted the list of the transcription factor genes enriched in the pooled samples. Third, they applied CRISPR-based F0 knockout to functionally identify transcription factor genes involved in cell fate decisions of photoreceptor subtypes. To benchmark this approach, they initially targeted foxq2 and nr2e3 genes, which have been previously shown to regulate S-opsin expression and S-cone cell fate (foxq2) and to regulate rhodopsin expression and rod fate (nr2e3). They then targeted other transcription factor genes in the candidate list and found that tbx2a and tbx2b are independently required for UV-cone specification. They also found that tbx2a expressed in the L-cone subtype and tbx2b expressed in L-cones inhibit M-opsin gene expression in the respective cone subtypes. From these data, the authors concluded that the transcription factors Tbx2a and Tbx2b play a central role in controlling the identity of all photoreceptor subtypes within the retina.

Overall, the contents of this manuscript are well organized and technically sound. The authors presented convincing data, and carefully analyzed and interpreted them. It includes an evaluation of the presented data on cell-type specific transcriptome by comparing it with previously published ones. I think the current transcriptomic data will be a valuable platform to identify the genes regulating cell-type specific functions, especially in combination with the fast CRISPR-based in vivo screening methods provided here. I hope that the following points would be helpful for the authors to improve the manuscript appropriately.

1) The manuscript uses the word "FØ" quite often without any proper definition. I wonder how "Ø" should be pronounced - zero or phi? This word is not common and has not been used in previous publications. I feel the phrase "F0 knockout", which was used in the paper cited by the authors (Kroll et al 2021), is more straightforward. If it is to be used in the manuscript, please define "FØ" and "CRISPR-FØ screening" appropriately, especially in the abstract.

2) Figure 1-supplement 1 shows that opn1mw4 has quite high (normalized) FPKM in one of the S-cone samples in contrast to the least (or no) expression in the M-cone samples, in which opn1mw4 is expected to be detected. The authors should address a possible origin of this inconsistent result for opn1mw4 expression as well as a technical limitation of using the Tg(opn1mw2:egfp) line for detection of opn1mw4 expression in the GFP-positive cells.

3) The manuscript lacks a description of the sampling time point. It is well known that many genes are expressed with daily (or circadian) fluctuation (cf. Doherty & Kay, 2010 Annu. Rev. Genet.). For example, the cone-specific gene list in Fig.2C includes a circadian clock gene, per3, whose expression was reported to fluctuate in a circadian manner in many tissues of zebrafish including the retina (Kaneko et al. 2006 PNAS). It appears to be cone-specific at this time point of sample collection as shown in Fig.2, but might be expressed in a different pattern at other time points (eg, rod expression). The authors should add, at least, a clear description of the sampling time points so as to make their data more informative.

Reviewer #3 (Public Review):

At the heart of this manuscript is a debate concerning the role of the orbitofrontal cortex (OFC) in goal-directed behavior. One commonly sees a paper in which Ostlund and Balleine placed large OFC lesions in behaviorally-experienced rats cited as irrefutable evidence that OFC is not involved in goal-directed behavior because these rats could perform typically in a simple devaluation task. Meanwhile, others have argued that the ventrolateral OFC (VLO) sits at a nexus between the medial PFC structures (which are attuned to reinforcer value, etc.) and the far lateral regions (which appear to be more specialized in Pavlovian associations) and may therefore play a role in goal-directed behavior (e.g., this argument is put forward in Gourley and Taylor, 2016, Nature Neuroscience). The present team published a crucial manuscript a couple of years ago showing that selective VLO lesions do indeed disrupt goal-seeking behaviors, particularly when value and contingency information needs to be integrated and/or updated (Parkes 2018). Because this sophisticated process is not tested in simple devaluation assays, it would have been missed in the older study. The Parkes 2018 paper, meanwhile, supports other investigations that also selectively manipulate the VLO and require animals to integrate new information into existing instrumental response strategies.

Here, the team first depleted NE fibers in the OFC and found that rats were unable to encode new associations in an instrumental reversal. This same deficit was not observed with parallel DA manipulation. They found that LC-OFC and not mPFC projections had the same effect. Throughout, important control experiments were conducted, and the tools being used were largely well-validated. The conclusions are sensible, and the writing is clear.

I would be curious about the authors' thoughts regarding the recent Duan ... Robbins Neuron paper (https://pubmed.ncbi.nlm.nih.gov/34171290/), in which marmosets displayed paradoxical responses to VLO inactivation and stimulation in contingency degradation tasks. Are there ways to reconcile these reports?

Reviewer #3 (Public Review):

The authors constructed a single-cell transcriptome atlas of bone marrow in normal and R-ISS-staged MM patients. A group of malignant PC populations with high proliferation capability (proliferating PCs) was identified. Some intercellular ligand receptors and potential immunotargets such as SIRPA-CD47 and TIGIT-NECTIN3 were discovered by cell-cell communication. A small set of GZMA+ cytotoxic PCs was reported and validated using public data.

For scRNA-seq data analysis, the authors did QC and filtering and removed low quality cells, including some doublets and followed by batch effect correction. Malignant PC populations were identified using the copy number analysis tool - "inferCNV".

The authors have done lots of analysis. But I think the results can be improved if they can do more analyses. I would recommend to 1) analyze doublets; 2) remove cell cycle effect; 3) GO and pathway analysis for genes with copy number change; 4) do cell-cell communication with more cell type/clusters.

Data analysis of public data was sufficient to prove the small set of GZMA+ cytotoxic PCs. More data analysis or wet experiment proof is required.

Reviewer #3 (Public Review):

The authors identified a splicing factor that regulates mitochondrial homeostasis by regulating the alternative splicing of the pro-apoptotic protein BAX, which induces basal upregulation of interferon stimulated genes and sensitizes cells to apoptotic cell death. They report that loss of Serine/Arginine Rich Splicing factor 6 (SRSF6) results in accumulation of an alternatively spliced form of BAX known as BAX-, which results in increased release of mitochondrial DNA (mtDNA). The released mtDNA is sensed by cGAS, which leads to upregulation of interferon stimulated genes via IRF3. Importantly, the increase in BAX- sensitizes macrophages to apoptosis and various pathogens decreased the expression of SRSF6 during infection, which served a protective role. Interestingly, Mycobacterium tuberculosis decreases SRSF6 expression, but this resulted in a replication advantage. Overall, these findings add new mechanistic insight into the role of alternative splicing in regulating immunity and cell death. This work can potentially open novel avenues of inquiry into the role of BAX in regulating apoptosis.

Strengths:

The paper is well written, and the major conclusions are rigorously tested by numerous experiments. The data supports the major conclusions, which are that loss of SRSF6 increases ISG and leads to accumulation of alternatively spliced BAX, sensitizing cells to death.

Weaknesses:

The authors make a very interesting discovery that SRSF6 KD sensitizes macrophages to a caspase independent death by up regulating an alternatively spliced variant of BAX, a protein that has a well-established role in mediating caspase dependent death, but they did not rigorously test whether it was truly caspase independent.

Reviewer #3 (Public Review):

In the manuscript, the authors provided the development of a sensitive and rapid diagnostic tool for detection of pathogenic bacteria in respiratory infections given the limitations of traditional cultures in the clinical settings. Rapid identification and treatment of bacterial infections can impact the prognosis in sepsis. This work highlights how a new rapid diagnostic tool may be beneficial in the treatment of patients with bacterial pneumonia given the time-consuming nature and low sensitivity of traditional culture methods.

Strengths:

The manuscript authors created a diagnostic tool using CRISPR-Cas12 with bacterial species-specific DNA-tags to 10 epidemic bacteria at their local intensive care unit (ICU). The appendix data provided detailed reports of the reaction conditions, sample preparations and reaction incubation time.

A 2-stage validation process was used. The initial validation stage compared the use of the novel diagnostic tool to traditional cultures from bronchoalveolar lavage samples from ICU patients. Once the accuracy of the diagnostic tool was evaluated, the second validation stage was pursued in the form of a randomized controlled trial at the ICU of the study. The second validation stage demonstrated that the proposed novel diagnostic tool had faster results and correlated with improved APACHE II scores and more effective antibiotic coverage rates in the experimental group.

The use of the novel diagnostic test highlighted limitations traditional culture modalities may have in identifying polymicrobial infections which were identified more frequently in the two validation stages

Weaknesses:

Although the study has many strengths, a potential weakness could lie in the unclear use of next-generation sequence (NGS) testing where samples were reported to be sent at random. However, similar to the novel diagnostic tool proposed in this manuscript, NGS testing has been noted to have high sensitivity and specificity and both had similar results in the manuscript.

Additionally, the novel diagnostic testing demonstrated increased detection of polymicrobial infection when compared to traditional cultures; however, clinical evaluation will remain important to help decipher potential "false positive" results or identification of non-pathogenic colonization.

Based on the author's proposed aims to develop a rapid and sensitive diagnostic tool for bacterial pathogens in pneumonia; the authors demonstrated a highly sensitive and specific test when compared to gold-standard testing. Random samples were assessed against NGS testing technology with similar reported results. The development of this rapid, sensitive diagnostic tool can have wide-spread clinical implications to guide management in patient care where earlier time to effective treatment can have important impacts on prognosis.

Reviewer #3 (Public Review):

In the manuscript by Gao et al, the authors were trying to achieve an understanding of how Kiaa1024L/Minar2 is necessary for hearing in vertebrates. It is known that the Kiaa1024L/Minar2 mutation causes deafness in mice but not much beyond that is known.

Strengths:<br /> - In this manuscript, they were successful in making two zebrafish mutant zebrafish strains in the Kiaa1024L/Minar2 gene using Crispr/Cas9. The mutant(s) has defects in hearing (using the C-start assay and determining thresholds) and reduced hair cell numbers in the ear (phalloidin labeling to determine hair cell density in utricle and saccule) and the lateral line (including using the AM1-43 assay). From these data, they demonstrate that hair cells are defective in these mutants.

- The authors show that Lamp1-GFP labeled lysosomes change in size in the minar2fs139 mutant. In addition, they show that GFP-Minar2 localizes to lysosomal membranes in cultured cells (human and monkey).

- They performed primary amino acid sequence analysis on Minar2 and showed that it contained a putative CSD of caveolin, which is known to interact with cholesterol. They then show that when Minar2 is expressed in cells in culture, there is an increase in cholesterol detection in the region that contained Minar2, supporting the idea that cholesterol interacts with Minar2.

The experiments in figure 5 seem to show that lowering cholesterol levels using pharmacology exacerbates hair cell defects in a minar2 mutant.

Weaknesses:<br /> 1. The authors attempt to show localization (Fig 2 A and B) of Minar2 to the stereocilia and the apical region of hair cells using GFP-MINAR2 fusion protein expression in hair cells of transgenic animals. Although this is a typical way of demonstrating localization, it is usually used to validate location after a similar pattern has been shown using an antibody (usually in mice.) So, special precautions must be taken when interpreting this kind of transgenic data. According to the authors, GFP-MINAR2 localized to the stereocilia and the apical region of hair cells. This needs to be validated by some other means. I can also see the localization of the green signal at the basolateral area of the cells in Fig 2a. Moreover, it's important to note that other mislocalized fusion proteins localize to the apical region of hair cells.

2. Figure 2C and D. The defects in the hair bundles are plausible but not convincing. Electron microscopy should be used to validate. Also, are hair bundle defects seen in the neuromast? EM would be easier to do there.

3. Fig 1A do prim 1- and prim 2-derived neuromasts express minar2? Do anterior neuromasts express minar2?

4. It's my impression that the authors don't take into account that there is much more plasma membrane in the stereocilia than in the basolateral membrane. So, this statement, "These data suggested that there are high levels of accessible cholesterol located to the stereocilia membranes, while the accessible cholesterol levels are marked lower in the basolateral membranes in the hair cells" based on Figure 4 needs to be reconsidered. The authors need to show that the little reporter that is present in the basolateral membrane is not equal to the reporter present in a single sheet of the plasma membrane in a stereocilium. I can see basolateral labeling in the lateral line hair cells.

5. It's not clear if there is a paralog of the Kiaa1024L/Minar2 gene.

Reviewer #3 (Public Review):

The manuscript by Sureshchandra et al is a very extensive analysis of monocyte function and their molecular landscape in cord bloods from lean and obese mothers. They aimed to analyze the effects of pre-pregnancy BMI on the functioning of the innate immune system in newborns in a very extensive way. The combination of functional and molecular analyses strengthens their observations and shows many different sides of monocyte activation. I think this approach needs to be praised and should be an inspiration to many others who study monocyte function. This allows for a broad view on the matter and also shows where potential targeting will be necessary in the future. Overall, the manuscript and particularly the methods section is very well written and extensive, making it easy to study how robust the data are.

Reviewer #3 (Public Review):

Through a series of rigorous in vitro studies, the authors determined McdB's domain architecture, its oligomerization domains, the regions required for phase separation, and how to fine-tune its phase separation activity. The SEC-MALS study provides clear evidence that the α-helical domains of McdB form a trimer-of-dimers hexamer. Through analysis of a small library of domain deletions by microscopy and SDS-PAGE gels of soluble and pellet fractions, the authors conclude that the Q-rich domain of McdB drives phase separation while the N-terminal IDR modulates solubility. A nicely executed study in Figure 4 demonstrated that McdB phase separation is highly sensitive to pH and is influenced by basic residues in the N terminal IDR. The study demonstrates that net charge, as opposed to specific residues, is critical for phase separation at 100 micromolar. In addition, the experimental design included analysis of McdB constructs that lack fluorescent proteins or organic dyes that may influence phase separation. Therefore, the observed material properties have full dependence on the McdB sequence.

Studies of proteins often neglect short, disordered segments at the N- or C- terminus due to unclear models for their potential role. This study was interesting because it revealed a short IDR as a critical regulator of phase separation. This includes experiments that remove the IDR (Fig 2 & 3) and mutate the basic residues to show their importance towards McdB phase separation. In a nice set of SDS-PAGE experiments, the authors showed that as the net charge of the IDR decreased the construct became more soluble.

One challenge is in the experimental design when mutating residues is to assess their impact on phase separation. The author's avoided substitutions to alanine, as alanine substitutions have synthetically stimulated phase separation in other systems. The authors, therefore, have a good rationale for selecting potentially milder mutations of lysine/arginine to glutamine. A potential caveat of mutation to glutamine is that stretches of glutamines have been associated with amyloid/prion formation. So, the introductions of glutamines into the IDR may also have unexpected effects on material properties. Despite these caveats, the authors show mutation of six basic residues in the short IDR abolished phase separation at 100 mM.

Computational studies (Fig 7) also suggest that this short N-IDR region may play a role as a MORF upon potential binding to a second protein McdA. The formulation of this hypothesis is strengthened by the fact that for other ParA/MinD-family ATPases, the associated partner proteins have also been shown to interact with their cognate ATPase via positively charged and disordered N-termini. This aspect of understanding McdB's N-IDR as a MORF is at a very early stage. This study lacks experimental evidence for an N-IDR: McdA interaction and experimental data showing conformational change upon McdA binding. However, the computation study sets up the future to consider whether and how the phase separation activity of McdB is related to its structural dynamics and interactions with McdA.

In summary, this study provides a strong foundation for the contribution of domains to McdB's in vitro phase separation. This knowledge will inform and impact future studies on McdB regulating carboxysomes and how the related family of ParA/MinD-family ATPases and their cognate regulatory proteins. For example, it is unknown if and how McdB's phase separation is utilized in vivo for carboxysome regulation. However, the revealed roles of the Q-rich domain and N-IDR will provide valuable knowledge in developing future research. In addition, the systematic domain analysis of McdB can be combined with a similar analysis of a broad range of other biomolecular condensates in bacteria and eukaryotes to understand the design principles of phase separating proteins.

Reviewer #3 (Public Review):

In this manuscript, the authors aim to identify the regulators of epithelial invasiveness upon Lethal giant larvae (Lgl), a basolateral polarity protein, knockdown in the follicular epithelium of the Drosophila ovaries, which can serve as a model system to investigate cellular plasticity when apical-basal polarity is lost. Knockdown (KD) of Lgl causes a multilayered epithelium and through extensive single cell RNA-seq analyses, the authors demonstrate that Lgl-KD triggers the appearance of groups of cells exhibiting tumor-associated molecular signatures and invasive behaviour. Overall, the manuscript is technically sound and the combination of computational and experimental approaches results in a thorough characterisation of the earliest steps of epithelial de-stabilisation upon the loss of apical-basal polarity. In my view, the aims set by the authors are met and the experimental data provided support the claims. Interpretations are balanced and the display items are presented logically and informatively for even non-experts. Together, this work will set the basis for further investigations using apical-basal destabilisation of the follicle epithelium as a model of epithelial tumorigenesis.

Reviewer #3 (Public Review):

Barsi-Rhyne reports a novel mode of engagement of beta arrestins as endocytic adaptors and associates this novel mode together with the previously known canonical mode to the regulation of endocytosis and signaling by class A versus class B receptors. The manuscript is very well written, very good to read, almost flawless, extremely interesting, and highly relevant to the GPCR field with very well-crafted figures and fantastic microscopy.

Reviewer #3 (Public Review):

The authors use a previously developed technology, CRISPR activation screening, in which pooled sgRNAs are used to guide an RNA-associated regulatory complex (MS2-p65-HSF1 transcriptional activators) to promoter regions resulting in increased expression of a specific target gene. The authors screen two different pooled libraries TM1 (single pass) and TM2+ (multiple pass) with 20 different recombinant biotinylated soluble ligands and identified 22 novel interactions. These interactions were further characterized by SPR and cell-based binding experiments; however, several of the interactions are low affinity and were not characterized for any activity or function beyond the relatively weak biochemical binding. Therefore, while the data provide evidence of potential novel interactions, the biological relevance remains unclear.

Reviewer #3 (Public Review):

Aside from one critical reservation, I thought this paper was excellent. The figures are clear, the manuscript is well-written, the scope of the study is well-defined (i.e. it characterizes traveling beta), and the authors were circumspect in all aspects of the work, with the authors' consideration of wave propagation along different cortical meshes being but one example in a generally deft and careful approach.

However, the inverse problem remains the inverse problem, and I believe there is one thorny issue to treat regarding the 3D geometry of the central sulcus as it pertains to synchronized beta events before I can accept the authors' conclusions. After this subtle issue is treated, I believe the work will be an important step forward and generally impactful on the community interested in human brain rhythms.

The authors were gracious enough to raise the issue of spatially synchronized events themselves in their discussion: Their argument, with which I mainly agree, is that the beamformer method essentially removes synchronous components from consideration, leaving the traveling component for analysis.

However, synchronization across the sulcus introduces a further bias into event detection by means of physical source-cancellation. I will here defer to Ahlfors et al (2010), who state that "Substantial cancellation occur also for locally extended patches of simulated [cortical] activity, when the patches extended to opposite walls of sulci and gyri."

With that in mind, let's look at Figure 1, where the authors seem to show a higher density of beta events relatively deep in the sulcus compared to the sulcal walls. This is certainly an interesting result if true! But even given only the occasional synchronization of mesoscale cortical neighborhoods, it appears that events in the sulcal walls will still be systematically undersampled and those deep in the sulcus oversampled here, by vice or virtue of cortical geometry as it pertains to the magnetic field.

This spatial sampling bias could impact nearly all aspects of the event propagation analysis that follows, and so I believe it must be considered in some detail before I can fully agree with the manuscript's conclusions.

Reviewer 3 (Public Review):

This is an interesting study that describes a single cell RNAseq analysis of human menisci. The study describes cell profiling of healthy and degenerated menisci divided into two zones, inner and outer meniscus.

Reviewer #3 (Public Review):

This article by Roberts, Hayden and colleagues expands on an interesting high-throughput experimental approach developed by Kobori and Yokobayashi (2016; Angew Chem) by determining the relative activity for every possible single and double mutant of five known self-cleaving ribozymes. While this approach is not in itself new, the fact that the authors analyze their data by looking at epistasis (non-additive effects between pairs of mutations) provides an additional opportunity for extracting meaningful structural information that is proposed to be similar to chemical or enzymatic probing experiments obtained on these self-cleaving ribozymes. In fact, this type of high throughput mutagenesis analysis might provide data closer to comparative sequence analysis and as such, might provide even more reliable structural information than structural probing experiments, especially when a relative activity can be properly assessed for the studied RNAs.

(1) Overall, the experiments have been carefully performed and the data seem to be highly reliable.<br /> (2) The strength of this article is that it demonstrates the generality of the approach initially developed by Kobori and Yokobayashi (2016; Angew Chem) by validating its usefulness in identifying most (if not all) the structural features of the studied ribozymes. The determination of positive and negative epistasis is very useful as it can facilitate the identification of base pairs covariations that are indicative of RNA structural elements.<br /> (3) At the present time, the authors have not really discussed how their data analysis compares to comparative sequence analysis. This aspect is important.<br /> (4) It is necessary to mention more clearly that this article builds on the method of Kobori and Yokobayashi (2016). Overall, with the exception of a few experimental details, the experimental method described herein is almost identical to the one of Kobori and Yokobayashi (2016) and this should be better emphasized.<br /> (5) Most importantly, this article provides an analysis of self-cleaving ribozymes for which the three-dimensional structures are known. Considering the scope of this article, instead of mostly focusing on the 2D structural aspect, it would be absolutely necessary to provide more 3D structural information.<br /> (6) When a self-modifying enzymatic activity is associated with the studied RNA, a relative activity could potentially be derived from high throughput sequencing. Could the authors expand on the generality and requirement of their high throughput approach for the study of RNA?

Reviewer #3 (Public Review):

Menjivar et al. present an analysis of the role of immunosuppressive Arginase 1 in myeloid cells in pancreatic cancer. They show that depletion of Arg1 in macrophages leads to attenuation in progression from PanIN to PDAC and use single cell analysis to understand underlying changes in immune activation, including an increase in cytotoxic T cells. Interestingly, the authors observed what seems to be a compensatory upregulation in Arg1 in epithelial cells and used arginase1 inhibitor to assess the therapeutic potential of targeting Arg1 systemically. This study is overall well performed and generates novel mouse models to study immunosuppression in pancreatic cancer. While the notion that Arginase1 is immunosuppressive is not novel, the observation that Arg1 is upregulated in epithelial cells is interesting. There are several instances of overstating conclusions that, if addressed in the main text (not just relegated to the discussion section), could significantly strengthen the manuscript.

Reviewer #3 (Public Review):

The central conclusion of this beautiful experimental study is that bumblebees prefer flowers on the basis of their remembered ranking in their context, but are insensitive to their absolute properties. Thus, let's say that there 4 flower types, ranked as follows in nectar concentration: A>B>C>D. However, when the bee learns about these flowers, it does in either of two 'contexts', populated as follows: A & B, or C & D. Thus, the bee experiences that B is the worse option in the context in which it is found, and C is the better one in its own context. If, at a later time, the bee has to make a novel choice, this time between B and C, its memory for ranking leads it to prefer C over B, while its (putative) memory for nectar concentration should favour B over C. The authors find, in a variety of different treatments, evidence for the influence for ranking, but they do not find any evidence for sensitivity to absolute properties (i.e., concentration).

One difficulty that permeates the argument is the ubiquitous difficulty in proving the null hypothesis as true: lack of significant evidence for a putative effect in one or a few experiments, does not mean reliable absence of the effect.

Another difficulty is that in my view memory for absolute properties was not given a full chance: bees were always trained in situations where both dimensions (concentration and ranking) were present. In such situations, they preferentially used ranking. However, to learn ranking between flower types in sequential encounters, they must remember the absolute properties, so that in each encounter they contrast the present flower with the memory for others. Say the bee encounters a type B flower. How does it store its ranking if it doesn't remember the properties of A at all? To take this objection into account and still maintain the claim, it is necessary to say that it remembers the properties of A when in the A & B context, but it erases it from memory when in the context B & C.

Neglecting memory for concentration may be an overshadowing effect. Overshadowing is known in learning studies, and it means that, when more than one cue is paired with an outcome, the most salient between them may reduce learning about the predicting power of the other. In this case, bees may remember and use concentration when trained in contexts where there is only flower type, so that there is no chance of using ranking, and then offered choices between pairs of them. In this case, the bees would not have access to ranking, so that there would be a stronger opportunity for absolute memory to manifest itself.

In experiment 4, during training, they could move between two zones representing the 'contexts', each with 2 flower types, and they were then given choices between the 4 types, rather than just binary choices as previously. In this case, the bees did prefer the top-quality flower type (type A), which is consistent with memory for absolute concentration and with ranking, because A offered the highest concentration of the 4-type context. Why this happened is not clear, but it indicates that the context of choice may be crucial. It is known from other studies that the number of options at the time of choice can be very influential. For instance, in one study, it was shown that starlings appeared to be risk prone when offered a binary choice and risk averse when offered a trinary choice, even if the choices were all intermingled in the same sessions. In any case, this experiment raises doubts as to the claimed insensitivity to memory for nectar concentration. Another possibility is that the separation between contexts in this experiment (a partially avoidable wall) was not extreme as in the previous ones, so that the bees could now establish a ranking among the 4 types because they were all encountered intermingled to an extent.

There is one potential mechanism that may also be discussed. It is known from other species, that state at the time of learning influences subjective value of alternatives. To explain this effect I will exemplify the problem with a non-eusocial consumer. Say that food sources B and C are of equal caloric value. Say, further, that B is encountered when the subject is less food deprived than when it encounters C. Then the hedonic (conditioning) power of B will be lower, because it causes a smaller improvement in fitness (this was Daniel Bernoulli's argument regarding the concept of utility). In animal studies this effect is called State-Dependent Valuation Learning (SDVL). Since in the present experiments the context A & B was richer than the context C & D, the bees would have been in a consequently more favourable state (maybe carrying bigger sugar loads), so that each encounter with B would cause a smaller improvement than each encounter with C. This effect is totally different from remembering the ranking of flower types. The two alternative explanations for preference of C over B (ranking and SDVL) can, fortunately, be confronted because it is possible to change the state of the bees by a common 3rd source that could be used to equate or manipulate the average richness of the contexts.

All the reasons mentioned above should make it clear that this reviewer finds the study of very great interest and much merit, but considers that the conclusion for exclusive impact of ranking on preference should be tempered, or at least defended more strongly against these doubts.

Reviewer #3 (Public Review):

In this ms Li et al. examine the molecular interaction of Rabphilin 3A with the SNARE complex protein SNAP25 and its potential impact in SNARE complex assembly and dense core vesicle fusion.

Overall the literature of rabphilin as a major rab3/27effector on synaptic function has been quite enigmatic. After its cloning and initial biochemical analysis, rather little new has been found about rabphilin, in particular since loss of function analysis has shown rather little synaptic phenotypes (Schluter 1999, Deak 2006), arguing against that rabphilin plays a crucial role in synaptic function.

While the interaction of rabphilin to SNAP25 via its bottom part of the C2 domain has been already described biochemically and structurally in the Deak et al. 2006, and others, the authors make significant efforts to further map the interactions between SNAP25 and rabphilin and indeed identified additional binding motifs in the first 10 amino acids of SNAP25 that appear critical for the rabphilin interaction.

Using KD-rescue experiments for SNAP25, in TIRF based imaging analysis of labeled dense core vesicles showed that the N-terminus of SN25 is absolutely essential for SV membrane proximity and release. Similar, somewhat weaker phenotypes were observed when binding deficient rabphilin mutants were overexpressed in PC12 cells coexpressing WT rabphilin. The loss of function phenotypes in the SN25 and rabphilin interaction mutants made the authors to claim that rabphilin-SN25 interactions are critical for docking and exocytosis. The role of these interaction sites were subsequently tested in SNARE assembly assays, which were largely supportive of rabphilin accelerating SNARE assembly in a SN25 -terminal dependent way.

Regarding the impact of this work, the transition of synaptic vesicles to form fusion competent trans-SNARE complex is very critical in our understanding of regulated vesicle exocytosis, and the authors put forward an attractive model forward in which rabphilin aids in catalyzing the SNARE complex assembly by controlling SNAP25 a-helicalicity of the SNARE motif. This would provide here a similar regulatory mechanism as put forward for the other two SNARE proteins via their interactions with Munc18 and intersection, respectively.

While discovery of the novel interaction site of rabphilin with the N-Terminus of SNAP25 is interesting, I have issues with the functional experiments. The key reliance of the paper is whether it provides convincing data on the functional role of the interactions, given the history of loss of function phenotypes for Rabphilin. First, the authors use PC12 cells and dense core vesicle docking and fusion assays. Primary neurons, where rabphilin function has been tested before, has unfortunately not been utilized, reducing the impact of docking and fusion phenotype.

In particular the loss of function phenotype in figure 3 of the n-terminally deleted SNAP25 in docking and fusion is profound, and at a similar level than the complete loss of the SNARE protein itself. This is of concern as this is in stark contrast to the phenotype of rabphilin loss in mammalian neurons where the phenotype of SNAP25 loss is very severe while rabphilin loss has almost no effect on secretion. This would argue that the N-terminal of SNAPP25 has other critical functions besides interacting with rabphilin. In addition, it could argue that the n-Terminal SNAP25 deletion mutant may be made in the cell (as indicated from the western blot) but may not be properly trafficked to the site of release.

Reviewer #3 (Public Review):

The manuscript by Le T.D.V. et al used in vitro cell culture and inhibitors for cellular signaling molecules and found that GLP-1 receptor activation stimulated the phosphorylation of Raptor, which was PKA-mediated and Akt-independent. The authors reported the physiological function of this GLP-1R-PKA-Raptor in liraglutide stimulated weight loss. This timely study has high significance in the field of metabolic research for the following reasons.

(1) The authors' findings are significant in the field of obesity research. GLP-1 receptor (GLP-1R) is a successful target for diabetes (and weight loss) therapeutics. However, the mechanisms of action for the weight-loss effect of GLP-1 agonists are not fully understood. Therefore, mechanistic studies to elucidate the signaling pathways of GLP-1 receptors pertaining to weight loss at the cellular level are timely.

(2) G protein-coupled receptors (GPCRs) induces various signaling activities, which could be cellular and tissue specific. As these are an important protein family for drug targeting, understanding the basic biology of these receptors is of interest to a broad readership.

(3) The authors have made important discoveries that Exendin-4 stimulated mTORC1 signaling was essential for the anorectic effect induced by Exendin-4. The study reported in this current manuscript provides more details of brain GLP-1R signaling pathways and is innovative.

Overall, the authors have presented sufficient background in a clear and logically organized structure, clearly stated the key question to be addressed, used the appropriate methodology, produced significant and innovative main findings, took potential caveats into consideration, and made a justified conclusion.

The manuscript can be further strengthened with more clarification on the following points.

1. In Figure 1 panels B and C, please provide the quantification for pCREB/CREB. In Figure 1 panel D, please provide the quantification for pAkt/Akt.

2. The western blots to assess the signaling activities revealed the phosphorylation status of the key signaling molecules at a single time point. Whether the overall signaling dynamics have been affected is unclear.

3. Figure 3 panels A and B demonstrated the remarkable importance of the Ser791 Raptor. However, this PKA-resistant mutant did not completely abolish the weight loss effect of Liraglutide. The authors pointed out the importance of AMPK in mTORC1 signaling. Other pathways that may complement GLP-1R-PKA-Raptor signaling can be further discussed.

4. Food intake was decreased on day 2 in Figure 3D but became comparable between WT and S791A Raptor groups on the following days. Could this be due to some compensatory mechanisms?

Reviewer #3 (Public Review):

The authors identify tse1, a gene located in the type 6 secretion system (T6SS) locus of the bacterium Pseudomonas chlororaphis, as necessary and sufficient for induction of Bacillus subtilis sporulation. The authors demonstrate that Tse1 is a hydrolase that targets peptidoglycan in the bacterial cell wall, triggering activation of the regulatory sigma factor sigma-w. The sporulation-inducing effects of sigma-w are dependent on the downstream presence of the sensor histidine kinases KinA and KinB. Overall, this is a well-structured paper that uses a combination of methods including bacterial genetics, HPCL, microscopy, and immunohistochemistry to elucidate the mechanism of action of Tse1 against B. subtilis peptidoglycan. There are some concerns regarding a few experimental controls that were not included/discussed and (in a few figures) the visual representation of the data could be improved. The structure of the manuscript and experiments is such that key questions are addressed in a logical flow that demonstrates the mechanisms described by the authors.

To begin, we have concerns regarding the sporulation assays and their results. The data should be presented as "Percent sporulation" or "Sporulation (%)" - not as a "sporulation rate": there is no kinetic element to any of these measurements, so no rate is being measured (be careful of this in the text as well, for instance near lines 204). More importantly, there is no data provided to indicate that changes in percent spores are not instead just the death of non-sporulated cells. For example, imagine that within a population of B. subtilis cells, 85% of the cells are vegetative and 15% are spores. If, upon exposure to tse1, a large proportion of the vegetative cells are killed (say, 80% of them), this could lead to an apparent increase in sporulation: from 15% for the untreated population to ~50% of the treated, but the difference would be entirely due to a change in the vegetative population, not due to a change in sporulation. The authors need to clearly describe how they conducted their sporulation assays (currently there is no information about this in the methods) as well as provide the raw data of the counts of vegetative cells for their assays to eliminate this concern.

A related concern is regarding the analysis of the kinases and the effects of their deletions on the impact of Tse1. Previous literature shows that the basal levels of sporulation in a B. subtilis kinA or a kinB mutant are severely defective relative to a wild-type strain; these mutants sporulate poorly on their own. Therefore, the data presented on Lines 394+ and the associated Supplemental Figure regarding the sporulation defects of these two mutants are not compelling for showing that these kinases are required for this effector to act. It is likely that simply missing these kinases would severely impact the ability of these strains to sporulate at all, irrespective of the presence of Tse1, and no discussion of this confounding concern is discussed.

Another concern is regarding the statistical tests used in Figure 2. For statistical tests in A, B, and D, it should be stated whether a post-test was used to correct for multiple comparisons, and, if so, which post-test was used. For C, we suggest the inclusion of a mock control in addition to the two conditions already included (i.e., an extraction from an E. coli strain expressing the empty vector) to provide a stronger control comparison.

An additional concern regarding controls is that there is an absence of loading controls for the immunoblot assays. In Figure 5D and all immunoblot assays, there is no mention of a loading control, which is a critical control that should be included.

Some of the visualizations could be improved to help the reader understand and appropriately interpret the data presented. For instance, in Figures 3 and 4 the scale bars are different across each of the Figure's imaging panels. These should be scaled consistently for better comparison. Additionally, the red false colorization makes the printed images difficult to see. Black-and-white would be easier to see and would not subtract from the images.

An additional weakness of the paper is that the RNA-seq data is not fully investigated, and there is an absence of methods included regarding the RNA-seq differential abundance analysis (it is mentioned on L379-380 but no information is provided in the methods). As stated by the authors, 58% of differentially regulated genes belonged to the w regulon, but the other 42% of genes are not discussed, and will hopefully be a target of future investigations.

Another methodological concern in this paper is the limited details provided for the calculation of the permeabilization rate (Figure 4, L359, L662-664). It is not clear how, or if, cell density was controlled for in these experiments.

Finally, one weakness of the paper is the broad conclusions that they draw. The authors claim that the mechanism of sporulation activation is conserved across Bacilli when the authors only test one B. subtilis and one B. cereus strain. They further argue (lines 469+) that Tse1 requires a PAAR repeat for its targeting, but do not provide direct evidence for this possibility.

Reviewer #3 (Public Review):

Leander et al used deep mutational scanning to assess the effect of nearly all possible point mutations on four homologous bacterial allosteric transcription factors (aTFs). In particular, they identified mutations that abrogated the transcription factor response to a small molecule effector. The authors go on to use machine learning to determine which physicochemical properties distinguish mutations with allostery-eliminating effects from those without an effect. They report that mutations that eliminate the allosteric response to small molecules are quite variable across homologs and that global features are more predictive of which mutations will break allostery relative to local properties. Overall, the experimental strategy is well-chosen, and a comprehensive comparison of mutational sensitivity across allosteric homologs is highly important to understand how conserved (or not) the implementation of allostery is across homologs. Moreover, the idea to use machine learning to assess which features are most predictive of "allosteric hotspots" is very nice, and provides some insight into what physical properties distinguish mutations that influence allostery. The authors include some interesting results on transfer learning (evaluating whether models trained on one protein predict allostery in another), and the use of alternate sequence representations (e.g. UniRep) in their machine learning analyses. However - at least in the manuscript's present form - the paper suffers from key conceptual difficulties and a lack of rigor in data analysis that substantially limits one's confidence in the authors' interpretations. More specifically:

1) A key conceptual challenge shaping the interpretation of this work lies in the definition of allostery, and allosteric hotspot. The authors define allosteric mutations as those that abrogate the response of a given aTF to a small molecule effector (inducer). Thus, the results focus on mutations that are "allosterically dead". However, this assay would seem to miss other types of allosteric mutations: for example, mutations that enhance the allosteric response to ligand would not be captured, and neither would mutations that more subtly tune the dynamic range between uninduced ("off) and induced ("on") states (without wholesale breaking the observed allostery). Prior work has even indicated the presence of TetR mutations that reverse the activity of the effector, causing it to act as a co-repressor rather than an inducer (Scholz et al (2004) PMID: 15255892). Because the work focuses only on allosterically dead mutations, it is unclear how the outcome of the experiments would change if a broader (and in our view more complete) definition of allostery were considered.

2) The experimental determination of which mutations impacted allostery is given only a limited description in the methods, but if we understand what was done, the analysis seems to neglect both (1) important caveats due to assay specifics and (2) more general limitations of deep mutational scanning data. In particular:<br /> a. The separation in fluorescence between the uninduced and induced states (the assay dynamic range, or fold induction) varies substantially amongst the four aTF homologs. Most concerningly, the fluorescence distributions for the uninduced and induced populations of the RolR single mutant library overlap almost completely (Figure 1, supplement 1), making it unclear if the authors can truly detect meaningful variation in regulation for this homolog.<br /> b. The methods state that "variants with at least 5 reads in both the presence and absence of ligand in at least two replicates were identified as dead". However, the use of a single threshold (5 reads) to define allosterically dead mutations across all mutations in all four homologs overlooks several important factors:<br /> i. Depending on the starting number of reads for a given mutation in the population (which may differ in orders of magnitude), the observation of 5 reads in the gated non-fluorescent region might be highly significant, or not significant at all. Often this is handled by considering a relative enrichment (say in the induced vs uninduced population) rather than a flat threshold across all variants.<br /> ii. Depending on the noise in the data (as captured in the nucleotide-specific q-scores) and the number of nucleotides changed relative to the WT (anywhere between 1-3 for a given amino acid mutation) one might have more or less chance of observing five reads for a given mutation simply due to sequencing noise.<br /> iii. Depending on the shape and separation of the induced (fluorescent) and uninduced (non-fluorescent) population distributions, one might have more or less chance of observing five reads by chance in the gated non-fluorescent region. The current single threshold does not account for variation in the dynamic range of the assay across homologs.<br /> c. The authors provide a brief written description of the "weighted score" used to define allosteric hotspots (see y-axis for figure 1B), but without an equation, it is not clear what was calculated. Nonetheless, understanding this weighted score seems central to their definition of allosteric hotspots<br /> d. The authors do not provide some of the standard "controls" often used to assess deep mutational scanning data. For example, one might expect that synonymous mutations are not categorized as allosterically dead using their methods (because they should still respond to ligand) and that most nonsense mutations are also not allosterically dead (because they should no longer repress GFP under either condition). In general, it is not clear how the authors validated the assay/confirmed that it is giving the expected results.<br /> 3) In several places, the manuscript lacks important statistical analyses needed to firmly establish the authors' claims<br /> a. The authors performed three replicates of the experiment, but reproducibility across replicates and noise in the assay is not presented/discussed<br /> b. In the analysis of long-range interactions, the authors assert that "hotspot interactions are more likely to be long-range than those of non-hotspots", but this was not accompanied by a statistical test (Figure 2 - figure supplement 1)

4) Data availability and analysis transparency need improvement. The raw fastq reads do not seem to be publicly available, nor did we see access to the code used to perform the analysis. If the code is not provided, the description of the analysis in the methods section needs to be more detailed for reproducibility.

Overall, these concerns with fundamental aspects of the data analysis make it challenging to assess the reproducibility of the results, the fidelity of the assay (in reporting allosterically dead mutations), and the extent to which the data robustly support the authors' claims.

Reviewer #3 (Public Review):

The authors assess response to ocean acidification with three populations of mussels encompassing two species: Mytilus trossulus from the intertidal and subtidal and M. galloprovincialis from a subtidal aquaculture farm. All three species received an ambient of low pH treatment prior to a freezing treatment. The authors find species differences in freeze tolerance in mussels, with intertidal M. galloprovincialis showing the least freeze tolerance. The authors go a step further and do a comprehensive assessment of the metabolic capacity and molecular components with analyses of amino acids, fatty acids, and osmolytes and anaerobic byproducts.

The authors hypothesized metabolic changes due to OA and cold temperatures, yet they demonstrated a significant amount of stasis with high similarity among species at the molecular level. The fatty acids in the intertidal M trossulus, the most freeze tolerant, did not change. Further, there is little explanation of molecular/metabolic changes that could explain their results. Because of this somewhat unexpected lack of signal of these stressors, I would like to see an enhanced explanation of animal homeostasis. The authors mention previous results relating to heat stress, and I thought it would be beneficial to discuss how the lack of a molecular response to freezing is related to the strong responses seen in heat stress.

The idea that species in fluctuating environments (here, the intertidal) might respond differently to those in constant environments (here, the subtidal) has been explored in multiple systems. These general concepts could be elaborated on more in the paper to increase the connection to other studies.

Reviewer #3 (Public Review):

This manuscript presents and analyzes a novel calcium-dependent model of synaptic plasticity combining both presynaptic and postsynaptic mechanisms, with the goal of reproducing a very broad set of available experimental studies of the induction of long-term potentiation (LTP) vs. long-term depression (LTD) in a single excitatory mammalian synapse in the hippocampus. The stated objective is to develop a model that is more comprehensive than the often-used simplified phenomenological models, but at the same time to avoid biochemical modeling of the complex molecular pathways involved in LTP and LTD, retaining only its most critical elements. The key part of this approach is the proposed "geometric readout" principle, which allows to predict the induction of LTP vs. LTD by examining the concentration time course of the two enzymes known to be critical for this process, namely (1) the Ca2+/calmodulin-bound calcineurin phosphatase (CaN), and (2) the Ca2+/calmodulin-bound protein kinase (CaMKII). This "geometric readout" approach bypasses the modeling of downstream pathways, implicitly assuming that no further biochemical information is required to determine whether LTP or LTD (or no synaptic change) will arise from a given stimulation protocol. Therefore, it is assumed that the modeling of downstream biochemical targets of CaN and CaMKII can be avoided without sacrificing the predictive power of the model. Finally, the authors propose a simplified phenomenological Markov chain model to show that such "geometric readout" can be implemented mechanistically and dynamically, at least in principle.

Importantly, the presented model has fully stochastic elements, including stochastic gating of all channels, stochastic neurotransmitter release and stochastic implementation of all biochemical reactions, which allows to address the important question of the effect of intrinsic and external noise on the induction of LTP and LTD, which is studied in detail in this manuscript.

Mathematically, this modeling approach resembles a continuous stochastic version of the "liquid computing" / "reservoir computing" approach: in this case the "hidden layer", or the reservoir, consists of the CaMKII and CaM concentration variables. In this approach, the parameters determining the dynamics of these intermediate ("hidden") variables are kept fixed (here, they are constrained by known biophysical studies), while the "readout" parameters are being trained to predict a target set of experimental observations.

Strengths:

1) This modeling effort is very ambitious in trying to match an extremely broad array of experimental studies of LTP/LTD induction, including the effect of several different pre- and post-synaptic spike sequence protocols, the effect of stimulation frequency, the sensitivity to extracellular Ca2+ and Mg2+ concentrations and temperature, the dependence of LTP/LTD induction on developmental state and age, and its noise dependence. The model is shown to match this large set of data quite well, in most cases.

2) The choice for stochastic implementation of all parts of the model allows to fully explore the effects of intrinsic and extrinsic noise on the induction of LTP/LTD. This is very important and commendable, since regular noise-less spike firing induction protocols are not very realistic, and not every relevant physiologically.

3) The modeling of the main players in the biochemical pathways involved in LTP/LTD, namely CaMKII and CaN, aims at sufficient biological realism, and as noted above, is fully stochastic, while other elements in the process are modeled phenomenologically to simplify the model and reveal more clearly the main mechanism underlying the LTP/LTD decision switch.

4) There are several experimentally verifiable predictions that are proposed based on an in-depth analysis of the model behavior.

Weaknesses:

1) The stated explicit goal of this work is the construction of a model with an intermediate level of detail, as compared to simplified "one-dimensional" calcium-based phenomenological models on the one hand, and comprehensive biochemical pathway models on the other hand. However, the presented model comes across as extremely detailed nonetheless. Moreover, some of these details appear to be avoidable and not critical to this work. For instance, the treatment of presynaptic neurotransmitter release is both overly detailed and not sufficiently realistic: namely, the extracellular Ca2+ concentration directly affects vesicle release probability but has no effect on the presynaptic calcium concentration. I believe that the number of parameters and the complexity in the presynaptic model could be reduced without affecting the key features and findings of this work.

2) The main hypotheses and assumptions underlying this work need to be stated more explicitly, to clarify the main conclusions and goals of this modeling work. For instance, following much prior work, the presented model assumes that a compartment-based (not spatially-resolved) model of calcium-triggered processes is sufficient to reproduce all known properties of LTP and LTD induction and that neither spatially-resolved elements nor calcium-independent processes are required to predict the observed synaptic change. This could be stated more explicitly. It could also be clarified that the principal assumption underlying the proposed "geometric readout" mechanisms is that all information determining the induction of LTP vs. LTP is contained in the time-dependent spine-averaged Ca2+/calmodulin-bound CaN and CaMKII concentrations, and that no extra elements are required. Further, since both CaN and CaMKII concentrations are uniquely determined by the time course of postsynaptic Ca2+ concentration, the model implicitly assumes that the LTP/LTD induction depends solely on spine-averaged Ca2+ concentration time course, as in many prior simplified models. This should be stated explicitly to clarify the nature of the presented model.

3) In the Discussion, the authors appear to be very careful in framing their work as a conceptual new approach in modeling STD/STP, rather than a final definitive model: for instance, they explicitly discuss the possibility of extending the "geometric readout" approach to more than two time-dependent variables, and comment on the potential non-uniqueness of key model parameters. However, this makes it hard to judge whether the presented concrete predictions on LTP/LTD induction are simply intended as illustrations of the presented approach, or whether the authors strongly expect these predictions to hold. The level of confidence in the concrete model predictions should be clarified in the Discussion. If this confidence level is low, that would call into question the very goal of such a modeling approach.

4) The authors presented a simplified mechanistic dynamical Markov chain process to prove that the "geometric readout" step is implementable as a dynamical process, at least in principle. However, a more realistic biochemical implementation of the proposed "region indicator" variables may be complex and not guaranteed to be robust to noise. While the authors acknowledge and touch upon some of these issues in their discussion, it is important that the authors will prove in future work that the "geometric readout" is implementable as a biochemical reaction network. Barring such implementation, one must be extra careful when claiming advantages of this approach as compared to modeling work that attempts to reconstruct the entire biochemical pathways of LTP/LTD induction.