Reviewer #1 (Public Review):

In this manuscript, McLeod and Gandon propose a framework for understanding multidrug resistance (MDR) evolution in a structured population in terms of linkage disequilibrium (LD) dynamics, and apply this framework to three concrete examples of MDR evolution. I was asked to evaluate this manuscript, as well as the authors' response to comments from previous reviewers. My expertise is in epidemiological modelling of antibiotic resistance; I am not hugely familiar with population genetics.

Overall, I think the authors address an important and interesting question, and I think the approach has the potential to generate valuable insights. I also think the authors addressed the previous reviewers' comments well. However, I have substantial concerns about the modelling framework and the interpretation of the results. In particular: i) there are some problems with the interpretation that LD arises from variation in susceptible density; ii) presenting these results as a re-interpretation and generalisation of Lehtinen et al. 2019 is incorrect; and iii) the modelling of additive transmission costs needs further thought/explanation.

1) Interpretation of results and re-interpretation of Lehtinen et al. 2019.

The authors present their results as a generalisation of the effect observed in Lehtinen et al. 2019. Both models show that variation in the strength of selection for resistance between populations can give rise to LD in a model of multiple resistances. In Lehtinen et al., this variation in selection is attributed to variation in clearance rate. The authors re-interpreting the effect as arising from variation in susceptible density instead. This re-interpretation is incorrect: the change in how costs of resistance are modelled (additive here, multiplicative in Lehtinen et al.) changes the evolutionary dynamics, so the two models capture different evolutionary effects. (See points 2 and 3 for further discussion of additive vs multiplicative costs).

One way to see this is to consider a simple model of single resistance as presented in Lehtinen et al. eqn 1, in which resistance is selected for when: B_r/a_r > B_s/(a_s + tau), where "B" is the transmission rate, "a" the clearance rate and tau the treatment rate. Re-arranging for tau shows how the threshold of selection for resistance depends on the strain's properties (B and a) under different assumptions about cost. With an additive cost in transmission (i.e. B_r = B_s - c), this threshold depends on both transmission rate and clearance rate, predicting LD if populations vary in either transmissibility or duration of carriage. With an additive cost in clearance, this threshold is independent of the strain's properties, predicting no LD. These are precisely the results the authors describe lines 268-277 and Figure 3.

However, if the costs are multiplicative, this threshold depends on clearance rate only, whether costs are modelled as part of clearance or transmission rate. This is why the model in Lehtinen et al. 2019 predicts LD when populations vary in duration of carriage, even when there is no transmission cost. The author's re-interpretation of the effect in Lehtinen et al. as arising from variation in the density of susceptibles, contingent on an explicit transmission cost, is therefore not correct. More generally, representing one model as a generalisation of the other is misleading.

I am also not sure about the authors' interpretation that the effects in the model with additive costs arise from variation in susceptible density. Variation in the density of susceptibles can also be generated by variation in the overall population density, so if I understand correctly, this interpretation would predict that LD would arise if the population density was different between populations? And that the selective pressure on single resistance would also depend on overall population density (argument stating line 261)? I am not able to reproduce this dependence of population density in a simple model. I would instead interpret the effect the authors observe as arising because the same additive transmission cost is much more significant if the baseline transmission rate is low (e.g. with c = 1, a strain with B_s 1 would never evolve resistance because B_r would be 0, which would not be the case for a strain with baseline transmission rate B_s = 3).

The problem with the interpretation in terms of susceptible density is clear in the section on serotype dynamics. The main text refers to serotype-specific susceptibles (S^x) (line 303) and explains observed effects in terms of variation in S^x. In the supporting information however, the authors present a model of serotype dynamics which does not have serotype-specific susceptible classes and the pool of susceptibles is the same for all serotypes (eqn 43). While I absolutely agree this is a better model to study transient effects than introducing a serotype-specific susceptible class, I don't understand what the authors mean by serotype-specific susceptible density in the main text.

2) The use of an additive transmission cost

The use of an additive transmission cost requires further consideration/discussion. An additive transmission cost is difficult to interpret epidemiologically and can lead to implausible consequences. For example, if costs are high enough compared to baseline transmission rate, additive costs with no epistasis would lead to a negative transmission rate for the dually resistant strains, which does not make sense (say B_ab = 2 and B_Ab = B_aB = 0.5, then B_AB = -1).

3) Why is epistasis defined in terms of an additive rather than multiplicative expectation?

I also have quite a basic question about the overall framework (eqn. 2). In the modelling framework, epistasis is the difference between the actual per capita growth rate of the dually-resistant infections and the expected growth rate, defined as the sum of the difference between the growth rates of the singly-resistant infections and the baseline rate. It was not obvious to me whether the expectation needs to be additive, or whether this is a question of definition (could the expectation be defined, for example, as a multiplicative rather than additive effect?). In particular, I was wondering about this in the context of the authors' suggestion that multiplicative costs are problematic because they give rise to epistasis - this seemed a little tautological to me because epistasis has been specifically defined as deviation from an additive expectation. I think a discussion about why epistasis is defined in terms of additive effects, and the implications for the derivation of the dynamics of D, would be very interesting and also helpful in making the paper more accessible.

Joint Public Review:

Chen et al. identify LIN37, a member of the DREAM transcriptional repressor complex as a new regulatory factor in DNA double-strand break (DSB) end resection. The study commences with a CRISPR-Cas9 screen for chromatin associated RPA in quiescent pre-B cells that lack DNA ligase 4. In addition to the established anti-resection components 53BP1-Shieldin, findings that validates the screen, the authors identify the transcriptional repression complex LIN37-DREAM. This well executed study makes a number of compelling observations. Namely, that LIN37 limits end-resection only in quiescent cells, that it is not epistatic with 53BP1-Shieldin, and loss of Lin37 allows expression of hundreds of genes including genes involved in DSB end resection and homologous recombination. yielding Rad51 filament formation and HR. All phenotypes were recapitulated by a LIN37 mutant that that does not interact with DREAM. Moreover, generality is shown across human and mouse cell types using either Cas9 breaks or IR.

The results presented in this manuscript are fascinating and should open a new avenue to study cell cycle dependent regulation of DSB repair. The combination of cell biological and end-seq approaches make a very convincing argument for this unanticipated finding. The conclusions drawn from this work are for the most part well-supported by the data, which are of high-quality, and the experiments are rigorously performed. What is unclear is whether these effects are direct or indirect, including whether other DREAM factors also participate in this end-resection suppression. It would also be nice to know the cellular consequences of dysregulated HR that occurs in quiescent LIN37-deficient cells.

Reviewer #1 (Public Review):

This paper shows that the sphingomyelin-degrading enzyme SMPDL3B is under transcriptional control of SIRT1 and c-Myc in ESCs, and that loss of SIRT1 lowers sphingomyelin content of ESCs. Sphingomyelin accumulation in SIRT1-deficient ESCs is associated with changes to membrane fluidity and the abundance of several differentiation markers. The paper also includes interesting data showing that maternal HFD feeding increases the SM content of SIRT1 KO embryos. The studies presented are thorough and the data are interesting.

Reviewer #1 (Public Review):

The authors used EEG-based multivariate pattern analysis and acute stress induction to assess the neural representations mediating a previously demonstrated influence of stress on the balance between goal-directed and habitual responding. They found that stress reduced neural outcome representations and enhanced response representations - results that are consistent with associative structures thought to mediate goal-directed and habitual response strategies, respectively. The study addresses an important and open question, and the combination of clinical, neural and behavioral assays is appealing. However, the interpretability, and thus impact, is threatened by an apparent lack of temporal synchrony between relevant measures, and by the potential effects of social feedback.

Specifically, it is hard to understand how neural and behavioral devaluation differences between groups can be stress related given that they emerge at a point when differences in stress measures (e.g., cortisol) are no longer present. It seems more likely that, at the time when devaluation insensitivity became more pronounced in the stress group, this group was being released from stress, perhaps experiencing corollary fatigue or buoyancy.

Another concern is that it is unclear whether the "Error" feedback screen was being employed during devaluation blocks. This is important, because most human psychology experiments use accuracy as the only incentive, and it appears to be a pretty effective motivator. Given that participants in the stress group had just been subjected to an aversive social stressor, they might have found the socially relevant error feedback more painful than the relatively minor response cost.

Reviewer #1 (Public Review):

The novelty is that the sensitivity to TRAIL and co-localisation in lipid rafts is maintained in oxaliplatin-resistant cells post-treatment (previously shown in cells treated simultaneously with Oxaliplatin and TRAIL), and that this has the potential to target colorectal cancer cells through lipid-based TRAIL delivery. While the implications of the findings are compelling, the study would benefit from clarifying several open questions from the findings and demonstrating robust methodology.

Specifically, the findings could be strengthened by improvements of the image and statistical analysis. The authors compare the area with stain positivity per cell, which assumes no morphological differences between OxR cells and parental cells and also relies on a threshold. To avoid this, robust methods would need to be used to compare the intensity distributions and pixel intensity spatial correlation. Furthermore, there is a lot of variability in the effect of reduced cell viability of circulating tumour cells across patients and draws. Inter- and intra-patient variation should be considered when statistically comparison the cell viability of circulating tumour cells to TRAIL-conjugated liposomes.

Really liked beginning, end was boring

Reviewer #1 (Public Review):

The study by Huang et al. report on direct recordings (using DBS electrodes) from the human habenula in conjunction with MEG recordings in 9 patients. Participants were shown emotional pictures. The key finding was a transient increase in theta/alpha activity with negative compared to positive stimuli. Furthermore, there was a later increase in oscillatory coupling in the same band. These are important data, as there are few reports of direct recordings from the habenula together with the MEG in humans performing cognitive tasks. The findings do provide novel insight into the network dynamics associated with the processing of emotional stimuli and particular the role of the habenula.

Recommendations:

How can we be sure that the recordings from the habenula are not contaminated by volume conduction; i.e. signals from neighbouring regions? I do understand that bipolar signals were considered for the DBS electrode leads. However, high-frequency power (gamma band and up) is often associated with spiking/MUA and considered less prone to volume conduction. I propose to also investigate that high-frequency gamma band activity recorded from the bipolar DBS electrodes and relate to the emotional faces. This will provide more certainty that the measured activity indeed stems from the habenula.

Figure 3: the alpha/theta band activity is very transient and not band-limited. Why refer to this as oscillatory? Can you exclude that the TFRs of power reflect the spectral power of ERPs rather than modulations of oscillations? I propose to also calculate the ERPs and perform the TFR of power on those. This might result in a re-interpretation of the early effects in theta/alpha band.

Figure 4D: can you exclude that the frontal activity is not due to saccade artifacts? Only eye blink artifacts were reduced by the ICA approach. Trials with saccades should be identified in the MEG traces and rejected prior to further analysis.

The coherence modulations in Fig 5 occur quite late in time compared to the power modulations in Fig 3 and 4. When discussing the results (in e.g. the abstract) it reads as if these findings are reflecting the same process. How can the two effect reflect the same process if the timing is so different?

Be explicit on the degrees of freedom in the statistical tests given that one subject was excluded from some of the tests.

Reviewer #1 (Public Review):

The authors demonstrate in this study that it is possible to train mice to perform a challenging tactile discrimination task, in a highly controlled manner, in a fully automated setup in which the animals learn to head-fix voluntarily. A number of well described tricks are used to prolong the self-fixation time and thereby obtain enough training time to reach good performance when the decision perceptual decision is difficult. In addition the study establish that this experimental design allows targeted silencing of relatively deep brain areas through a clear skull preparation.

It has already been demonstrated that mice can perform voluntary head-fixation and can do behavioral tasks in this context. However, this is the first time this methodology is applied to first to a tactile task and second to a task that mice learn is thousands of trials. Another advantage of the present technique is that it is fully automated and allows training without virtually any human intervention.

The demonstration that optogenetic silencing can be performed in this context is nice but not very surprising as already done in other contexts. Nevertheless it is an interesting application of self head-fixation. The authors should make sure that a maximum of information is available relative to the efficiency of the silencing (fraction of cells silenced) and about its impact on the behavior (does it result or not in a complete impairment?).

Reviewer #1 (Public Review):

In this ms, Voroslakos et al., describe a customizable and versatile microdrive and head cap system for silicon probe recordings in freely moving rodents (mice and rats). While there are similar designs elsewhere, the added value here is: a) a carefully designed solution to facilitate probe recovery, thus reducing experimental costs and favoring reproducibility; b) flexibility to accommodate several microdrives and additional instrumentation; c) open access design and documentation to favor customization and dissemination. Authors provide detailed description to faccilitate building the system.

Personally, I found this resource very useful to democratize multi-site recordings, not only for standard silicon probes, but also more novel integrated optoelectrodes and neuropixels. While there are other solutions, this design is quite simple and versatile. A potential caveat is whether it could be perceived as just an upgrade, given some similitudes with previous designs (e.g. Chung et al., Sci Rep 2017 doi: 10.1038/s41598-017-03340-5) and concepts (Headley et al., JNP doi: 10.1152/jn.00955.2014). However, the system presented in this paper provides added value and knowledge-based solutions to make silicon probe recordings more accessible.

DOI: 10.1016/j.devcel.2021.03.027

Resource: (ZFIN Cat# ZDB-ALT-101006-1,RRID:ZFIN_ZDB-ALT-101006-1)

Curator: @Naa003

SciCrunch record: RRID:ZFIN_ZDB-ALT-101006-1

What is this?

Reviewer #1 (Public Review):

The authors note how previous studies on myocardial infarction have usually studied individual tissues and not examined the cross talk between tissues and their dysregulation. To address this challenge they have therefore performed, in a mouse model of MI, an integrated analysis of heart, liver, skeletal muscle and adipose tissue responses at 6 and 24 hours. They have then validated their findings at 24 hours in two independent mouse model data sets.

A major strength is their comprehensive approach. They have used high throughput RNA seq and applied integrative network analysis. They show for multiple genes whether they are up regulated or down regulated in these four tissues at the 6 and 24 hour time points and whether the regulation directions are concordant or opposite and note in particular that for the liver both concordant and opposite effects occur. They identify key tissue specific clusters in each tissue and identify the key genes in each cluster. Finally they use whole body modelling to identify cross talk between tissues.

A further strength of this paper is the integration of transcriptomic data (differential expression, functional analysis and reporter metabolite analysis). The final strength is the very clear presentation of the findings and their implications such that the reader gets a very clear message and at the same time can go in to more detail if this is their area of research interest.

There are no major weaknesses. The authors have achieved their aims and the data supports their conclusions.

This work represents a major advance in both methodology and understanding of a multi tissues approach to the study of the metabolic impact of MI and the underlying up and down regulation of relevant genes.

The relevance of these findings in human MI will need to be tested and may ultimately have therapeutic implications.

Reviewer #1 (Public Review):

This study examines the evolution of tooth loss (endentulism) across amphibians, finding many cases of endentulism and the majority of losses in frogs and toads (Anura). The study is the first to characterize amphibian endentualism at this scale and the authors have collected an impressive dataset. The study shows correlations among endentulism, diet, and jaw length and discusses potential proximal and ultimate explanations for endentulism in anurans. There are important data provided in this manuscript (e.g. region-specific tooth loss - dentary, maxilla, premaxilla, palate), which could further illuminate developmental pathways responsible for endentulism as well as evolutionary correlates of region-specific tooth loss. The authors collated an impressive breadth of diet data but clearer documentation and examination of that data would allow readers to better evaluate the support for the relationship between diet and endentulism. Overall, this study reveals an interesting evolutionary pattern of endentulism, with the number of independent evolutionary cases of endentulism in amphibians (and anurans, in particular) dwarfing those found in other tetrapod clades.

Reviewer #1 (Public Review):

The authors examine measures of viral reservoir to understand how different antiviral treatment regimens impact residual virus in HIV infection. They find that NNRTI-based treatments are associated with lower viral reservoirs than PI-based regimens, suggesting they may have some advantage at reducing HIV levels long term.

Reviewer #1 (Public Review):

The study by Song and colleagues explores the role of circRNAs in fibrosis of the endometrium. Endometrial cells for patients with and without fibrosis were subjected to expression profiling analysis, and circPTPN12 and miR-21-5p were strongly separate in fibrosis in endometrial, with circPTPN12 acting as an inhibitory factor for miR-21-5p. Through the use of various molecular approaches, the authors further that miR-21-5p inhibition results in upregulation of ΔNp63α, and transcription factor that induces EMT. The role of circPTPN12 was also confirmed in vivo using a mouse model of mechanically induced endometrial fibrosis. The authors concluded that targeting the path circPTPN12/miR-21-5p/∆Np63α may be a therapeutic strategy for endometrial fibrosis.

The authors clearly and convincingly show the involvement of the circPTPN12/miR-21-5p/∆Np63α in EMT and its potential involvement in endometrial fibrosis. Whether or not this can be a therapeutic target is too preliminary at this point. First because the in vivo experiments confirm the link between circPTPN12/miR-21-5p/∆Np63α at the RNA level only (p63) and it would be more convincing to see protein data as well. The involvement of p63 in the process remains a little elusive in this paper. In addition, if the authors believe this pathway can be a real future target to treat endometrial fibrosis, they could better contextualise such a statement, specifically describe what kinds of therapeutic intervention they think of, like regression or prevention of fibrosis. These should be tested in vitro and in vivo. More evidence of the involvement of circPTPN12/miR-21-5p/∆Np63α and the correlation between the three players using clinical material is also necessary.

Reviewer #1 (Public Review):

-In the present paper the authors have attempted to develop a novel statistical method and sequence reporting tool that combines epidemiological and sequence data to provide a rapid assessment of the probability of HCAI among HOCI cases (defined as first positive test >48 hours following admission) and to identify infections that could plausibly constitute outbreak events.

-As healthcare-associated infections in hospitals present a significant health risk to both vulnerable patients and healthcare workers, significant improvements to provide a rapid assessment of the probability of HCAI among HOCI cases is of utmost importance in a pandemic setting.

-The strength of the paper is that they have successfully used a large number of virus sequence data from two UK cities with selected hospitals and developed a statistical method to bring these together with classical epidemiological data, which has resulted in a sequence reporting tool (SRT) that was evaluated in relation to:

-The IPC classification system recommended by PHE,

-The PHE definition of healthcare-associated COVID-19 outbreaks (using a 2 SNP threshold).

-They show the added value of combining the two systems. Obviously, this can only work prospectively in a setting like in the UK, where indeed a system like the COVID-19 Genomics (COG) UK initiative is effectively in place. They conclude that through their retrospective application to clinical datasets, to have demonstrated that the methodology is able to provide confirmatory evidence for most PHE-defined definite and probable HCAIs and provide further information regarding indeterminate HCAIs. Therefor, the SRT may allow IPC teams to optimise their use of resources on areas with likely nosocomial acquisition events.

-The acquisition of the extensive prospective datasets necessary to use the system requires a non-negligible investment that is possible in a setting in which sequencing routine and phylogenetic analyses can be carried out in real time. The added value of the methodology should eventually justify the investment.

Reviewer #1 (Public Review):

In this manuscript, Katrukha et al. use advanced microscopy techniques to quantify the organization of microtubules within neuronal dendrites. This is a challenge due to the tight bundling of microtubules along neuronal processes, and they tackle it using two techniques: STED microscopy and Expansion Microscopy. They thus measure by two independent procedures the density of microtubules along proximal dendrites and the proportion of acetylated and tyrosinated microtubules, showing that dendritic microtubules are either acetylated or tyrosinated, but rarely present both or none of these post-translational modifications.

The manuscript is a significant methodological advance, with a combination of new sample preparation and image analysis procedures that provide images of dendritic microtubules with enhanced quality. These enhancements don't allow the tracing of a significant number of individual microtubules. They are used for refined intensity-based statistics that provide a worthy insight into the organization of dendritic microtubules. Here the approaches are robust, with clear explanation of the quantitation workflow using open-source tools that are made available. One can regret that these measurements don't address the longitudinal dimension of microtubules, which could help revisiting and answering a couple of important questions (existence of modification domains along single microtubules, microtubule orientation...).

Nonetheless, the authors bring forward very good imaging and solid quantification workflows that allow them to convincingly answer the main question they ask: what is the radial organization of dendritic microtubules, including their post-translational modification state?

Reviewer #1 (Public Review):

This work is a powerful example of thinking across silos. It combines much knowledge of innate and adaptive immunity, with primate evolution of certain antigens lost only in certain primate lineages and tests an important idea about host-mediated, antibody dependent shaping of gut microbiota using laboratory mice with different engineered genetic alterations. Gut microbiota are all the rage these days, but is often forgotten that these microbial communities represent formidable danger that is really too close (one epithelial layer away) for comfort. The authors demonstrate in laboratory mice, how antibodies against non-self sugar molecules present on bacteria can shape the microbiome. Claims and conclusions seem justified by the data presented.

Reviewer #1 (Public Review):

Facial muscles control the execution of essential tasks like eating, drinking, breathing and (in most mammals) tactile exploration. The activity of motor neurons targeting different muscles are coordinated by premotor regions distributed throughout brainstem. The precise identity of these cells and regions in adults is presently unclear, largely due to technical challenges. In the current work, Takaoh and colleagues develop an elegant strategy to label premotor neurons that target select muscles and register these cells on a common digital atlas. Their work confirms and also extends previous studies in neonates and provides a useful resource for the field.

Reviewer #1 (Public Review):

The study by Diboun et al. aims to investigate methylation profiles in Paget's disease of bone patients. Many of the genes identified near areas of differentially methylated sites were known to be involved in osteoclast differentiation, viral infection and mechanical loading. These gene pathways are known to play a role in the pathogenesis of PDB. The strength of this study is that it is the first study to look at changes in methylation profiles in Paget's disease of bone patients. Additionally, the genes identified as having differentially methylated sites suggest that environmental factors such as host immune responses may be altered and play a role in the pathogenesis of PBD. The main weakness of this study is that the cells that were analyzed for changes in methylation sites were not osteoclasts the cells of interest in PBD. While many of the genes identified have been shown to play a role in regulation of the skeletal system, results should be interpreted with caution until they are validated in bone tissue.

DOI: 10.1016/j.xpro.2021.100465

Resource: (ZFIN Cat# ZDB-ALT-070316-1,RRID:ZFIN_ZDB-ALT-070316-1)

Curator: @Naa003

SciCrunch record: RRID:ZFIN_ZDB-ALT-070316-1

What is this?

DOI: 10.1016/j.xpro.2021.100465

Resource: (ZFIN Cat# ZDB-ALT-141023-1,RRID:ZFIN_ZDB-ALT-141023-1)

Curator: @Naa003

SciCrunch record: RRID:ZFIN_ZDB-ALT-141023-1

What is this?

Reviewer #1 (Public Review):

In this technically difficult study of a crucial and understudied area of the human anterior temporal lobe (ATL), the authors set out to investigate the possibility that representations in this area are dynamic, in keeping with its putative role as a semantic hub. In short, they report evidence for stable representations in posterior areas and dynamic representations in anterior areas.

The major strength of this paper is in the nature of the physiological data (ECOG) and the complexity of the associated modeling and computational work. In particular, the consideration of and attempt to model dynamic representations is a real strength.

The major weakness is a slight lack of direct statistical tests to back up certain claims. For example, there is a discussed difference between the posterior and anterior electrode, but not much of direct statistical comparison of those areas. The model which has the best performance clearly changes over time, but there are not direct statistical comparisons of the models performance over that period.

Overall, there is some evidence for a dynamic representation in this area, and the analyses here do point at the need for a more thorough (i.e., considering the possibility of dynamic change) and generally applied approach to studying representations.

Reviewer #1 (Public Review):

The protein kinase RIPK3 was widely known to promote a form of lytic cell death termed necroptosis. However, RIPK3 could also promote apoptotic cell death under certain conditions. However, the mechanism by which RIPK3 promotes apoptosis and the physiological relevance of this apoptotic activity were not understood. In this study, the authors provided answers to these two questions.

Strengths:

The authors found that a specific phosphorylation on RIPK3 plays a critical role in the switch of RIPK3 into an apoptosis-inducing protein. The authors provided strong evidence to support their conclusion using mouse genetics and demonstrated a role for this RIPK3 activity in reproductive physiology.

Weaknesses:

Although the authors succeeded in finding the protein phosphorylation that controls the form of cell death mediated by RIPK3, key questions remained as to how this modification prevents RIPK3 from promoting necroptosis. Also, the authors implied that the kinase activity of RIPK3 is critical in this switch to apoptosis. However, the phenotypes of mice that lack RIPK3 kinase activity do not match that of the mice that harbor mutations that mimic this phosphorylation.

Overall, this work should provide useful information for future studies to further examine the mechanism by which RIPK3 controls different types of cell death in normal and pathophysiology.

Reviewer #1 (Public Review):

The largest concern with the manuscript is its use of resting-state recordings in Parkinson's Disease patients on and off levodopa, which the authors interpret as indicative of changes in dopamine levels in the brain but not indicative of altered movement and other neural functions. For example, when patients are off medication, their UPDRS scores are elevated, indicating they likely have spontaneous movements or motor abnormalities that will likely produce changed activations in MEG and LFP during "rest". Authors must address whether it is possible to study a true "resting state" in unmedicated patients with severe PD. At minimum this concern must be discussed in the manuscript.

This reviewer was unclear on why increased "communication" in the medial OFC in delta and theta was interpreted as a pathological state indicating deteriorated frontal executive function. Given that the authors provide no evidence of poor executive function in the patients studied, the authors must at least provide evidence from other studies linking this feature with impaired executive function.

In this article, authors repeatedly state their method allows them to delineate between pathological and physiological connectivity, but they don't explain how dynamical systems and discrete-state stochasticity support that goal.

Reviewer #1 (Public Review):

The authors generated new transgenic fly lines with the human dopamine transporter (hDAT-WT) and the hDAT with the R445C mutation (hDAT-R445C). Studies in the hDAT-R445C flies show a decrease of tissue DA content and a loss of TH+ PPL1 neurons indicating an effect of the DAT mutation on dopamine neuron phenotype or cell survival rather than general DA levels per se. The motor phenotypes observed in the fly include a decrease in the time to initiate flight and in the velocity of locomotion (vigor) but not in the velocity of locomotion initiation or grooming behavior. These behaviors are consistent with the bradykinesia observed in patients. This model system could potentially be used to assay for specific modulators of the mutant to restore surface expression, TH expression and motor behavior.

In the recombinant cell culture system (HEK Cells), the major consequence of the mutation is a decrease in cell surface expression (there is a decrease in conversion to the mature form). A change in the Km is difficult to ascertain with such a dramatic change in the cell surface expression level but looks to be dramatically decreased (higher affinity). These data differ somewhat from those reported in the study by Ng et al, 2014 where the Bmax for CFT was slightly reduced and the affinity was significantly decreased (Km was ~8 fold higher) as was the Ki for DA inhibition of CFT. It should be noted that the decrease in cell surface expression of R445C reported by Ng et al was also not as dramatic as what the same group demonstrated for the other mutation, R87L, that was compound heterozygous in this family. Differences in the transport properties between the two studies should be discussed.

X-ray crystallography and molecular modeling provide novel insights into how the mutation (and other substitutions at this site) affects structure-function relationships of the transporter with respect to gating, uptake and efflux. This information could be used to design modulators of the transporter mutants to rescue cell surface expression or function.

The behavioral effect of CQ on the mutant flies was on the time to flight initiation, which decreased. Locomotion was not tested.

The value of the study is the creation of the flies for screening and the crystallography and molecular modeling studies which examined the impact of this residue on function in detail. The weakness of the study was the limited characterization of the transport properties and cell surface expression in the flies. Being able to tie together the different studies into a cohesive understanding of what happens in patients and thus what needs to be corrected in patients is an important goal of the study. Some of the key questions needed to achieve this understanding were not fully addressed.

Reviewer #1 (Public Review):

One proposed function of biomolecular condensates is in controlling the membrane dwell time to modulate activity of signaling factors. This has been well quantified in vitro but is challenging to quantitatively demonstrate in cells because of substantial cell-to-cell variability in behavior. This is important because cell-to-cell variations can obscure quantitatively measuring the functional impacts of clustering.

In this manuscript, Chen et al. describe the application and adaptation of a micro-patterned substrate that addresses this intrinsic heterogeneity problem by analyzing the activity from clustered (condensed) and non-clustered (diffuse) in the same cell. This was achieved by creation of discrete zones of mobile (membrane-bound) and immobile (polymer-bound) ligands. Using this substrate, the authors examine receptor clustering upon binding to mobile ligands. This clustering drives receptor phosphorylation, increases molecular dwell time of key downstream signaling effectors, and drives local actin polymerization. Importantly, this patterning of substrate allows the authors to de-couple the effects of receptor binding from receptor clustering within individual cells. While there is not a major new conceptual insight in the study, the technical platform allows for a critical quantitative analysis of kinetic proof-reading in signaling pathways that appear phase-separated in cells.

The results are convincing and conclusions are well supported by the data. The impact of this work include providing quantitative, in vivo evidence for functions of membrane-associated condensates and may extends to other questions related to signaling where it is critical to assess diffusive versus non-diffusive ligands within the same cell.

Reviewer #1 (Public Review):

Hutcherson and Tusche address an important question: what is the role of dorsolateral prefrontal cortex in normative decision-making? Some have argued that dlPFC plays a role akin to cognitive control - overriding non-normative choices or holding in mind and enhancing the weight of normative goals. Others have argued that dlPFC reflects the accumulation of evidence, akin to a drift diffusion process, during decision-making, and that any apparent special role in normative choices follows from this. Hutcherson and Tusche provide evidence in favor of the second view from three neuroimaging studies across two domains where norms are relevant (altruistic choice and healthy food choice). The key prediction that distinguishes the evidence accumulation hypothesis is that, when normative considerations are weighted more strongly (for example, due to regulatory focus instructions), non-normative choices should be associated with stronger activation than normative ones. The authors observe moderate support for this prediction.

Reviewer #1 (Public Review):

This manuscript explores the effect of loss of cholinergic innervation on microglia homeostatic phenotype. The authors show that loss of cholinergic tone "primes" microglial towards an activated phenotype, which makes them more prone to an exacerbated response to an inflammatory stimulus administered when microglial cells have already reverted to a "basal state". They further show that this overresponse is due to impaired nicotinic signalling mediated by the α7-nAChR expressed in microglial cells. The authors conclude that cholinergic signalling normally contributes to maintain microglia cells in a resting state. In its absence or reduction, microglial cells are primed and enter an acute proinflammatory state upon a second stimulus.

This is an interesting and well conducted study that provides a molecular mechanism underlying the feed-forward contribution of microglial cells to conditions such as brain aging or neurodegenerative pathologies such as Alzheimer's disease. A few aspects of the manuscript could however be improved:

• Figure 2D. In all graphs there are two samples that do not show any increase in cytokine content with respect to saline treated animals? Are these the same animals? Do the authors have an explanation for their lack of response?

• Can the authors provide double immunostaining analysis to show that Iba1 positive cells express α7-nAChR? This will nicely support FACS results given that FACS represent after all a stress for the cells.

• Figure 5. The data are somewhat confusing and in part they seem to repeat those reported in Fig 2 and 3. They are not easy to follow for non-specialists in the field and will benefit from re-writing. Furthermore, why looking at circulating levels of the cytokines? It would be more appropriate to determine cytokine content in the brain

Reviewer #1 (Public Review):

N-glycosylation, the covalent attachment of glycans to selected asparagine residues of target proteins, is a post-translational protein modification that occurs across evolution. However, in contrast to the relatively well described eukaryotic and bacterial N-glycosylation processes, the parallel event in Archaea is less well defined. In this manuscript, Meyer and colleagues report on Agl24, a new component of the N-glycosylation pathway of Sulfolobus acidocaldarius, an archaeal species that lives in hot and acidic conditions. Reminiscent of what is seen in eukaryotic N-linked glycans, the N-linked glycan of S. acidocaldarius also relies on a chitobiose (two N-acetylglucosamines) core. Agl24 was shown to add the second such sugar. This activity is similar to that catalyzed by eukaryotic Alg13/14 and bacterial MurG. Accordingly, similarities among these enzymes were first used to predict Agl24 function. Such activity was subsequently confirmed using various experimental approaches. Finally, the evolutionary implications of Agl24 contributing to the assembly of a chitobiose moiety much like Alg13/14 in eukaryotes were considered. Phylogenetic analyses lend support to the idea that eukaryotic N-glycosylation originated from a sub-set of Archaea.

Reviewer #1 (Public Review):

The authors of this study investigated the evolutionary process of the mammalian group of species including moles, shrews, hedgehogs, and solenodons with molecular approaches, with a reference to their diverse lifestyles. They first unveiled the among-species relationships and the chronological pattern of diversification by comparing molecular sequences of commonly shared genes. The highlight of the study is the inference of net surface charge and three-dimensional structure of the oxygen-storing muscle protein myoglobin, which reflected the varied lifestyles, with the Russian desman, the smallest endothermic diver, exhibiting a prominently altered disposition of myoglobin, possibly resulting from the adaptation to a semi-aquatic lifestyle.

Reviewer #1 (Public Review):

Maimon-Mor et al. examined the control of reaching movement of one-handers, who were born with a partial arm, and amputees, who lost their arm in adulthood. The authors hypothesized that since one-handers started using their artificial arm earlier in life then amputees, they are expected to exhibit better motor control, as measured by point-to-point reaching accuracy. Surprisingly, they found the opposite, that the reaching accuracy of one-handers is worse than that of amputees (and control with their non-dominant hand). This deficit in motor control was reflected in an increase in motor noise rather than consistent motor biases.

Strengths:

• I found the paper in general very well and clearly written.

• The authors provide detailed analyses to examine various possible factors underlying deficits in reaching movements in one-handers and amputees, including age at which participants first used an artificial arm, current usage of the arm, performance in hand localization tasks, and statistical methods that control for potential confounding factors.

• The results that one handers, who start using the artificial arm at early age, show worse motor control than amputees, who typically start using the arm during adulthood, are surprising and interesting. Also intriguing are the results that reaching accuracy is negatively correlated with the time of limbless experience in both groups. These results suggest that there is a plasticity window that is not anchored to a certain age, but rather to some interference (perhaps) from the time without the use of artificial arm. In one-handers these two time intervals are confounded by one another, but the amputees allow to separate them. I think that the results have implications for understanding plasticity aspects of acquiring skills for using artificial limbs.

Weaknesses:

• While I found that one of the main conclusion from the paper is that the main factor that is related to increased motor noise is the time spent without the artificial arm, it felt that this was not emphasized as such. These results are not mentioned in the abstract and the correlation for amputees is not shown in a figure.

• The suggested mechanism of a deficit in visuomotor integration is not clear, and whether the results indeed point to this hypothesis. The results of the reaching task show that the one-handers exhibit higher motor noise and initial error direction than amputees. The results of the 2D localization task (the same as the standard reaching task but without visual feedback) show no difference in errors between the groups. First, it is not clear how the findings of the 2D localization task are in line with the results that one-handers show larger initial directional errors. Second, I think that these results suggest that the deficiency in one-handers is with feedback responses rather than feedforward. This may also be supported by the correlation with age: early age is correlated with less end-point motor noise, rather than initial directional error. Analyses of feedback correction might help shedding more light on the mechanism. The authors mention that the participants were asked to avoid doing corrective movement and imposed a limit of 1 sec per reach to encourage that. But it is not clear whether participants actually followed these instructions. 1 sec could be enough time to allow feedback responses, especially for small amplitude movements (e.g., <10 cm).

Reviewer #1 (Public Review):

This manuscript reports a novel and original approach to examine the possible mutational paths underlying directed protein evolution.

The authors conclude from their experiments that "Necessity was almost entirely absent" (line 209). Indeed, the vast majority of states evolved in just one replicate from one starting point. But this is the problem of a half-full glass: is it half full or half empty? If I understand Fig.4F correctly, one can still detect amino acid changes that recapitulate historical substitutions, and others that revert to the historical state, so it does not seem that necessity is "almost entirely absent". Furthermore, several of the amino acid changes that were detected may not have any effect on NOXA or BID biding, maybe they occurred because of mutational bias, drift or hitchhiking. If this is the case, then one cannot compare all acquired states in each trajectory and conclude about the importance of chance, as in this sentence for example: "Pairs of trajectories launched from the same starting point differed, on average, at 78% of their acquired states, indicating a strong role for chance" (line 219). There are causal mutations that arose repeatedly during PACE replicates from each starting genotype and these mutations do indeed confer the selected-for specificity in their "native" background (as is nicely shown in Figure 6A-B). So this, to me, is evidence for necessity.

Loosing a binding property can probably occur via multiple ways, which are likely to be more numerous than gaining binding for a given protein. It would be nice to discuss this point in more detail.

The experiments presented are limited to one protein family and to the binding properties to two different proteins. In living organisms, each protein is likely to exhibit particular properties such that it can bind or not bind to hundreds of different proteins, and not just two as tested here. So the constraints present in living organisms may be much larger than the ones present within this experimental evolution set up. Furthermore, the tested proteins probably encounter other constraints in their native environment besides affinity for other proteins, and it is yet unclear whether the variant forms obtained here via experimental evolution would be fine to replace the endogenous proteins in living organisms. It is therefore difficult to generalize from the obtained results to all types of evolutionary changes. In general, the conclusions should be toned down and focused on this particular example.

Reviewer #1 (Public Review):

The manuscript by MeHeust reports identification of flavinylation proteins that can potentially function as cellular redox mediators related to electron transfer systems in prokaryotes.

The work is useful and informative. The authors used bioinformatic approach to illustrate wide distribution of these proteins in a variety of prokaryotes. Although exact functions of these proteins are not known, this work should inspire further investigation by researchers in the fields of redox enzymology and bioenergetics.

Reviewer #1 (Public Review):

The authors sought to understand the relationship between sequence conservation and biological function for a protein complex that undergoes conformational changes during its functional cycle. This included understanding the extent to which phylogenetic comparisons can guide identification of functionally important residues for a specific family member. The particular focus was on the bacteriophage clamp-clamp loader complex, with the long-term goal of understanding structure-function principles that might facilitate the design of novel AAA+ ATPase proteins.

A systematic mutagenesis screen was used to determine the relative fitness for single site mutations throughout the bacteriophage T4 clamp-loader and clamp proteins. A feature of the screen is selection for fitness in an (almost) authentic biological context. The vast majority of residues are highly permissive to substitution, which is notable because the considerable conformational changes required during the loader-clamp reaction cycle might have been expected to place more constraints throughout the structure. It is demonstrated that tolerance to mutation within the T4 proteins does not correlated well with conservation across 1,000 other bacteriophage sequences, thereby illustrating the importance of specific context and limits of inference from phylogenetic comparisons. The only critical residue distant from a catalytic active site or binding surface is a glutamine, whose importance was not previously noted, but imparts rigidity to the structure by coupling functionally-important clusters through a hydrogen bonded network. Inspection of distantly related AAA ATPases indicates that this residue is important for many, although not all members of this large and diverse family of molecular machines.

One major concern is that the levels of protein expression and folding are not verified. This is concerning for the Gln118 mutation because lack of fitness could result trivially from misfolding or accelerated degradation that might result from increased flexibility and conformational stability. Moreover, the authors' finding that it was not possible to purify Gln118 mutant proteins for biochemical studies is consistent with this sort of trivial explanation for apparent lack of biological function.

Reviewer #1 (Public Review):

Using a third odor for memory test, the authors found that single-trial conditioning produces a protein synthesis-dependent LTM leading the avoidance to both CS+ and CS- for more than 7 days. By acutely blocking neurotransmissions from target neurons, they showed that the merged LTM requires outputs from TH-positive dopaminergic neurons during training, and from αβ Kenyon cells and α2sc mushroom body output neurons during testing. Several lines of evidence support their claim that the long-term avoidances of CS+ and CS- after single-trial conditioning are based on the same memory component: 1, Five independent disruptions of the system produced the same level changes of avoidance from CS+ and CS-; 2, Re-exposure to either CS+ or CS- alone abolished both CS+ avoidance and CS- avoidance; 3, The same PPL1 DANs, αβ KCs, and α2sc MBONs involved in both avoidances; and 4, Similar responses of functional depression to CS+ and CS- occurred in the same α2sc MBONs. Based on these results, the authors suggest that animals can develop distinct memory strategies for occasional and repeated threatening experiences.

While the entire manuscript is well-written and the data are well presented, the conclusion has several weaknesses. First, memories for CS+ and CS- are clearly distinct from each other at least during the first 24 hours after training (Figure 1B). During this period, CS+ memory shows a gradual decline similar to conventional LTM. Oppositely, CS- memory shows a gradual increase. When these flies were tested immediately after training, they seemed to approach CS-, a result similar to the previous study using a similar third-odor test showed that multiple-trial conditioning induces "two independent LTMs of opposite valence for avoiding CS+ and approaching CS-". How does "the approaching CS- memory" turn into "the avoiding CS- memory"? Or, "the avoiding CS- memory" is derived independently, similar to the anesthesia-resistant memory?

If initial behavior responses to the CS+ and CS- memories are opposite and thus separated, where does each of them occur? Where and how are they merged after one day? The authors claimed that both CS+ and CS- memories require the same PPL1 DANs, αβ KCs, and α2sc MBONs. But, they manipulate PPL1 DANs with TH-Gal4 that expressed in many other dopaminergic neurons. Similarly, the αβ KCs include at least three major cell populations, each has hundreds of neuron with distinct functions. Thus, while both CS+ and CS- memories require the same family of neurons, it remains uncertain whether these events actually occur in the same neurons.

In general, the identified merged LTM is original and brings a new concept to the field of learning and memory. The finding suggests that two forms of protein-synthesis-dependent LTM induced respectively after occasional or repetitive experiences are encoded in the brain by different neuronal mechanisms. It appears that merged LTM after single-trial training remembers the event with limited details and repetitive spaced trials of training then adds more details to the classical LTM.

Reviewer #1 (Public Review):

The study is focused on neural deficits in Smith-Lemli-Opitz syndrome (SLOS) that is caused by loss of function of 3b-hydroxysterol-D7 -reductase (DHCR7) and results in lower cholesterol. Individuals with SLOS have cognitive impairment and the authors use mouse models and human iPSCs to investigate the effects of the SLOS mutation on neural progenitor proliferation and neurogenesis. Data show that the loss of DHCR7 leads to premature differentiation of cortical progenitors and altered cortical development. However, the work offers little mechanistic insight.

Reviewer #1 (Public Review):

The regulation of highly dynamic interactions is for many biological processes of great importance. The authors study the regulatory interaction of the single transmembrane helix protein Phospholamban with the P-type ATPase SERCA which is responsible for removing calcium ions from the sarcoplasm and restoring its concentrations in the sarcoplasmic reticulum. The inhibitory interaction between both proteins is relieved by phosphorylation of a single residue in the cytoplasmic domain of Phospholamban. The authors show by a combination of solid state NMR as well as MD simulations that phosphorylation results in a order to disorder transition in the cytoplasmic part which leads to an re-arrangement of electrostatic networks which is propagated into weakened hydrophobic interactions between the transmembrane parts, thus activating SERCA. Phospholamban has been studied extensively by solid state NMR, liquid state NMR or hybrid methods. For example the phosphorylated form was studied previously, showing that it interacts differently with lipids (doi: 10.1021/bi0614028) and that Ser-16 phosphorylation alters the structural properties of the cytoplasmic domain with respect to the lipid bilayers (doi:10.1016/j.bbamem.2009.12.020). There have also been EPR and other studies, in principle showing the same effect. The current paper adds to this a new solid state method that shows additional details that could not be investigated previously. The work confirms less well determined previous models. The major new aspect is an MD simulation that provides a more detailed view than what was previously possible.

Reviewer #1 (Public Review):

The authors address the broad question of what is responsible for the large diversity of presynaptic function at synapses arising from a single type of neuron. They use a variety of sophisticated and complementary approaches to address the functional and molecular heterogeneity of hippocampal pyramidal cell to fast-spiking interneuron synapses. The rigorous functional and molecular analysis is clearly described and compelling. The conclusions are consistent with the current view that each presynaptic active zone contains a variable number of release sites, and this variability makes a substantial contribution to the heterogeneity in postsynaptic response amplitude at unitary synaptic connection. Using state-of-the-art imaging approaches, the authors report variability in the content of Munc13-1, a core component of release sites, between release sites. Although these results and conclusions are well-supported, the functional significance of Munc13-1 variability at release sites is unclear.

Reviewer #1 (Public Review):

The authors have done a great job in carefully labeling the β-catenin with fluorescent protein SGFP2 and quantitatively measuring the β-catenin behavior during Wnt pathway activation with advanced biophysical methods. This is an excellent effort on quantitative biological studies. The knock-in constructs, the cell lines the authors made are great resources for the Wnt field. And the quantification like the β-catenin concentration, β-catenin diffusion coefficient are great knowledge for future studies. The finding that S45F mutation lead to higher fraction of the slow-moving complexes is interesting. Other areas could borrow the research ideas and methods used in this manuscript. My primary concern is the difficulty of interpreting some of the quantitative results in the biological context.

The authors have concluded that β-catenin has two major populations: free population and slow-diffusing complexed population. The authors have concluded with FCS that the diffusion coefficient of free β-catenin to be 14.9 um2/s (line 259) and the complexed β-catenin to be 0.17 um2/s (line 327). Similar to the authors' argument in the manuscript, this difference means about a 100-fold change of the complex length scale. If the complex is linear, this means a 100-fold change in molecule size, but if the complex is spherical, this means a one-million-fold increase of the molecule size. Furthermore, in the next section, with the N&B method, the authors have suggested that "few, if any, of these complexes contain multiple SGFP2-CTNNB1 molecules" (line 366). When combining the two parts of information, it is hard to imagine a complex that contains one thousand to one million molecules only have one or a few β-catenin subunits. From the biology point of view, APC is the backbone of the destruction complex, which has several β-catenin binding sites by itself. Additionally, APC also contains several Axin1 binding sites where each Axin1 can also recruit one β-catenin. It is unlikely that one APC complex contains only one β-catenin, not mentioning the potential oligomerization of APC. The conclusion that most of the β-catenin containing complexes has only one β-catenin could either be real or due to the misinterpretation of experimental data.

Reviewer #1 (Public Review):

This manuscript by Kurashina et al. describes a novel post-mitotic role in synaptic patterning for a cell fate determining gene, unc-4, and its co-repressor, unc-37. The DA neurons in C. elegans are cholinergic motor-neurons that exhibit unique synaptic tiling of their dorsal axonal segments. Mizumoto et al has previously shown that Semaphorin-Plexin signaling is required to establish the tiling between DA8 and DA9, by functioning in cis in the DA9 neuron. Using temperature sensitive mutant of unc-4, as well as a combination of CRISPR/Cas9 genome editing with the AID system for specific temporal degradation, the authors nicely examine the spatiotemporal requirement of unc-4, and show that unc-4 is required only post-mitotically for synapse tiling, but not during the development of the DA neurons. Interestingly, activity of the corepressor unc-37 is required both during development and postmitotically for correct tiling. unc-4 and unc-37 are suggested to function by inhibiting the canonical wnt signaling. Overall this is an interesting study which sheds light on our understanding of the post-developmental role of cell fate genes in synapse patterning. I only have one major issue that requires some clarification. The authors present in their introduction the results from Kerk et al, regarding the role of unc-4 as a cell fate determining gene for the VAs and DAs. Kerk et al have shown that UNC-4 is specifically required for the expression of DA genes, without affecting ACh pathway genes. Table 1, however, doesn't fully recapitulate the same results and actually shows that unc-4 and unc-37 mutants do not exhibit significant cell fate defects. The authors use these results to argue in the discussion that the synaptic patterning defects can occur independent of the cell fate transformation. The issue of unc-4 as a cell fate determining gene of A type motor neurons needs to be more clearly addressed. The authors should test whether acr-5 expression is elevated in DAs in unc-4 and unc-37 mutants (Winnier 1999, Kerk 2017). In addition, they should also analyze these DB markers in the temp shift experiments (do the VB-DB markers show up in the post embryonic knockout? And if so, will silencing them specifically in DA neurons rescue the tiling defects?). The discussion, accordingly, should also address these issues.

Reviewer #1 (Public Review):

Higashi et al. present a molecular mechanism of how the cohesin complex, a supramolecular assembly of several proteins, can topologically embrace DNA and actively extrude DNA into loops. The loop-extruding activity of cohesin and of related condensin complexes have been proposed to represent a cornerstone of genome organization. While recent in vitro studies demonstrate that cohesin and condensin complexes are capable of extruding loops, the molecular mechanisms driving loop extrusion, i.e. how ATP energy is utilized, and what underlies the processivity of the loop extrusion, remains enigmatic. The cohesin complex consist of two long flexible protein "arms" connected at the 'hinge' ends. The other, 'head' ends are linked by the kleisin protein and also can dimerize to form an ATP-binding chamber. Defining how transitions in the cohesin complex structure and its ATPase activity underlies known cohesin functions has been the object of numerous studies for over two decades. Here, the authors build upon these studies.

The authors start by analyzing available structural data for cohesin domains and associated loading factors. First, by combining the structure of the cohesin-head-domain complex engaged with DNA in ATP-bound state and the corresponding free crystal structures, they show that the 'head module' in the ATP-bound state can tightly wrap around DNA, and upon ATP hydrolysis the DNA-embracing cavity will dilate. In other words, the complex transitions from a 'DNA-gripping state' into a 'DNA-slipping' state after ATP is hydrolyzed. Next, they show that the other DNA-binding module, the 'hinge module', does not change its interaction with the DNA after ATP hydrolysis. The authors also conclude that ATP hydrolysis weakens the interaction between the head and hinge modules, suggesting that the cohesin ring alternates between folded (with head and hinge closed) and unfolded ('free' hinge) states. The authors next carried out FRET experiments to provide experimental evidence for the predicted change in spatial arrangement between the head and hinge modules. Based on this structural analysis, they propose that whether DNA is passed (or not) through the 'kleisin gate' before binding to the head module (into the gripping state) determines if the DNA will be released inside the cohesin ring (i.e. 'topological entry') or if the DNA will remain loosely associated with the head module (i.e. 'loop extrusion') upon ATP hydrolysis. In the latter case, repetitive simultaneous binding of DNA to the head and hinge modules in a folded state followed by relaxation of the cohesin ring while DNA remains bound to the hinge module, may result in a overall 'inward' directed motion of the DNA thread relative to the head domain, i.e. loop extrusion. Stochastic simulations of a coarse-grain model further support that such a model can give rise to loop extrusion.

The real strength of the paper is in its combination of several pieces of structural and biophysical data that results in a compelling mechanism for cohesin function. The outcome is a united model for cohesin's two characteristic activities - topological engagement of the DNA and DNA loop extrusion. Importantly, the authors explore the role of ATP hydrolysis in driving conformational changes, and, thus, the translocation of DNA, as well as the role of the DNA binding kinetics. The authors go on to relate these findings to the consequences for cohesin function inside cells, where it must content with chromatized substrates. For example, they suggest that while a single nucleosome probably can be bypassed by the cohesin complex, an array of the nucleosome may present a significant hindrance.

Given its interdisciplinary nature and important conclusions, I believe that this paper will be of broad interest to scientists across disciplines and will influence and stimulate future consideration of how cohesin contributions to the spatiotemporal organization of chromatin.

Reviewer #1 (Public Review):

Redmond et al. use single-cell and single-nucleus RNA-sequencing to reveal the molecular heterogeneity that underlies regional differences in neural stem cells in the adult mouse V-SVZ. The authors generated two datasets: one which was whole cell RNA-seq of whole V-SVZ and one which consisted of nuclear RNA-seq of V-SVZ microdissected into anterior-posterior and dorsal-ventral quadrants. The authors first identified distinct subtypes of B cells and showed that these B cell subtypes correspond to dorsal and ventral identities. Then, they identified distinct subtypes of A cells and classified them into dorsal and ventral identities. Finally, the authors identified a handful of genes that they conclude constitute a conserved molecular signature for dorsal or ventral lineages. The text of the manuscript is well written and clear, and the figures are organized and polished. The datasets generated in this manuscript will be a great resource for the field of adult neurogenesis. However, the arguments and supporting data used to assign dorsal/ventral identities to B cells and A cells could be strengthened, and more rigorous data analysis could result in new biological insights into stem and progenitor cell heterogeneity in the V-SVZ.

Reviewer #1 (Public Review):

This study focuses on the consequences of deleting the GluA4 subunit of AMPA receptors for cerebellar synaptic transmission and cerebellar-dependent behaviors. The manuscript is well organized and the information is clearly presented. The first aim of the study is to investigate the effect of the deletion at the level of synaptic function. This is well achieved by a combination of patch-clamp recordings from cerebellar slices and modeling. It is found that deletion of the GluA4 subunit results in a strong decrease in synaptic currents from mossy fibers (MF) to granule cells (GC) as well as in two «compensatory» changes pertaining to NMDA Rs and tonic inhibition. As a consequence, MF-GC transfer is strongly reduced at high frequencies but less affected al low frequencies. The second part of the work investigates the effect of the GluA4 deletion on cerebellar-dependent behaviors. GluA4 knock-out mice are found to have no deficits in locomotion but exhibit a total absence of associative learning in an eye-blink conditioning paradigm. Both, at the slice level and at the behavioral level the strength of this work resides on the quality of the data and the rigorous analysis. A shortcoming of the work stems from the «compensatory» changes which complicate interpretation. However modeling strategies are implemented incorporating those changes and they are able to well predict the observed alterations in GC firing pattern, thus limiting the negative impact.

Joint Public Review:

The way homologous chromosomes identify one another and become paired is an intriguing phenomenon that has a long history of study, yet the molecular mechanism remains unclear. Recent studies have led to a phenomenological button model for homolog pairing, which hypothesizes that pairing is initiated at discrete sites along the length of each chromosome. The authors aimed to rigorously investigate this idea using biophysical modeling and live imaging. They first constructed a simple polymer model with buttons distributed along the chain that possess locus-specific interactions, and thoroughly investigated its property via stochastic simulation in 3D. Their study confirms that homolog-specific interactions are necessary for homolog pairing. They also tested the effect of time, interaction strength, initial inter-homolog distance, and button density. The authors went on to perform live imaging of pairing dynamics at two selected loci, using the fluorescent signal from nascent mRNA at the corresponding locus. They fitted the model to the experimentally quantified pairing probability of the selected loci over a 6-hour developmental window, and used the constrained model to predict the individual pairing dynamics. The predicted inter-homolog distance post pairing agrees very well with experimental observation.

Their study supports a button mechanism for homolog pairing, where stable pairing is initiated by reversible random encounters that are propagated chromosome-wide. This work suggests that active processes are not necessary to explain pairing and paves the way for further investigating the molecular mechanism of such a pairing phenomenon.

Reviewer #1 (Public Review):

The authors have identified an entire set of genes for the synthesis of sulfated exopolysaccharides (EPS) in the cyanobacterial model Synechocystis 6803. They show convincingly that the respective gene products are involved in the production of these compounds and they have extensively characterized the regulation of these genes. Among the regulators they found a STAND protein. STAND proteins include animal and plant regulators of programmed cell death but were rarely characterized in bacteria. Last but not least they come up with an entirely new model for the buoyancy regulation of cyanobacteria (as light-dependent aquatic organisms it is important for cyanobacteria where they are in the water column). The authors suggest a mechanism in which EPS-entrapped cells together with extracellular gas bubbles derived from photosynthesis form multicellular complexes that float at certain depths. This would be a very important function and explain the extensive regulatory and signaling apparatus in controlling the synthesis of these sulfated EPS.

Reviewer #1 (Public Review):

Major Comments/Concerns

On line 101 - The use of only the longest transcript for each gene could miss important functional sections of the genome. This could create bias against genes with many isoforms and miss exons that do not happen to lie in the longest transcript. How different would the resulting profiles of conservations be if all coding regions or exons of every gene were used?

On line 106 - Does this approach create good specificity to our gene of interest rather than just broad functional similarity? For example, with this approach, are there any major neuronal function genes that have NPP very different from MeCP2? Could authors provide a more objective evaluation to baseline/null?

Minor Comments/Concerns

On line 132 - It seems fair to examine this set of genes first, but I am not sure this approach to filtering in particular moves us further towards finding a therapeutic for Rett. These genes could be all good potential targets, and your subset of focus are just the best ones for current validation.

Figure 2C could be made with all 390 co-evolved genes to strengthen the argument that chr19p13.2 is an important region for MeCP2s role.

Figure 3, 4, 5, 6 - Dynamite plots. While the stats tests are great for understanding the impact of different treatments, box plots or jittered dots would be even more clear.

Reviewer #1 (Public Review):

The energy released upon zippering of SNARE complexes from the N-terminus to the membrane-proximal C-terminus is widely believed to provide the driving force for membrane fusion, and the cis-SNARE complexes resulting after fusion are disassembled by formation of a 20S complex with Sec17 and Sec18, followed by ATP-hydrolysis by Sec18. This paper now shows that membrane fusion still occurs when the hydrophobic interactions that drive C-terminal zippering of the yeast vacuolar SNARE complex is completely prevented by C-terminal truncation of two of the SNAREs and replacement of the hydrophobic residues at the C-terminus of the SNARE domain of a third SNARE with polar residues, and that such fusion requires Sec17, Sec18 and non-hydrolyzable ATP homologues, in addition to the HOPS tethering complex, which mediates SNARE complex assembly. The results also show that Sec17 plays a key role in fusion through hydrophobic residues in an N-terminal loop that are known to interact with membranes. These results suggest that the core membrane fusion machinery is formed by the SNAREs, Sec17 and Sec18 rather than by the SNAREs alone, and that fusion is driven by a combination of SNARE C-terminal zippering and perturbation of the lipid bilayers by the hydrophobic loops of Sec17. These conclusions are strongly supported by a variety of membrane fusion experiments. FRET assays to SNARE complex assembly also support the conclusions but are less convincing.

Reviewer #1:

The authors investigate a role for a candidate new inhibitor of USP28 in destabilizing c-MYC to reduce the growth of lung squamous carcinomas. They demonstrate that c-MYC levels are higher in lung squamous cell carcinomas (LSCC) versus lung adenocarcinomas (LADC), and depletion of c-MYC reduces LSCC cell growth. The deubiquitinase USP28 is known to stabilize c-MYC; the authors show that depletion of USP28 also decreases c-MYC protein levels. USP28 action opposes that of a ubiquitin complex targeted by the FBXW7 tumor suppressor. The authors create a new mouse model in which FLP recombinase initially causes deletion of FBXW7 and activation of KRAS to cause tumorigenesis with LSCC and LADC, followed by tamoxifen-dependent CRE recombinase deletion of USP28. Loss of USP28 in this model reduced numbers of LSCC but not LADC, and led to decreased expression of c-MYC and other short-lived proteins such as c-JUN and deltap63. A limitation of the data shown is that tumor number calculations are shown for a relatively small number of mice. Deletion of USP28 also did not restrict LADC growth in a second mouse model, with tumors forming based on activation of KRAS and loss of TP53. The authors then describe a compound, FT206, which they show is a specific inhibitor of USP28 among other ubiquitinases. They demonstrate that this compound reduces expression of c-MYC, c-JUN, and deltap63, but do not demonstrate this effect is directly mediated through USP28. They also show FT206 reduces growth of LSCC but not LADC in the KRAS/FBXW7 tumor model, and in human LSCC xenografts. These latter data suggest the compound FT206 may be useful as a lead compound. However, the current data are not sufficient to demonstrate FT206 binding and biological effect is specific for USP28, as the compound may also bind and regulate other non-deubiquitinase proteins.

Reviewer #1 (Public Review):

In this manuscript, Ma, Hung and colleagues rewind the tape to explore the genetic landscape that precedes carbapenem resistance of Klebsiella pneumoniae strains. The importance of this work is underscored by the paucity of new drugs to treat CPO (carbapenemase producing organisms). 'Given the need for 35 greater antibiotic stewardship, these findings argue that in addition to considering the current 36 efficacy of an antibiotic for a clinical isolate in antibiotic selection, considerations of future 37 efficacy are also important.' And so I would say the major weakness of the paper is the aspirational nature of how this work could be used by clinicians in antibiotic selection or treatment of the patient.

The strains selected for these experiments and the evolutionary in vitro models are both well considered. One idea that has stuck with me from the figures of a review article by Kishony (https://pubmed.ncbi.nlm.nih.gov/23419278/, figure 4) is the concept of constraining the evolutionary pathways or fitness landscape for antibiotic resistance. Are there any peaks that a microbial strain reaches that optimize resistance to one AbX but basically leave it inherently unable to evolve resistance to another AbX? This could have application for dual drug therapy or pulsed therapy. When you sequence the isolates that have increased their MIC do you find 'unrelated' mutations in genes that would control protein synthesis or other functions that might be compensatory mutations. Developing a clearer understanding of the rewiring of the bacterium's basic processes might also elucidate both integrated functions and potential weaknesses. You mention mutations in wzc, ompA, resA, bamD.

Point of discussion. Classic ST258 carries blaKPC on pKpQIL plasmid. Your ST258 strain (UCI38) carries blaSHV-12 on pESBL. Am I to assume that pESBL is in lieu of pKpQIL? Transformation of CPO have many variables and in vitro data does not always mirror what is observed in vivo. So the findings of Fig 2f might need to be considered under different laboratory conditions (substrate, temperature) [https://pubmed.ncbi.nlm.nih.gov/27270289/].

Reviewer #1 (Public Review):

Mandal and colleagues identified novel functions of Relish in the hematopoietic niche development and its coordinative role in innate immunity. The authors found that Relish is expressed in the PSC, which is essential for various developmental functions, including the maintenance of hematopoietic progenitors, the number of PSC cells, expression of Wg, and the PSC actin cytoskeletal structure. Furthermore, Relish acts as an inhibitor of JNK signaling and functions downstream of the ecdysone pathway in the PSC. The authors moved on to find the developmental and physiological relevance of this phenomenon and discovered that Relish is downregulated upon bacterial infections to accommodate immune responses. These findings show that Relish plays a critical role in hematopoiesis as a downstream of hormonal control and in switching between the developmental and physiological mode of the PSC.

Conclusions are well-supported by data, and experiments were carefully performed and analyzed. Given that most of the studies on Relish describe its function in innate immunity and that it is the first study showing critical roles of Relish in blood development, this study will draw broad attention and contribute to understanding insect hematopoiesis and immunity.

Reviewer #1 (Public Review):

Yan et al. take a comprehensive look at structural variants in the 1000 Genomes Project high-coverage dataset, using recent developments that can link short- and long-read data. Combined with genomic simulations, they identify and characterize the timing and origin of a likely selected region in Southeast Asian populations. The combination of multiple data types adds depth to the interpretation.

The study is timely, combing recently released data and methods, and had interesting biological implications. Tree main areas would help interpretation and robustness of the paper:

1) Further context and interpretation of the original SV set found is needed, for example comparisons to previous work to identify clearer "positive controls" or sanity checks on the method, and to understand what the contribution of the method/dataset/paper is.

2) The above is particularly important across ancestries/populations which differ in their LD levels. How does population-specific LD patterns impact the ability to detect these SV patterns? and therefore to make cross-population comparisons or infer differences in frequency that are central to the selection scan and the 220 highly differentiated SVs of interest. Perhaps this is in the original methods paper, but is central to this paper so should at least be explained or analyzed.

3) The genomic simulations to infer the strength selection was a nice addition, a step beyond common empirically-driven work. It would help to know how to interpret the ABC model in the context of the later finding that the region was introgressed from Neanderthals--the model seems to not include this aspect.

Reviewer #1 (Public Review):

This MS combines two-photon glutamate sensing (using the iGluSnFR fluorescent probe), two-photon glutamate uncaging, two-photon calcium imaging and electrophysiology to investigate whether synaptically released glutamate activates receptors outside the synapse of release, and at neighboring synapses. The data themselves are very impressive. The authors arrive at the revolutionary conclusion that synaptically released glutamate is able to activate both NMDA and even AMPA receptors at neighboring synapses, remarkably strongly. I say revolutionary, because previous modelling has yielded diametrically opposite conclusions. The reflex would be to prefer experiment over theory, yet the modelling was based upon quite strongly constrained physical parameters that would be quite incompatible with the interpretations reported here. However, I believe the authors have failed to take into account significant technical limitations inherent in the technologies they apply. These include spatial averaging of fluorescence, possible saturation of iGluSnFR and diffusive exchange of (caged) glutamate during uncaging. As a result, the conclusion is wholly unproven. Indeed, I believe it highly probable that all of the data in favor of distal activation will prove to be consistent with synapse specificity and the presence of technical artifacts related to spatial averaging of fluorescence signals and diffusive exchange of (caged) glutamate during uncaging.

Reviewer #1 (Public Review):

In this work the authors address inter-subunit interactions leading to ESCRT-III function during MVB sorting in a yeast model system. ESCRTs mediate function in multiple biological processes, however the fundamental question of how ESCRT-III mediates membrane remodeling is not understood. As such this represents a topic of considerable interest despite significant technical limitations surrounding the issue. Random and rational mutagenic strategies, including compensatory mutations, are combined with protein-protein interaction studies and in vivo functional assays to identify residues within Vps24 and Vps2 mediating associations with each other and Snf7. Based on these analyses the authors put forth a series of "rules" governing ESCRT-III assembly and function. While beneficial to our conceptual understanding of ESCRT-III these rules fall short in explicitly defining the structural basis of assembly and function including explaining requisite heterodimerization of Vps24-Vps2. This work represents a significant step forward in addressing this challenging question, the experimental design and implementation are convincing, however the limitations of this work could be conveyed more clearly.

Reviewer #1 (Public Review):

For bacteria, yeast and mammalian cells, energy depletion has been linked to a vitrification of cytosol and protein aggregation. Previous studies have postulated this is in part due to acidification and the shift in pH to match a large set of proteins pIs resulting in large-scale protein aggregation as well as changes in crowding of the cytosol. Additionally, a more direct role for ATP in protein aggregation has been proposed through its chemical properties as a hydrotrope. The appeal of this hypothesis is that the steady-state levels of ATP far exceed the Kd of most enzymes pointing to a potential non-enzymatic role for the high levels.

In this study, the authors take advantage of a FRET-reporter for ATP that they developed previously called "Queen". They then manipulate ATP levels using mutants in AMP kinase(Snf1) or Adenyl kinase (Adk1) and find null mutants indeed have lower concentrations of ATP and experience sudden drops in ATP levels which the authors term ATP catastrophe. These mutants also show genetic interactions with protein folding/glycosylation pathways and are sensitive to conditions that generate proteotoxic stress. Hsp104 forms foci in the genetically induced lowered ATP levels as well as exogenous ectopic aggregation prone proteins such as alpha-synuclein. The authors attempt to show that the cause of aggregation is due to limiting ATP directly by adding excess adenosine to the media and showing this diminished the formation of foci, potentially due to the ability of increased exogenous to raise ATP levels according to previous reports.

The issue of whether ATP levels play a direct or indirect role in preventing protein aggregation is extraordinarily challenging to address. While ATP can act as a hydrotrope, the formation of aggregates could be due to limitations of the activity of chaperones and helicases which would not be surprising role for ATP in the cell. While the experiments are carefully performed, well analyzed and fairly interpreted; questions still remain about the impact of these experiments on understanding how ATP impacts cytosol.

Reviewer #1 (Public Review):

Cucinotta et al. examine the widespread, transient transcription of genes that occurs within minutes of refeeding quiescent Saccharomyces cerevisiae cells, focusing on the role of the RSC remodeler complex in this process. A range of appropriate genomic approaches are used to characterize the initial burst of transcription, changes in localization of RSC and RNA Pol II, and changes in the occupancy and positioning of nucleosomes during the first minutes after nutrient repletion. Several new insights are reported including the role of RSC in maintaining promoters in state that is ready to respond rapidly to nutrient repletion, the relocalization of RSC into genes following initiation of transcription, a role for TFIIS in exiting quiescence that was not apparent in log phase, the timing of histone acetylation in response to transcription, changes in chromatin architecture during the exit from quiescence, and the effects of chromatin changes on transcription start site selection and repression of antisense transcription from downstream nucleosome depleted regions. Given how little is known about the emergences of cells from quiescence and how common and important this transition is in long-term viability, development, and carcinogenesis, these insights are certain to have broad impact. The data are of high quality and the manuscript is very clearly written, with good correlation between the level of support provided by the data and the strength of the conclusions drawn. Only minor issues remain to be addressed.

Reviewer #1 (Public Review):

In this study, Leydon et al., use an elegant multi-component genetic system to address the mechanisms of repression by the Arabadopsis TOPLESS (Tpl) protein. Taking advantage of the genetic tools and knowledge of the structure of the Tpl protein the authors determine two short alpha helical regions that act as independent repression domains. They provide evidence that the target of one of these domains is the N-terminal region of the Med21 subunit of the mediator complex. Chromatin immunoprecipitation experiments, anchor-away loss of function and co-immunoprecipitation assays indicate that Tpl mediated repression involves formation of a promoter complex comprising the mediator complex along with several general transcription factors, but lacking RNA polymerase II. The authors also show that Tpl-Med21 interactions are involved in Tpl mediated repression in plants.

Reviewer #1 (Public Review):

This paper was a pleasure to read. It is a tour-de-force study that is well-written, clear, and transparent. The study recounts how the HMA domain became integrated into the Pik NLRs and how it evolved higher affinity binding to a pathogen effector. Strikingly the authors demonstrate adaptability of distinct regions of the HMA:effector interface on two Pik NLRs, driving the convergent evolution of high-affinity binding to the effector. The study furthermore provides a framework for understanding protein evolution in the context of host-microbe interactions. The breadth and depth of the experiments that support the authors conclusions is extraordinary in my view.

Reviewer #1 (Public Review):

The study by Hendley et al takes advantage of duct-specific DBA-lectin expression to purify pancreatic ductal populations that were then subjected to scRNA-seq analysis. The ability to enrich for this relatively low abundant pancreatic cell population resulted in a more robust dataset that had been generated previously from whole pancreas analyses. The manuscript catalogs several different gene clusters that delineate heterogeneous subpopulations of three different pancreatic ductal subpopulations in mice: mouse pancreatic ductal cells, pancreatobiliary cells, and intra pancreatic bile duct cells. Additional comparisons of the resulting data sets with published embryonic and adult datasets is a strength of the study and allows the authors to subclassify the different ductal cell populations and facilitates the identification of potentially novel subpopulations. Pseudotime analysis also identified gene programs that led the authors to speculate the existence of an EMT axis in pancreatic ducts. Overall, the data analyses is strong, but the authors tend to draw conclusions that are not fully supported by the presented data.

The second half of this study focuses on three candidate proteins that were identified in the transcriptome analysis - Anxa3, SPP1 and Geminin. Crispr-Cas9 was used to delete each gene in an immortalized human duct cell line (HPDE). Deletion of each gene resulted in increased proliferation; SPP1 mutant cells also displayed abnormal morphology. Additional functional studies of the cell lines or in mouse models suggested a role for SPP1 in maintaining the ductal phenotype and Geminin in protecting ductal cells from DNA damage, respectively. Although the provided phenotypic analysis suggest important functional roles for these proteins, follow up studies will be required to fully understand the role of these genes in homeostatic or cancer conditions.

Strengths:

1) Enrichment of pancreatic ductal populations enhanced the robustness of the scRNA-Seq dataset

2) Quality of the sequencing data and extensive computational analysis is extremely good and more comprehensive than previously published datasets

3) Comparative analysis with existing mouse and human data sets

4) Use of human ductal cell lines and mouse models to begin to explore the function of candidate ductal genes.

Weaknesses:

1) There are many suppositions based on gene expression changes that are somewhat overstated.

2) The conclusion that there is an EMT axis in pancreatic ducts is not fully supported by the gene expression and immunofluorescence data

3) A good rationale for choosing Anxa3, SPP1 and Geminin for additional functional analysis is not provided. In addition, it isn't clear why Anxa3 function isn't pursued further.

4) Although extensive models (transplanted cells for SPP1 and mouse conditional KOs for Geminin) were generated, the functional analysis for each gene is preliminary; additional longer term studies will be necessary to fully understand the role of these proteins in pancreatic duct development and cancer.

Reviewer #1 (Public Review):

The authors develop a mechanistic model for inferring infectiousness profile from data on times of symptom onset in pairs of infector-infectee. The novelty of their approach lies in assuming that infectiousness of an infected individual depends also on the whether or not they have symptoms. The authors fit a data set of time of symptom onset in 191 transmission pairs to a model that assumes that infectiousness varies along the incubation period. They compare the model fit to fits from models and find that their model of differential infectiousness explains the data better than the other models considered.

This is a carefully constructed study, and the conclusions are well supported by the analysis carried out. My only concern is that the data used were obtained during the early stage of the pandemic (January to February 2020). As the pandemic was growing in most countries during this time, we are more likely to have observed shorter serial intervals. Similarly, as isolation of infected individuals would prevent them from transmitting further, longer serial intervals are likely to be under-represented in the data. Indeed, the longest serial interval in the data used was 5 days. It would be interesting to understand whether the conclusions about the proportion of onward transmissions averted by contact tracing and subsequent isolation still hold as the pandemic progresses, and we continue to observe longer serial intervals. If the authors are unable to find more recent data, this caveat should be clearly discussed.

Reviewer #1 (Public Review):

Stokes, et al. describe the effects of isoflurane on metabolism in post-natal day 7 mice, and older mice. They demonstrate that blood levels of glucose and ß-hydroxybutyrate fall quickly in response to isoflurane, and that the magnitude of the decrease increases with the length of the exposure. Mice 30 days post-natal do not exhibit these changes in response to isoflurane. The authors document the much higher circulating levels of ß-hydroxybutyrate in the post-natal day 7 mice, highlighting the importance of this substrate for supporting the energetics of the developing brain. Important control experiments, administering 100% oxygen without anesthetic to post-natal day 7 mice, as well as administering anesthetics to 30 day old mice on a ketogenic diet, did not result in significant decreases in glucose and ß-hydroxybutyrate blood levels. Remarkably, they observed significant decreases in response to very small, subanesthetic doses of isoflurane, halothane and sevoflurane in post-natal day 7 mice. Administration of bolus glucose corrects the glucose level for these mice under anesthesia, but not the level of ß-hydroxybutyrate, while administration of bolus ß-hydroxybutyrate corrects both levels.

The authors then proceed to a series of measurements in an attempt to determine a direct target of volatile anesthetics on metabolism, focusing on hepatic metabolism. This is something of a Procrustean bed, given that the there is ample evidence that volatile anesthetics affect a large number of different membrane bound processes. Nonetheless, these experiments provided valuable data demonstrating anesthetic induced decreases in fatty acid oxidation. This reviewer finds the arguments regarding impairment of the citric acid cycle a bit unconvincing: 7 and 30 day old mice exhibit the same increase in citrate and isocitrate levels, yet only the 7 day old mice show elevated lactate levels. Rather than exhibiting increased metabolic flexibility, as the authors suggest, this finding seems to argue that 7 day old mice have less metabolic flexibility. The authors demonstrate that several perturbations of fatty acid metabolism can result in depression of ß-hydroxybutyrate, leading them to focus on carnitine palmitoyl transferase-1. They demonstrate that inhibition of this enzyme produces a decrease in ß-hydroxybutyrate; however, they also find that mice with a knockout of this enzyme do not have decreased ß-hydroxybutyrate levels.

The authors are circumspect in their conclusions regarding the targets responsible for the metabolic changes observed in neonatal mice in response to anesthetics. They do correctly highlight the potential importance of these metabolic effects. It will be crucial for future research to determine whether these effects can be directly correlated to measures of cerebral function during anesthesia, e.g., EEG or evoked potentials, and to measures of neuropathological change. Of great interest to clinicians will be demonstration of whether co-administration of glucose or ß-hydroxybutyrate together with anesthetics can abrogate such changes.

Reviewer #1 (Public Review):

In this manuscript, Zilova et al. show that primary embryonic cells derived from blastula-stage Medaka and Zebrafish embryos can self-organize into retinal organoids. When aggregates of 1000-2000 primary embryonic cell are embedded in Matrigel addition, they form a neuroepithelium under the control of Rx3 which develops into a retinal organoid. The process mirrors some aspects of embryo development. Moreover, another interesting finding is that Rx3 expression is initiated in the absence of Matrigel at day 0, which indicates that the retinal fate occurs by default and is not dependent on extracellular matrix components. The authors compare the ability of cells from Mesaka and zebra fish and show that both are competent to form organoids, though each does it with the time scale of the embryo of origin. The authors show that by reducing the number of Medaka cells to aggregate (500-800 cells), Rx2 and Rx3 are expressed only in restricted regions of the small aggregates, presumably where they organize into discrete circular Rx2 and Rx3 positive neuroepithelial units that develop into structure resembling retinal epitjhelia with some diversity of retinal cell types including amacrine, ganglion, photoreceptor, bipolar and horizontal cells.

This is a novel and original piece of work that reveals the capacity of fish primary embryonic pluripotent cells to behave like mammalian embryonic stem cells and organize optic cup organoids.

Reviewer #1 (Public Review):

The complexity of the infection model developed by the authors is to be praised as it allows the dissection of host-pathogen interactions with multiple players coming together, namely human epithelial cells, endothelial cells and neutrophils, UPEC, urine, antibiotics and mechanical forces at play during bladder filling and micturition. This is truly a tour de force and should provide the authors (and other labs potentially able to recapitulate it) with an unprecedented model to study UTIs and their response to antibiotics. Notably, authors have been able to document the formation of NETs in response to UPEC infection in this model. One small caveat was the choice of antibiotics used to treat the infection in their model. Is Ampicillin really a drug of choice, both because of its inability to reach intracellular niches and it not being a drug of choice in the clinic?

Reviewer #1 (Public Review):

In their manuscript, Lawrence et al. investigate the direct effects of the microtubule-associated protein, SSNA1, on microtubule (MT) dynamics and damage using purified proteins and TIRF microscopy. Prior work on this protein showed that SSNA1 self-assembles into higher-order filaments and binds longitudinally along stabilised MTs, inducing MT branching and nucleation. In this study, they find that SSNA1 promotes templated MT nucleation, consistent with prior results, but further define the effect of SSNA1 on MT dynamics. SSNA1 overall dampens MT dynamics by reducing both growth and shrinkage rates, suppressing catastrophe frequency, and increasing rescues. The authors also quantify SSNA1 on GMPCPP over a timecourse both at single-molecule and multi-molecule concentrations. On dynamic MTs, SSNA1 recognizes the growing end and promotes end curvature, but it did not recognize the curves of taxol-stabilised MTs, leading the authors to conclude that it likely induces curvature, rather than recognizes it. Perhaps this is the mechanism by which SSNA1 prevents catastrophe, a role which the authors demonstrate for SSNA1 after both tubulin dilution or stathmin sequestration of tubulin. The most interesting part of this study is found in Figure 4, where the authors show that SSNA1 prevents MT severing by spastin and also localizes to sites of lattice damage. The authors conclude that SSNA1 is a MT stabilizing protein and a sensor of MT damage. The results on MT dynamics do not provide much insight into the mechanism of this protein, which isn't even found to colocalize with MTs in vivo (SSNA1 instead accumulates at branchpoints in neurons). The role of SSNA1 in lattice damage recognition is the highlight of this paper, and also correlates well with its in vivo localization pattern, indicating this could be a true function of this protein. This damage recognition ability could potentially be the first step that leads to SSNA1-induced MT nucleation and branching from an existing MT.

Reviewer #1 (Public Review):

The authors interrogated an underexplored feature of CRISPR arrays to enhance multiplexed genome engineering with the CRISPR nuclease Cas12a. Multiplexing represents one of the many desirable features of CRISPR technologies, and use of highly compact CRISPR arrays from CRISPR-Cas systems allows targeting of many sites at one time. Recent work has shown though that the composition of the array can have a major impact on the performance of individual guide RNAs encoded within the array, providing ample opportunities for further improvements. In this manuscript, the authors found that the region within the repeat lost through processing, what they term the separator, can have a major impact on targeting performance. The effect was specifically tied to upstream guide sequences with high GC content. Introducing synthetic separator sequences shorter than their natural counterparts but exhibiting similarly low GC content boosted targeted activation of a reporter in human cells. Applying one synthetic separator to a seven-guide array targeting chromosomal genes led to consistent though more modest targeted activation. These findings introduce a distinct design consideration for CRISPR arrays that can further enhance the efficacy of multiplexed applications. The findings also suggest a selective pressure potentially influencing the repeat sequence in natural CRISPR arrays.

Strengths:

The portion of the repeat discarded through processing normally has been included or discarded when generating a CRISPR-Cas12a array. The authors clearly show that something in between-namely using a short version with a similarly low GC content-can enhance targeting over the truncated version. A coinciding surprising result was that the natural separator completely eliminated any measurable activation, necessitating the synthetic separator.

The manuscript provides a clear progression from identifying a feature of the upstream sequences impacting targeting to gaining insights from natural CRISPR-Cas12a systems to applying the insights to enhance array performance.

With further support, the use of synthetic separators could be widely adopted across the many applications of CRISPR-Cas12a arrays.

Weaknesses:

The terminology used to describe the different parts of the CRISPR array could better align with those in the CRISPR biology field. For one, crRNAs (abbreviated from CRISPR RNAs) should reflect the final processed form of the guide RNA, whereas guide RNAs (gRNAs) captures both pre-processed and post-processed forms. Also, "spacers" should reflect the natural spacers acquired by the CRISPR-Cas system, whereas "guides" better capture the final sequence in the gRNA used for DNA target recognition.

A running argument of the work is that the separator specifically evolved to buffer adjacent crRNAs. However, this argument overlooks two key aspects of natural CRISPR arrays. First, the spacer (~30 nts) is normally much longer than the guide used in this work (20 nts), already providing the buffer described by the authors. This spacer also undergoes trimming to form the mature crRNA. Second, the repeat length is normally fixed as a consequence of the mechanisms of spacer acquisition. At most, the beginning of each repeat sequence may have evolved to reduce folding interactions without changing the repeat length, although some of these repeats are predicted to fold into small hairpins.

Prior literature has highlighted the importance of a folded hairpin with an upstream pseudoknot within the repeat (Yamano Cell 2016), where disrupting this structure compromises DNA targeting by Cas12a (Liao Nat Commun 2019, Creutzburg NAR 2020). This structure is likely central to the authors' findings and needs to be incorporated into the analyses.

Many claims could better reflect the cited literature. For instance, Creutzburg et al. showed that adding secondary structures to the guide to promote folding of the repeat hairpin enhanced rather than interfered with targeting. Liu et al. NAR 2019 further showed that the pre-processed repeat actually enhanced rather than reduced performance compared to the processed repeat. Finally, the complete loss of targeting with the unprocessed repeat appears represent an extreme example given multiple studies that showed effective targeting with this repeat (e.g. Liu NAR 2019, Zetsche Nat Biotechnol 2016).

Relating to the above point, the vast majority of the results relied on a single guide sequence targeting GFP. While the seven-guide CRISPR array did involve other sequences, only the same GFP targeting guide yielded strong gene activation. Therefore, the generalizability of the conclusions remains unclear.

Reviewer #1 (Public Review):

This manuscript is a meta-analysis of literature, predominantly that from evolutionary psychology. The background seems well-explained, and the discussion and literature review well-written. The authors have done an impressive job of collating and synthesising a truly vast amount of literature that (as they demonstrate) is often pretty ambiguous in its results. The results are well-presented and well-reasoned, without overstating the evidence. The entire manuscript is clear and easy to read and follow. Table 1 makes it particularly easy to follow. I appreciate their emphasis that the various hypotheses about sexual dimorphism are not mutually exclusive, and that this study does not seek to explicitly test either one of them.

There is enough evolutionary anthropology inserted here to see that the authors have a passing familiarity with it, although I would encourage them to dig much more deeply into this literature in framing their work. In short, there is a tension between evolutionary psychology and evolutionary anthropology that can be very fruitfully explored with the results of this analysis, and the authors only scratch the surface of this at the end of the manuscript.

Something that seems crucial here, and in this literature more generally, is the likelihood that men have a number of different effective strategies. The background and discussion do a good job of discussing the various possibilities, and how combinations of possibilities that include both female choice or male-male competition could explain human mating behavior. However, it does not really dig into what the implications might be for how multiple, distinct strategies could impact different aspects of the data. What comes to mind is orangutans, in which the large, masculine males appear to obtain mating opportunities primarily through female choice, while the smaller males that have not developed the large body sizes and facial flanges may obtain additional mating opportunities through sexual coercion. In a large sample or meta-analysis like this, a combination of strategies in human males that are at odds with one another, yet both highly effective, may have results that tend to cancel one another out - is there any evidence of this? Getting more into the primate literature here could be useful.

The authors point out that there could be a strong confounding effect with the way testosterone operates developmentally. Testosterone during adolescence translates well to the development of masculine characteristics, but does not necessarily predict testosterone later in life (hence, the expression of masculine features may not actually relate well to circulating testosterone that could be at least partially drive male-male competition). The authors did an excellent job of discussing these potential confounding effects, but I would have found potential issues like this (and like the one above) to be presented usefully in a table that lays out the different potential confounding issues, and then discusses what the predictions should be in the meta-analysis results for each one.

The meta-analysis seems well-designed, and the methods appropriate. However, it did feel a bit like data mining with so many different variables run against one another. I do not think this is actually the case, and the authors do justify each of their decisions. In fact, one of the main outcomes of this work is that they show how few of these parameters actually relate strongly to one another. However, the authors might want to be aware that this study could be read as data-mining because of the search for significance amongst so many different variables, and offset this with explicit discussion and framing up front that they intend to examine how effective the various study parameters actually are at uncovering the relationships they seek to uncover. This is something the authors discuss very articulately at the end, but I would appreciate seeing this up front as one of the goals of the paper.

Reviewer #1 (Public Review):

Summary: The study by Steenkiste focuses on the formation of adaptor protein complexes at sites of integrin receptor adhesion in the modulation of in vitro membrane ruffling and cell movement. The authors are studying the role of BCAR3 (also termed AND34 or NSP1) protein regulation by post-translational mechanisms (ubiquitin degradation and tyrosine phosphorylation). This is one of many adaptor proteins localized to adhesion sites. Studies are being performed on MCF10A or Hela cells to knockdown (siRNA) or over-express tagged protein constructs. By proteomics, a new phosphorylation site was identified (BCAR3 Y117). Mutagenesis showed that BCAR3 Y117 is important for enhancement of in vitro cell movement under conditions where the cullin-5 E3 ligase has also been reduced by siRNA expression.

Opinion: The authors provide support for a "co-regulatory" model whereby the recruitment of BCAR3 to adhesions acts in part to modulate another adaptor protein tyrosine phosphorylation, p130Cas. This is associated with enhanced cell migration. The data presented are generally supportive of the conclusions and consistent with previous studies of BCAR3 and p130Cas. However, an unresolved issue is why cell phenotypes are dependent on cullin-5 knockdown or otherwise investigated by BCAR3 mutant over-expression. Cul5 loss can alter multiple aspects of cell signaling and the transient knockdown or inducible over-pression assays are a limited primary means of investigation. As multiple protein domains and post-translational modifications modulate the BCAR3-p130Cas complex, the authors did not establish a strong mechanistic linkage between newly-identified BCAR3 Y117 phosphorylation, SOCS6 binding, and a CUL5-dependent cell phenotype. Additionally, some of the experimental conditions (+/- EGF in growth media) are difficult to connect to EGF receptor activation and or signaling.

Reviewer #1 (Public Review):

In the current manuscript, Frey et al. describe a convolutional neural network capable of extracting behavioral correlates from wide-band LFP recordings or even lower-frequency imaging data. Other publications (referenced by the authors) have employed similar ideas previously, but to my knowledge, the current implementation is novel. In my opinion, the real value of this method, as the authors state in their final paragraph, is that it represents a rapid, "first-pass" analysis of large-scale electrophysiological recordings to quickly identify relevant neural features which can then become the focus of more in-depth analyses. As such, I think the analysis program described by the authors is of real value to the community, particularly as it becomes more commonplace for labs to acquire multi-site in vivo recordings. However, to maximize its utility to the community, several aspects of the analysis need clarification.

Reviewer #1 (Public Review):

Pulgar et al. describe an interesting mechanism explaining how directed motion of group of cells maintain their migratory path as a group of cells. Incomplete delamination allows here to maintain coordinated cell movements amongst the DFC. The story is self-contained, logical, well-written and just in general very nice. The mechanism described belongs to the so-called mechanical drag which is a new type of multicellular locomotion and may be a general feature involved in many morphogenetic systems.

The major strength of the study is the extensive use of live imaging and analysis of dynamic events. The study provides a nice cellular mechanism in the process they described. The molecular mechanism would be the only weakness of the study.

An overall very exciting study.

Reviewer #1 (Public Review):

*A summary of what the authors were trying to achieve.

The study takes advantage of the interesting plant genus Leucadendron to compare gene expression between male vs. female in species with more or less sexual dimorphism. This question was addressed in a somewhat comparable manner in only one previous paper by Harrison et al. 2015 across six bird species. The overarching question is the role of natural selection in sexual dimorphism.

*An account of the major strengths and weaknesses of the methods and results.

-Beside the genus-wide comparison of whole transcriptomes across related species, which makes in itself a strong dataset, the major strength of the analysis is the phylogenetic framework that allows the authors to track the evolution of sex bias through several tens of million years of evolutionary history. Despite ancestral dioecy in the genus, very few genes show consistent sex bias across several species, with sex-bias being mostly species-specific. Two striking negative results will be of special interest to the community : 1) species with more pronounced sexual dimorphism at the morphological level do not tend to exhibit more pronounced sex-biased gene expression 2) the few genes that do show sex-biased expression were apparently recruited among those with the highest expression variance to begin with, strongly suggesting that sexual selection has not been the main force driving their expression divergence.

-In my view, the main limitation of the work is the use of leaf rather than reproductive tissues, making the comparison to other studies less straightforward to interpret. It is especially important that the expectations for somatic vs gonadic tissues be made a lot clearer in the text. Also, the fact that a single leaf phenotype is measured (specific leaf area) seems arbitrary : one could imagine sexual dimorphism on many other characteristics, yet they are not considered here. The text on p.324 mentions "striking convergence in aspects of morphological dimorphism across the genus", but there is no way for the reader to appreciate the extent of this convergence. Finally, it would be useful to at least make some mention of the sex-determination system in these species, since the expectations would differ if some of the sex-biased genes were linked to sex chromosomes.

*An appraisal of whether the authors achieved their aims, and whether the results support their conclusions.

The analysis is mostly sound, but I am a bit concerned by the arbitrary threshold used to define SBGE. The text on p.305 says that "This result is extremely robust to the choice of threshold", but 1) the results are not reported so it is impossible for the readers to evaluate the basis of this assertion and 2) it is not clear whether robustness of the other results has been evaluated at all. This aspect clearly deserves more attention.

*A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community.

This work will be of interest to the community, as rapid rates of expression evolution would generally be interpreted as the consequence of sex bias, whereas the phylogenetic analysis presented here instead supports the idea that the expression of genes that end up being sex biased were instead intrinsically less constrained to begin with.

Reviewer #1 (Public Review):

This manuscript by Lauer et al follows up on previous articles that ask the question whether there are funding disparities at the National Institutes of Health for African American or Black (AAB) investigators. The investigators breakdown the analysis by race, topic of proposal, and NIH institute-Center (IC) to which an application was assigned. They conclude that the most important factor in determining funding is the Institute assignment with lower funding rates related to the funding capacity of a particular Institute (e.g National Eye Institute vs Minority Health and Health Disparities). The present study is a welcome addition to this debate since if biases do exist, NIH needs to address these. The strengths of this manuscript are the detailed breakdown of the available data in order to evaluate for biases, the availability of data for multiple years (2011-2015) and the consideration of alternate explanations (e.g new applications vs resubmissions; single vs multi PI, etc). A weakness of the data is that if their conclusion is that Institute assignment was the main determinant of funding rates, why wasn't the approach for Institute assignment discussed? Are there possible biases in this assignment besides keyword searches? There is also the question of whether there is circular logic operating here. The Minority Health and Health Disparities received the most AAB applications but had one of the lowest funding rates. Wouldn't this Institute be expected to be one in which AAB applicants would try to direct their application to? This manuscript is sure to generate additional discussion on this topic which is an important step in trying to address the issue of potential funding disparities. However as the authors point out the fact that only 2% of the applications submitted to the NIH were from AAB investigators is of concern.

Reviewer #1 (Public Review):

Ekeng et al. have sequenced and analyzed 46 Vibrio cholerae whole genome sequence data. The authors demonstrated a predominant lineage (T12) where all isolates from 2018-2019 fall. Their analysis suggest continuous transmission through repeated reintroduction of the same lineage back into the population. The work is interesting and the conclusions of this paper are mostly well supported by data. The present study reinforce the need of more genome sequence data and a strong surveillance network to interpret the data.

This is a successful model of regional coordination to have genomic surveillance data from a region where surveillance data was inadequate. The manuscript should be modified to focus on strengthening of genomic surveillance further.

Reviewer #1 (Public Review):

Halliday et al. developed a framework to disentangle the total effect of environment on disease into a direct effect and indirect effects by environment-induced change of host community and by modifying the relationships between host community and disease.

Applying this framework, the authors studied the direct and indirect effects of elevation on plant leaf disease in the Swiss Alps. They focused on host community structures as mediator of indirect effects. Host community structures were measured by host species richness, phylogenetic diversity, and community pace of life. One important finding is that the positive effect of host community pace-of-life on disease weakened as elevation increased, suggesting an important, but less appreciated, mechanism on how elevation can indirectly influence plant disease. However, since the major findings were based on the analyses with elevation but not specific environmental variables, it does not have that strong implications about the influence of global climate change on disease as the authors stated.

The developed framework on environmental effects on disease, the well-designed filed study and the large-scale dataset would all make this paper an important contribution to the field.

Overall, the statistical analyses were reasonable. However, accurate interpretations of some results would require more clarifications on the analyses.

Reviewer #1 (Public Review):

The authors of this paper seek to understand how HIV infects cells. HIV is a retrovirus that harbors a core of RNA nucleic acid in complex with important replication enzymes such as reverse transcriptase. After infection, reverse transcriptase converts the RNA into DNA, which is then integrated into the chromosome. The authors used advanced imaging techniques to visualize the DNA that is made by reverse transcription. They used fluorescent readout markers of proteins to also look at the viral proteins that are brought into the cell and track with the viral DNA during the virus infection.

From this work the authors conclude that reverse transcription is completed in the cell nucleus, that intact or nearly intact cores are the substrate for nuclear import, and that virus core uncoating likely occurs in the nucleus, immediately preceding the integration step. Moreover, by using electron tomography, they drill down to the sub-micron level to glean an ultrastructural view of the viral complexes that are performing these important HIV infection steps. Some of these complexes appear to be novel, and thus the work will be of interest to other scientists in this field.

Weaknesses of the study include insufficient control samples for some of the experiments and also clarifying some of the approaches used and some of their interpretations of the data (detailed below). The authors of this paper could have also done a better job of citing papers published by other scientists who came up with very similar conclusions and/or used very similar techniques.

Reviewer #1 (Public Review):

The centrality of RAS proteins in human malignancies has long been established, but many issues regarding their regulation and functions remain unresolved. The results of this paper provide strong supporting evidence for an emerging model that posits that activated KRAS can only be tolerated by cells up to a certain point, after which the stress it imposes outweigh its transforming potential. These restrictions impose limits on the amount of KRAS expressed in tumor cells and are also consistent with the frequent coupling of KRAS mutations with loss of the tumor suppressor p53, as the latter relieves the stress signals induced by KRAS.

Reviewer #1 (Public Review):

Summary and Strength:

Single-cell RNA sequencing is the most appropriate technique to profile unknown cell types and Koiwai et al. made good use of the suitable tool to understand the heterogeneity of shrimp hemocyte populations. The authors profiled single-cell transcriptomes of shrimp hemocytes and revealed nine subtypes of hemocytes. Each cluster recognizes several markers, and the authors found that Hem1 and Hem2 are likely immature hemocytes while Hem5 to Hem9 would play a role in immune responses. Moreover, pseudotime trajectory analysis discovered that hemocytes differentiate from a single subpopulation to four hemocyte populations, indicating active hematopoiesis in the crustacean. The authors explored cell growth- and immune-related genes in each cluster and suggested putative functions of each hemocyte subtype. Lastly, scRNA-seq results were further validated by in vivo analysis and identified biological differences between agranulocytes and granulocytes. Overall, conclusions are well-supported by data and hemocyte classifications were carefully performed. Given the importance of aquaculture in both biology and industry, this study will be an extremely useful reference for crustacean hematopoiesis and immunity. Moreover, it will be a good example and prototype for cell-type analysis in non-model organisms.

Weaknesses:

The conclusions of this paper are mostly well supported by data, but some aspects of data analysis QC and in vivo lineage validation need to be clarified.

1) It is not a trivial task to perform genome-wide analyses of gene expression on species without sufficient reference genome/transcriptome maps. With this respect, the authors should have de novo assembled a transcriptome map with a careful curation of the resulting transfrags. One of the weaknesses of this study is the lack of proper evaluation for the assembly results. To reassure the results, the authors would need to first assess their de novo transcripts in detail and additional data QC analysis would help substantiate the validity.

2) The authors applied SCTransform to adjust batch effects and to integrate independent sequencing libraries. SCTransform performs well in general; however, the authors would need to present results on how batch effects were corrected along with before and after analysis. In addition, the authors would need to check if any cluster was primarily originated from a single library, which could be indicative of library-specific bias (or batch effects).

3) Hem6 cells lack specific markers and some cells in this cluster are scattered throughout the other clusters (Fig. 1 & 2). Based on the pattern, it is possible that these cells are continuous subsets of other clusters. It would be good if the authors could group these cells with Hem7 or other clusters based on transcriptomic similarities or by changing clustering resolution. Additionally, they may also be a result of doublets, and it is unclear whether doublets were removed. Hem6 cells require additional measures to fully categorize as a unique subset.

4) The authors took advantage of FACS sorting, qRT-PCR, and microscopic observation to verify in silico analyses and defined R1 and R2 populations. While the experiments are appropriate to delineate differences between the two populations, it is not sufficient to determine agranulocytes as a premature population (Hem1-4) and granulocytes as differentiated subsets (Hem5-9). To better understand the two groups (ideally nine subtypes), additional in vivo experiments would be essential. For example, proliferation markers (BrdU or EdU) could be examined after FACS sorting R1 and R2 cells to show R1 cells (immature hemocytes) are indeed proliferating as indicated in the analyses.

5) FACS-sorted R1 or R2 population does not look homogeneous based on the morphology and having two subgroups under nine hemocyte subtypes may not be the most appropriate way to validate the data. The better way to prove each subtype is to use in situ hybridization to validate marker gene expressions and match with morphology.

Reviewer #1 (Public Review):

This manuscript, which follows on from a recent eLife paper documenting the relevance of the multi-basic cleavage site (MBCS) in the spike (S) protein of SARS-CoV-2, shows that growing SARS-CoV-2 on relevant epithelial cell lines or differentiated stem cell-derived culture systems prevents the emergence of MBCS mutations than impact on properties of S that contribute to cell tropism and the viral entry mechanism.

The paper builds on the authors previous work and that of others, and in some respects the results are not surprising. Nevertheless, the paper sets out a number of important findings. 1) That SARS-CoV-2 grown in Vero cells rapidly acquire MBCS mutations, where as virus grown in airway epithelial cells or Vero-TMPRSSR2 cells do not; 2) that deep sequencing is necessary to see mutations that are not apparent in consensus sequence reads, 3) that factors such as the addition of fetal calf serum can influence the selection of mutant phenotypes and 4) that cultures derived from differentiated stem cells can provide reproducible systems for virus culture. Together, the work sets out clear guidelines for the production of SARS-CoV-2, and potentially other viruses, avoiding the pitfalls that can arise from growing viruses in permissive transformed cell lines.

The data and manuscript are clearly presented, and my concerns are minimal. Overall, the paper will make a useful addition to the SARS-CoV-2 literature and will be of value to researchers working not just of SARS-CoV-2 but on many other viruses.

Reviewer #1 (Public Review):

Mature mammalian hair cells in the cochlea do not regenerate after damage. The outer hair cells of the cochlea, which function to amplify sound, are particularly susceptible to damage. Ectopic activation of two key transcription factors for outer hair cell formation, Atoh1 and Ikzf2, in damaged adult cochlea is sufficient to convert supporting cells into hair cells expressing Prestin, which is an essential protein mediating outer hair cell functions. Although there is no functional recovery in these transgenic mice based on auditory brainstem response, this study paves the way for future design of models for hearing recovery. The main concern is the identity of the OHC-like cells drawn from the small sample size in the scRNA-seq experiments.

Reviewer #1 (Public Review):

The presented manuscript takes a comprehensive and elaborated look at how T cell receptors (TCR) discriminate between self and non-self antigens. By extending a previous experimental protocol for measuring T cell receptor binding affinities against peptide MHC complexes (pMHC), they are able to determine very low TCR-pMHC binding affinities and, thereby, show that the discriminatory power of the TCR seems to be imperfect. Instead of a previously considered sharp threshold in discriminating between self and non-self antigen, the TCR can respond to very low binding affinities leading to a more transient affinity threshold. However, the analysis still indicates an improved discrimination ability for TCR compared to other cell surface receptors. These findings could impact the way how T cell mediated autoimmunity is studied.

The authors follow a comprehensive and elaborated approach, combining in vitro experiments with analytical methods to estimate binding affinities. They also show that the general concept of kinetic proofreading fits their data with providing estimates on the number of proofreading steps and the corresponding rates. The statistical and analytical methods are well explained and outlined in detail within the Supplemental Material. The source of all data, and especially how the data to analyze other cell surface receptor binding affinities was extracted, are given in detail as well. Besides being able to quantify TCR-pMHC interactions for very low binding affinities, their findings will improve the ability to assess how autoimmune reactions are potentially triggered, and how potent anti-tumour T cell therapies can be generated.

In summary, the study represents an elaborated and concise analysis of TCR-pMHC affinities and the ability of TCR to discriminate between self and non-self antigens. All conclusions are well supported by the presented data and analyses without major caveats.

Reviewer #1 (Public Review):

The authors demonstrate applications including fluorescent marking of membranes with GFP or monomeric RFP, reporter alleles for convenient assessment of differentiation status based on fluorescence, and targeted gene knockout. They also demonstrate conditional gene knockdown and induction with tight control achieved by engineering a protein destabilizing domain. The design of the constructs is clever and imparts the ability to leverage iterative FACS to enrich successfully targeted cells, particularly useful when targeting alleles that are not actively expressed by the progenitors. The work is well done and clearly presented.

Reviewer #1 (Public Review):

In this study, Moncla et al. used genomic data to analyse a mumps outbreak in Washington, in order to draw inferences about the epidemiological factors driving the outbreak. Some important strengths of the analysis include sophisticated sequencing and modeling techniques to reconstruct chains of transmission during the outbreak, which support the conclusions that the mumps virus was introduced several times in Washington from other North American regions during the outbreak, and that the Washington Marshallese community was particularly at risk of mumps infection and transmission during this time. Limitations of the analysis include potential for sampling bias, where the sample may not be entirely representative of mumps outbreak cases, and a sample size that is too low to allow sufficient statistical power to assess the impacts of age and vaccination status on transmission. The work has potential public health impacts in terms of identification of a vulnerable community and points to social networks as the primary risk factor for potential future respiratory virus outbreaks. The analysis methods could be potentially applied for the phylodynamic analysis of other infectious disease outbreaks.

Reviewer #1 (Public Review):

It is difficult to overestimate the importance of this paper. The full connectome of the Drosophila central complex is both the beginning and the end of an era. It provides the first comprehensive dataset of arguably the most enigmatic brain region in the insect brain. This endeavor has generated ground truth data for years of functional work on the neural circuits the connectome outlines, and constitutes an unparalleled foundation for exploring the structure function relations in nervous systems in general. This will be of great importance far beyond work on the Drosophila brain, and will have far reaching implications for comparative research on insect brains and likely also smoothen the path toward understanding navigation circuits in vertebrate nervous systems. Based on presented data, the paper develops overarching ideas (at exquisite detail) of how sensory information is transformed into head direction signals, how these signals are used to enable goal direction behavior, how goals are represented, and how internal state can modulate these processes. The connectome enables the authors to base these ideas and their detailed models on actual biological data, where earlier work was forced to indirectly infer or speculate. While significantly going beyond models of central-complex function that existed previously, the authors have to be much credited for incorporating huge amounts of existing knowledge and data into their interpretations, not only work from Drosophila, but also from many other insects. This makes this paper not only an invaluable resource on the connectome of the Drosophila central complex, but also a most comprehensive review on the current state of the art in central-complex research. This unifying approach of the paper clearly marks a reset of central-complex research, essentially providing a starting point of hundreds of new lines of enquiry, probably for decades to come.

Given the type and amount of data presented, the paper is clearly overwhelming. That said, it also clearly needs to be presented in the way it was done, mostly because no single aspect of the function of this neuropil makes as much sense in isolation as it makes sense when viewed in conjunction of all its other functions. The complexity of the neural circuits discussed is clearly reflected in the enormous scope of the paper. Nevertheless, the authors have done a fantastic job in breaking the circuits and their function down into digestible bits. The manuscript is very systematic in its approach and starts with sensory pathways leading to the CX, covering the clearly delineated head direction circuits and then moving on to the more complex and less understood parts, always maintaining a clear link between structure and function. As function is necessarily based on previous work, including that from other species, the results part is interwoven with interpretation, but this is clearly necessary to keep the text readable. The authors have made considerable efforts to provide additional introductions and summaries whenever needed, almost creating nested papers embedded within the overall paper.

The figures are equally overwhelming as the text at first sight, but when taking the time to digest each one in detail, they present the data in a rich and clear manner. The figures are often encyclopedic and will serve as reference about the central complex for years. The summary graphs that are presented in regular intervals are welcome resting places for the reader, helping to digest all the detailed information that has preceded or that will follow.

The analysis performed in the paper is excellent, comprehensive and should set the standard for any future work on this topic. Also, the text is very honest about the limits of the conclusions that can be reached based on this kind of data, which is important in generating realistic and feasible hypotheses for future experiments.

Reviewer #1 (Public Review):

Brascamp and colleagues address pupil-size changes around perceptual switches in perceptual multistability. Several previous studies have found pupil dilation around or after the switch and some have found pupil constriction, though the latter was typically less robust. Moreover, while most previous studies included some controls for the effect of reporting and for the physical stimulus change, to my knowledge, so far, no study has fully crossed the factors report/no-report and endogenous/exogeneous switch. In the present study, this gap is filled using a binocular-rivalry stimulus and an OKN-based no-report paradigm. This allows the authors to isolate the constriction component from the dilation component and interestingly they find the constriction more robustly tied to the perceptual switch, while the dilation component is mostly related to the response. Experiments are soundly conducted and analysed and results are interpreted with appropriate care. Since the results challenge frequent interpretations as to why perceptual switches in multistability may cause pupil-size changes, the paper is of high relevance to the fields of pupillometry and multistability, but also to other areas where pupillometry is used as index of perceptual and cognitive processes. I only have some minor questions and requests for clarification with regard to result presentation and interpretation.

Reviewer #1 (Public Review):

Strengths and Weaknesses. The authors did quite a lot to establish gene expression and function of the annelid's trunk cells and compare them to photoreceptors of the annelid's eye. They isolated the cells with FACS and characterized gene expression in detail, they knocked down r-opsin with TALEN in the trunk and found a significant difference in a crawling response, and they express the opsin in cell culture to confirm wavelength and G-protein sensitivity. As a potential link between light sensitivity and mechano-sensitivity, they report r-opsin function and light intensity influence expression of atp2b2, a gene that modulates neuronal sensitivity in other organisms. Wavelength and G-protein activation data are valuable because I can think of few or no other organisms in the entire group of lophotrochozoan animals, where this level of experimental manipulation could be done. In short, a strength of this manuscript is the detailed characterization of the trunk receptor cells, which express r-opsins. The authors have brought much evidence to the claim that these TRE cells have both light and mechano-sensitive gene expression and function. Based on these findings in an annelid worm, I believe the paper is a significant advance, and of interest to a broad audience by adding to a growing set of discoveries of similar hybrid sensory cells.

If a hybrid mechano/photo-receptor is indeed an ancient cell type in bilaterians, this would bring many evolutionary implications for sensory biology. However, in these evolutionary interpretations is where I find a weakness of the manuscript. Namely, with only a handful of species shown thus far to have the hybrid cell type - and many differences in detail about these cell types in different organisms - we can not yet make firm conclusions about whether the multi-functional cells were ancestral. I believe other interpretations are equally valid (and still interesting) and should be given more consideration. Namely, it seems possible that photo- and mechan- sensory processes "joined forces" (e.g. through separate co-option events) in new cell-types, multiple times during evolution. The current manuscript loosely indicates ancestral multi-functionality is more parsimonious. However, no detail is given about that. I suppose the authors mean a single origin of hybrid cell types requires fewer evolutionary transitions than multiple origins. However, such a parsimony count does not count the transitions requiring loss of phototransduction in mouse hearing and do not count transitions to loss of mechanosensitivity in eye photoreceptor cells.

substitutes

Replacement

Barriers

A lot more barriers for suppliers to jump through

unintended consequences

Reviewer #1:

The authors have acquired a substantial multimodal dataset and have used careful statistical approaches throughout. The data are acquired and analysed using appropriate methods.

Overall, this is an impressive body of work that aims to answer an interesting question. However, a number of questions over the methods and interpretation make the authors' conclusions difficult to justify.

When comparing between older and younger adults it would also be helpful to know the amount of grey matter in the voxels of interest. It might be expected that older adults might have more atrophy and therefore lower GABA+, than younger adults and this should be controlled for in the statistical models. The authors have put assumptions into their quantification, which are reasonable but are still assumptions. It would be helpful to directly test for a difference in grey matter fraction in the voxel between the two groups, and include this in the model if necessary.

The authors then look at behaviour, where they use a previously described task which consists of bimanual tapping, with switching between two patterns. The results are complex as there are a number of behavioural metrics, and no clear pattern emerges. While older adults produced more errors in continuation, they also produced more fully correct switching transitions. Older subjects were slower than younger adults in all trials. While this task produces a very rich dataset, which is helpful for analysing complex behaviour, it is not clear how each metric should be interpreted in terms of the underlying neural mechanisms, and how they can be usefully combined, could be given.

In terms of connectivity, the authors found no significant group X task difference between in-phase and anti-phase conditions. They therefore look at the groups and tasks separately. They show different changes in connectivity between age groups in different frequency bands, for example between left and right M1 in the alpha/mu and beta, between EMG and left M1 in the theta band. I am not sure that describing EEG-EMG connectivity as cortico-spinal is strictly accurate - there may be a number of other factors in this -corticomuscular would seem to be more precise. The frequency bands used are not typical, and it would be helpful to have an a priori explanation of which are being tested and why - as well as details about correction for multiple comparisons across these bands.

Finally, the authors bring their GABA, behaviour and connectivity metrics together in a number of mediation analyses. They demonstrate a relationship between cortico-cortical connectivity and behaviour, which is mediated by age.

The authors describe their finding of higher GABA+ in the occipital cortex as a posterior-anterior gradient, which I think is not justified by the results - there could be a number of other reasons for this, for example that different functional networks have different GABA+ levels, which is not related to their anatomical position. With only three voxels it is difficult to make a general claim such as this, and this should probably be reworded.

The authors state that higher GABA+ indicated neural system integrity and better functioning in the older group. This seems to be rather over-interpreting their results - there are many other metrics of integrity and functioning that have not been assessed here. I would suggest rewording.

Consequence: finding a different market from a safe one to where you can male a higher profit

Insentive to decrease the supply

Competition

trying to make money by saying that this 'might' be a soultion but not actually caring if it was or not

physical money cost on society

Reviewer #1 (Public Review):

This paper presents the exciting statement that increasing viral loads within a community can be used as an epidemiological early-warning indicator preceding increased positivity. It would be interesting to support this claim to present both Ct and positivity on the same graph to demonstrate that indeed, declining Ct can be used as an early marker of a COVID-19 epidemic wave. Percentage of positive test data should not only include the ones obtained in the present study but should be compared with "national data" as the present study design includes a bias in patients selection that might not reflect the "true" situation at the time. Only with this comparison, we could claim that the present study design could predict COVID-19 epidemic waves. A correlation of Ct with clinical evidence to rank the confidence of positive results is also included and further support the high specificity of the RT-PCR for detecting SARS-CoV-2 (99.995%).

In a serological investigation, it was observed that some of these RT-PCR-positive cases do not appear to seroconvert and that possible re-infections might occur despite the presence of anti-spike antibodies. Although, reported on few individuals and therefore to be taken with extreme caution, this add some piece of information to the current unknown of the serological response of COVID-19 patient and would be of uttermost importance in the context of the current vaccination campaign.

Reviewer #1 (Public Review):

Sensorimotor integration is required for the accurate execution of volitional movements, but the neural circuits underlying sensorimotor integration are still not fully understood. The whisker system of the rodent has emerged as one model of sensorimotor integration with many recent studies focused on the synaptic organization of the underlying circuitry. Here, Yamawaki et al report results regarding the synaptic organization of the ascending sensory pathways related to mouse forelimb somatosensory and motor cortex. Using anatomical and functional approaches, they elucidate the circuitry from the cuneate nucleus through thalamus to forelimb S1 and M1. This work complements recent studies in the mouse of other aspects of the forelimb sensorimotor pathways and leads to informative comparisons to the circuit organization of the whisker system. The studies are well executed and well explained. The use of multiple approaches compensates for the limitations of each individual technique, although some limitations such as any effects of viral tropism are difficult to overcome. Overall, this work contributes to a better understanding of the wiring diagram of sensorimotor circuits in the mouse.

Joint Public Review:

Worker bees perform specialised tasks: young workers nurse larvae, older ones forage for either nectar or pollen. Behaviours - including these specialist ones - arise when a stimulus (nectar, pollen or larvae) exceeds a certain 'response threshold' of the organism. This threshold can be modulated by neuropeptides to alter behaviour.

The study first shows that response thresholds to task-related stimuli differ among nurse bees, nectar and pollen foragers. Pollen foragers are most responsive to sucrose and pollen, and nurse bees most responsive to chemical stimuli of larvae. Then, taking a proteomic approach, they identify a neuropeptide, Tachykinin related protein (TRP), to be expressed in a task-specific pattern: low in nurse bees and highest in the nectar foragers.

This work provides valuable resource information on the abundance of brain neuropeptides in two species of bees. The study is exceptional in its breath of techniques used and the addition of manipulative experiments which are difficult to do in honey bees. Through their studies the authors identify a neuropeptide that modulates response thresholds of bees.

The study would have been exceptional if the authors had included studies on the expression of the tachykinin receptor. The level of tachykinin expression increases between nurse bees and foragers, but does not involve changes in spatial expression (Takeuchi et al., 2004 ref. 56). So, it is likely that the specificity of the effects of tachykinin are due to differences in the spatial expression of the receptor.

Reviewer #1 (Public Review):

The authors developed a very interesting tool, named NICEdrug.ch, used it to identify drug metabolism and toxicity, and finally predicted druggability of disease-related enzymes and reposition drugs. Comprehensive integration effort based on publicly available datasets and several previous methods developed by the authors (e. g. BridgeIT, BNICE.ch, ATLAS of Biochemistry) results with a resource named NICEdrug.ch. The idea is interesting and addresses a very important problem in the field. The manuscript is clearly written, provides enough analysis of overall challenges and an overview of the most important results. Also, it presents figures that are remarkable.

Reviewer #1 (Public Review):

Kinetochores are huge protein assemblies on chromosomes which are used as attachment point for microtubules and allow microtubules to pull chromosomes into daughter cells during cell division. The proteins that form the kinetochore are well known, but the temporal regulation of the assembly of all these proteins into functional kinetochores is less understood.

In this paper the authors have identified phosphorylation sites in the 'CCAN' of budding yeast, the 'inner', i.e. chromatin-proximal, part of the kinetochore. They characterize in detail the function of phosphorylation of Ame1 (CENP-U in humans), which is part of CCAN. The data support the idea that a cluster of phosphorylation sites in Ame1 is phosphorylated by mitotic CDK1 and serves as phospho-degron for the E3 ligase SCF/Cdc4.

The authors show phosphorylation of these CDK1 consensus sites in vivo and their phosphorylation by CDK1/Clb2 in vitro. Genetic experiments and molecular dynamics simulations support the idea that phosphorylation sites on Ame1 can serve as phospho-degron for SCF/Cdc4. Even the non-phosphorylatable mutant of Ame1 is stabilized in an SCF mutant background, though, suggesting that this phospho-degron is not the only way in which SCF influences kinetochore protein levels.

Mutants in the characterized phosphorylation sites do not impair budding yeast growth. This suggests that the degron characterized in this paper may be important for fine-tuning, but is not essential for the proper execution of mitosis. The observations overall add to prior evidence that kinetochore assembly can be regulated by phosphorylation and/or ubiquitination.

Interestingly, the authors find that phosphorylation of Ame1 by CDK1 in vitro is impaired when Ame1 binds Mtw1, another kinetochore protein. The fact that Mtw1 seems to shield these sites from phosphorylation leads the authors to put forward an interesting model: they propose that cell cycle-dependent phosphorylation and SCF-dependent degradation of kinetochore subunits allows for excess subunits during kinetochore assembly in S-phase (which will speed up assembly) while depleting any excess subunits after assembly, when the kinetochore needs to be functional.

This is an interesting model. The in vivo evidence is still limited, though. For now, it remains unknown whether the phosphorylation status of kinetochore-bound and free Ame1 is indeed different, whether more soluble Ame1 exists in S-phase, whether too early degradation of Ame1 (or possibly other kinetochore proteins) indeed impairs kinetochore assembly, or whether a failure to remove the soluble pool after assembly leads to mitotic defects. It is an attractive proposal, though, that can now be further explored experimentally.

In addition to the specific characterization of Ame1 sites, the paper also includes comprehensive data on CCAN phosphorylation sites obtained by mass spectrometry which can serve as basis for future studies.

Reviewer #1 (Public Review):

The manuscript is somewhat readable but the many acronyms for the cell types in model and biology make it difficult to follow. Is there a reason why the biological neuron names cannot be used in the model? The presentation of data in figures can be more powerful. In many cases, the data in figures and the supplemental videos show apparently different results. This can be an artifact of how the videos were made and if yes, these can be improved. Tail tip coordinates can be plotted to show the behaviors in much better detail.

Especially for beat and glide swimming, the points regarding burst firing, inhibition, etc. have not been robustly made.

Reviewer #1 (Public Review):

The study presents relatively high and robust sensitivity of Abbott ID NOW for the detection of SARS-CoV-2 (COVID-19) in an ambulatory population, utilizing the RT-PCR methodology as a comparative correlation. The study was well designed and enrolled both symptomatic and asymptomatic populations to provide sufficient statistical power for the comparative analysis of the methodologies, as well as to represent accurately the patient populations. This is a useful and timely study that has a great impact in clinical setting for the rapid detection of COVID-19.

Reviewer #1 (Public Review):

The goal of this manuscript is to develop gene-agonistic approaches for promoting cone survival in retinal degenerative diseases. Based on their previous studies, the authors tested a total of 20 genes by subretinal delivery using an AAV vector which utilized a cone-specific promoter. Most of these genes augmented glucose utilization. Interestingly, only Txnip showed a positive result by prolonging cone survival (tested up to 50 days in rd1 retina). Txnip therapy also appears to be effective in rd10 and rho-/- retina. Additional strength of this study is the use of Txnip C247S allele that blocks its association with thioredoxin. Furthermore, additional work on how Txnip may contribute to cone survival by better utilization of lactate for energy is well presented though the conclusion on "heathier" mitochondria require additional data. This manuscript is potentially of great interest. The data are extensive and biological implications of the study are clear. However, the broad conclusions with respect of Txnip therapy for RP (or even AMD) are less than justified based on the data. Two weaknesses are apparent: the first is related to the method of quantification using whole mount retina, and the second related to the duration of the study. Immunostainings of retinal sections (and even TEMs) are critical to elucidate the structure of surviving cone photoreceptors (specially in the absence of rods) and their relationship to other cells (e.g., RPE, bipolar cells, glia). Similarly, Prusky's OMR can't be equated to visual acuity. The authors need to show cone structure/function at P50 and beyond (how long do the cones survive?) in rd1 and other models before claiming the potential benefit of Txnip for retinal and macular degeneration.

Reviewer #1 (Public Review):

Oon and Prehoda report pulsatile contraction of apical membrane in the process of Par protein polarization in Drosophila neuroblasts. This explains how/why actin filament was required to localize/polarize Par complex. Specifically, using spinning disc confocal microscopy with high temporal resolution, they found the directed actin movement toward the apical pole, which nicely correlates with concentration of aPKC. They also show that myosin II is involved in this pulsatile movement of actin filament. This very much resembles the observation in C. elegans embryos, and nicely unifies observations across systems. Although descriptive in nature, I think this is an important observation and indicates a universal mechanism by which cells are polarized. I think this is a well executed study.

Reviewer #1 (Public Review):

The primary objective of this manuscript was to examine if multi-kinase inhibitor YKL-05-099 can inhibit salt inducible kinases (SIKs) with the goal to examine a new class of bone anabolic agents for the treatment of osteoporosis. They found that YKL-05-099 was successful in increasing anabolism and, surprisingly, decreasing bone resorption, leading them to investigate why this inhibitor differed from the effects of deletion of SIK2 and SIK3. They found that YKL-05-099 also inhibited the CSF1 (M-CSF) receptor, thus, inhibiting osteoclast activity. This is an interesting manuscript but there are some flaws in the conduct of the experiments and in the analyses which lessen its impact. Nevertheless, it opens the way for another possible oral therapeutic for osteoporosis.

Reviewer #1 (Public Review):

One of the most consistent and thus surprising patterns revealed by experimental evolutionary studies is the observation of a very predictable pattern of increase in fitness of replicate populations. The fitness increase tends to be very rapid at the beginning and then slows down but continues to increase for tens of thousands of generations (e.g. the Lenski LTEE). The studies from the Desai group specifically two: one by Kryazhmisky et al and one by Jonnson et al further established that the pattern of decrease in the fitness gain is due to really counterintuitive patterns of global epistasis. In particular it is not due to the evolution running out of adaptive mutations but rather to the fact that the same adaptive mutations are less beneficial on fitter backgrounds (Kryazhmisky et al). Johnson et al further found that the fitter backgrounds are more fragile with deleterious mutations being more deleterious on fitter backgrounds. All of this is rather bizarre at first glance as the microscopic epistasis is known to be highly idiosyncratic.

This paper, along with one by Lyons et al (Nat Ecol Evol 2020), resolves this paradox and shows that the observed pattern of global epistasis is in fact directly dependent on microscopic epistasis being widespread, involving multiple loci - with most parts of the organisms being connected in an "everything affecting everything" pattern, and being idiosyncratic. The Lyons et al paper focused on the data showing the epistasis is in fact idiosyncratic - their key observation - and provided an intuition for why such widespread idiosyncrasy would result in the observed pattern of global epistasis. Although neither set of authors seems to use this term, this should fit the notion of the Anna Karenina principle: "All happy families are alike; each unhappy family is unhappy in its own way." That is, in order for the right things to happen, most things need to go right, but in order for things to fail, anyone of many such things can go wrong. The more adapted systems are more fragile and more difficult to improve, because in both cases it is easier to disrupt what is already working.

The Reddy and Desai paper takes this notion and develops a very simple and transparent quantitative theory of this principle that generates specific quantitative predictions about the dynamics of adaptation that we, as a field, will spend considerable time now testing. The work has the potential to become a seminal paper in the field.

Reviewer #1 (Public Review):

This study builds upon previous findings by the authors and others that the Hedgehog (Hh) co-receptor Ihog not only binds Hh to trigger Hh signal transduction, but also engages trans-homophilic interactions in cell-cell adhesion. Using experimental manipulation and mathematical modeling, the authors assessed the role of Ihog trans-homophilic binding in stabilizing cytoneme structure and the relative strengths of Ihog-Ihog and Hh-Ihog binding. These findings led to a model whereby the weaker Ihog-Ihog trans interaction promotes direct membrane contacts along cytonemes and that Hh-Ihog binding releases Ihog from trans Ihog-Ihog complex. The studies are well designed and executed, and the findings are convincing.

AssayGeneralClass: BAO:0003061 reporter protein

AssayMaterialUsed: CLO:0036938 tumor-derived cell line

AssayDescription: Stable expression of wild type and variant PALB2 cDNA constructs in Trp53 and Palb2-null mouse cell line containing DR-GFP reporter; I-SceI endonuclease introduces a double-stranded break in the reporter construct and efficient repair results in GFP expression, which is detected by flow cytometry

AssayReadOutDescription: Homology directed repair (HDR) activity fold change, measured as GFP-positive cells and normalized relative to wild type PALB2 (set to 5.0) and the p.Y551X truncating variant (set to 1.0).

AssayRange: scaled score

AssayNormalRange: >4.4

AssayAbnormalRange: ≤1.7 for "deleterious" variants and ≤2.4 for "hypomorphic"variants

AssayIndeterminateRange: >2.4-<4.4

ValidationControlPathogenic: 7

ValidationControlBenign: 4

Replication: At least 2 independent clones per variant, each analyzed in duplicate

StatisticalAnalysisDescription: Not reported

AssayResult: 5

AssayResultAssertion: Normal

StandardErrorMean: 0

ControlType: Normal; wild type PALB2 cDNA

AssayResult: 0.5

AssayResultAssertion: Abnormal

StandardErrorMean: 0.03

AssayResult: 0.5

AssayResultAssertion: Abnormal

StandardErrorMean: 0.05

AssayResult: 0.6

AssayResultAssertion: Abnormal

StandardErrorMean: 0.15

AssayResult: 0.6

AssayResultAssertion: Abnormal

StandardErrorMean: 0.07

Comment: This variant was reported as c.2145_2146delT p.(Asp715Glufs2), however the numbering implies the deletion of two nucleotides. The deletion of TA, c.2145_2146delTA, gives the reported protein change (p.(Asp715Glufs2), and was assumed to be the intended variant. Use this evidence with caution.

AssayResult: 0.8

AssayResultAssertion: Abnormal

StandardErrorMean: 0.14

AssayResult: 0.8

AssayResultAssertion: Abnormal

StandardErrorMean: 0.13

AssayResult: 1

AssayResultAssertion: Abnormal

StandardErrorMean: 0

AssayResult: 1.5

AssayResultAssertion: Abnormal

StandardErrorMean: 0.16

AssayResult: 1.7

AssayResultAssertion: Abnormal

StandardErrorMean: 0.34

AssayResult: 1.7

AssayResultAssertion: Abnormal

StandardErrorMean: 0.84

AssayResult: 2.4

AssayResultAssertion: Abnormal

StandardErrorMean: 0.22

Comment: This variant is reported as a potential hypomorphic variant.

AssayResult: 3

AssayResultAssertion: Indeterminate

StandardErrorMean: 0.32

AssayResult: 3.6

AssayResultAssertion: Indeterminate

StandardErrorMean: 0.28

AssayResult: 3.6

AssayResultAssertion: Indeterminate

StandardErrorMean: 0.01

AssayResult: 3.6

AssayResultAssertion: Indeterminate

StandardErrorMean: 0.11

AssayResult: 3.7

AssayResultAssertion: Indeterminate

StandardErrorMean: 0.13

AssayResult: 3.8

AssayResultAssertion: Indeterminate

StandardErrorMean: 0.18

AssayResult: 3.9

AssayResultAssertion: Indeterminate

StandardErrorMean: 0.04

AssayResult: 4