Reviewer #1 (Public Review):

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

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

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

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

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

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

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

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

Reviewer #1 (Public Review):

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

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

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

Specific comments:

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

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

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

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

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

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

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

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

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

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

Reviewer #1 (Public Review):

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

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

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

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

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

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

Reviewer #1 (Public Review):

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

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

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

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

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

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

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

Reviewer #1 (Public Review):

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

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

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

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

Reviewer #1 (Public Review):

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

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

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

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

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

Summary:

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

Strength and weakness:

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

Significance of the work:

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

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

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

The research presents both strengths and weaknesses.

Strengths include:

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

Weaknesses include:

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

Reviewer #1 (Public Review):

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

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

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

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

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

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

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

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

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

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

Reviewer #1 (Public Review):

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

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

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

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

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

Main claim

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

The authors present a study of figure-ground segregation in different species. Figure-ground segregation is an important mechanism for the establishment of an accurate 3D model of the environment. The authors examine whether figure-ground segregation occurs in mice in a similar manner to that reported in primates and compare results to two other species (Tree shrews and mouse lemurs). They use both behavioral measures and electrophysiology/two-photon imaging to show that mice and tree shrews do not use opponent motion signals to segregate the visual scene into objects and background whereas mouse lemurs and macaque monkeys do. This information is of great importance for understanding to what extent the rodent visual system is a good model for primate vision and the use of multiple species is highly revealing for understanding the development of figure-ground segregation through evolution.

The behavioral data is of high quality. I would add one caveat: it seems unfair to report that the tree shrews could not generalize the opponent motion stimulus as it seems they struggled to learn it in the first place. Their performance was below 60% on the training data and they weren't trained for many sessions in comparison to the mice. Perhaps with more training the tree-shrews might have attained higher performance on the textures and this would allow a more sensitive test of generalization. The authors should qualify their statements about the tree-shrews to reflect this issue.

Reviewer #1 (Public Review):

This study used a multidimensional stimulus-response mapping task to determine how monkeys learn and update complex rules. The subjects had to use either the color or shape of a compound stimulus as the discriminative dimension that instructed them to select a target in different spatial locations on the task screen. Learning occurred across cued block shifts when an old mapping became irrelevant and a new rule had to be discovered. Because potential target locations associated with each rule were grouped into two sets that alternated, and only a subset of possible mapping between stimulus dimensions and response sets were used, the monkeys could discover information about the task structure to guide their block-by-block learning. By comparing behavioral models that assume incremental learning, quantified by Q-learning, Bayesian inference, or a combination, the authors show evidence for a hybrid strategy in which animals use inference to change among response sets (axes), and incremental learning to acquire new mappings within these sets.

Overall, I think the study is thorough and compelling. The task is cleverly designed, the modeling is rigorous, and the manuscript is clear and well-written. Importantly there are large enough distinctions in the behavior generated by different models to make the authors' conclusions convincing. They make a strong case that animals can adopt mixed inference/updating strategies to solve a rule-based task. My only minor question is about the degree to which this result generalizes beyond the particulars of this task.

Reviewer #1 (Public Review):

In the submitted manuscript, the authors observed that Glycine treatment could phenocopy deficiency of NINJ1, a recently discovered cell surface molecule critical for plasma membrane rupture, and also inhibit the aggregation of NINJ1. However, whether Glycine directly inhibits NINJ1 was not examined, and thus, the manuscript falls short of having a significant impact in the field.

Strengths of the manuscript:

1. Timely. There is great interest in understanding the mechanism of plasma membrane rupture.<br /> 2. The data provided using several mouse and human cell culture systems overall support the conclusion that Glycine targets NINJ1-mediated plasma membrane rupture (as the title says).

However, most of the presented data is predictable from previous publications. Direct evidence of the mechanism by which NINJ1 is inhibited by Glycine, or in other words, NINJ1 as the direct target of Glycine, was not provided in this manuscript. It is therefore still possible that Glycine acts indirectly upstream of NINJ1. This possible indirect mechanism can be inferred from previous reports where other amino acids such as Serine also could inhibit cell lysis (reviewed in PMID: 27066896).

Reviewer #1 (Public Review):

In order to study odor response dynamics in the olfactory peripheral organ, Kim et al. employs extracellular sensillum recording from the locust antenna to a set of 4 odors at different concentrations. Using spike sorting to assign odor responses to single olfactory sensory neurons (OSNs), the authors demonstrate that OSNs exhibit four distinct response motifs comprising two types of excitation, namely fast and delayed excitatory responses, as well as inhibitory responses in form of offset responses and inhibition. Notably, OSNs can switch between these four motifs depending on the odor applied. This finding is highly interesting and facilitates odor classification as demonstrated by computational modeling in this study. Furthermore, the authors demonstrate that each response motifs follows different adaptation profiles which further results in an increased coding space. The authors conclude and provide evidence with their model that the experimentally observed response dynamics also facilitate determining the distance to the odor source. The obtained results are novel and demonstrate a new dimension of odor response properties at the peripheral level. However, given that the authors used a very limited set of chemically similar odors and considering that the broad tuning and wiring of OSNs in the locust is special and follows different rules compared to the olfactory circuitry of OSNs in other insects (i.e. locust OSNs do not converge onto a single glomerulus but target multiple glomeruli), I wonder whether the observed distinct response motifs are a general phenomenon or a rather special case. I therefore recommend that the authors discuss their findings in the light of these key issues before general conclusions with regard to odor coding rules is being drawn. Do these response motifs also occur for highly ecologically relevant odors, such as PAN, where a rather specialized olfactory circuit would be assumed? Hence, the MS would benefit if those questions would be addressed as well. In addition, the computational modeling approach is written in specialized terms and is therefore difficult to grasp for readers lacking modeling expertise.

Reviewer #1 (Public Review):

The current manuscript by Schwager and colleagues describes a mechanism by which poorly migratory MDA-MB-231 cells can be metastatic. This study follows a recent paper from the same group (published in January) demonstrating that these poorly migratory cells are more metastatic than their highly migratory counterparts, and that this is due at least in part to E-Cadherin expression and the ability to form circulating tumour cell (CTC) clusters. In the current study, the authors show that the low migratory cells secrete unique EVs that can activate fibroblasts, concomitant with metastatic progression, and that this function is dependent on the presence of Tg-2. The novelty of this work is in the phenotypic heterogeneity of tumour cells, even within cell lines, and the importance the microenvironment in mediating metastasis associated with this diversity. While interesting, this work uses only one model, which was very recently published. The study, I think, would require repetition within additional models, as well as the inclusion of mechanistic studies designed to determine why the EV cargo differs between the highly and poorly migratory subclones.

Reviewer #1 (Public Review):

It has previously been shown that deletion of the GluA3 subunit in mice leads to alterations in auditory behavior in adult mice that are older than a couple of months of age. The GluA3 subunit is expressed at several synapses along the auditory pathway (cochlea and brainstem), and in ko mice changes in brainstem synapses have been observed. These previously documented changes may account for some of the deficits in hearing in adult ko mice.

In the current study, the authors investigate an earlier stage of development (at 5 wks) when the auditory brainstem responses (ABRs) are normal, and they ask how transmission persists at inner hair cell (ihc) ribbon synapses in GluA3 ko mice. They discovered that deletion of GluR3A significantly changed 1) the relative expression of Glu A2 (dramatically downregulated) and A4 subunits at SGN afferents, and 2) caused morphological changes in ihc ribbons (modiolar side) and synaptic vesicle size (pillar).

The changes documented in the 5 wk old GluA3ko mice were not necessarily predicted because in general the mechanisms involved in shuffling GluA receptors at this synapse (or other sensory synapses) are not completely understood; furthermore, much less is known about the role of differentiation of ihc-sgn synapses along a modiolar-pillar axis. With that said, the only shortcoming of the study is a lack of explanation for the observed changes in the synaptic structure; but this is not specific to this study.

Given the quality of the data and the clarity of presentation of results, this is a very valuable study that will aid and motivate researchers to further explore how auditory circuitry develops, and becomes differentiated, at the level of ihc-sgn synapses.

Reviewer #1 (Public Review):

In this study, Lefebvre et al. investigate the interplay between tissue geometry and the expression patterns of Runt and Tartan in establishing anisotropic myosin localization during germband extension in the Drosophila embryo. Using live and fixed light sheet imaging, computational analysis, and modeling, the authors establish a global time-resolved map of Runt expression and myosin localization during germband extension. They show that a posterior Runt stripe increasingly deviates from the dorsoventral (DV) axis during elongation, while myosin anisotropy in this region transiently deviates from the DV axis and then realigns with this axis after a delay. The authors attribute this delay to the timescale of myosin turnover and the realignment to an unidentified geometric cue. The authors develop a model that can largely account for myosin localization in wild-type, eve mutant, and twist mutant embryos using a myosin lifetime parameter representing myosin turnover. These results provide evidence for a static signal that aligns myosin anisotropy with the DV axis during elongation.

The strengths of this paper are the combination of modeling and quantitative measurements. Powerful in toto measurements show that myosin anisotropy becomes increasingly misaligned with Runt, an essential regulator of myosin planar polarity, at later stages of elongation in posterior regions of the embryo. In addition, the authors present a simple model in which changes in one parameter representing the myosin lifetime can recapitulate the relationship between myosin and edge orientation in wild-type, eve mutant, and twist mutant embryos.

The main weakness of the paper is that the authors do not directly test if their model correctly predicts the myosin lifetime in eve mutants, twist mutants, or in Fat2-RNAi embryos with altered geometry. As myosin turnover is the key parameter in their model, measuring myosin dynamics in these backgrounds would provide an important first test of their model. In addition, the authors should attempt to relate their measurements of myosin dynamics in wild-type embryos to the myosin lifetime value predicted by their model, and they should consider alternative explanations that could account for their observations in wild-type and mutant embryos.

Reviewer #1 (Public Review):

In this manuscript, Winter and colleagues define the sensitivity of cancer cells lacking the mitochondrial AAA+ ATAD1 to proteasome inhibition. They show that ATAD1 is often co-deleted with PTEN¬ in many different types of cancer. Using two complementary CRISPR screens in two distinct cell models, they identified the mitochondrial E3 ubiquitin ligase MARCH5 as a gene whose deletion is synthetically lethal with ATAD1. Since MARCH5 was previously reported to function to attenuate apoptotic signaling through mechanisms including promoting degradation of pro-apoptotic factors including BIM1, they sought to define the specific role of ATAD1 in regulating pro-apoptotic factor. They present evidence that ATAD1 extracts the pro-apoptotic protein BIMEL from mitochondria to facilitate its inactivation by mechanisms including degradation and inhibitory phosphorylation - a mechanism that appears enhanced during proteasome inhibition. This suggested that ATAD1-deficient cells could be preferentially sensitive to proteasome inhibitors. Consistent with this, expression of ATAD1 in ATAD1-deficient cells decreases sensitivity to proteasome inhibition. Similarly, depletion of ATAD1 in PC3 cells increased sensitivity to proteasome inhibition in xenografts, although somewhat curiously a corresponding increase in BIM was not readily observed (NOXA levels did increase). Finally, the authors show that prostate cancer patients with combined PTEN1/ATAD1 deletion show improved survival as compared to tumors where PTEN1 was deleted alone. Ultimately, these results support a model whereby ATAD1 promotes tumor cell survival and highlights that ATAD1 deletion may represent a vulnerability that can be exploited to treat tumors through the use of proteasome inhibitors.

Overall, this is an interesting and generally well-performed study that defines the mechanistic and functional implications of a genetic 'hitchhiker' in the context of cancer cell survival. The synthetic lethality for ATAD1 and MARCH5 observed using two different genetic approaches (deletion/overexpression) in two different cell models underscores a strong link between these two genes. Further, the data showing an important role for ATAD1 in regulating BIM mitochondrial localization/cytosolic phosphorylation are interesting. The evidence demonstrating relationships between ATAD1 and proteasome sensitivity is also convincing. However, there are some weaknesses. For example, the direct relationship between ATAD1-dependent prosurvival activities and BIM is not clearly defined. This is evident as BIM1 depletion did not influence ATAD1-deficient PC3 cells' sensitivity to bortezomib and BIM was not significantly impacted in the xenograft models. BIM deletion did partially rescue synthetic lethality in Jurkat cells deficient in both MARCH5 and ATAD1, indicating a potential role in those cells. While the authors do address this, these results do create a disconnect within the studies that complicates the overall interpretation, as the specific importance of BIM regulation by ATAD1 in different models is not consistent or always clear. Regardless, this study does reveal new insights into the genetic relationship between ATAD1 deficiency and proteasome inhibition that could have direct therapeutic potential to improve the treatment of patients. Further, considering that the anti-apoptotic roles for ATAD1 appear to extend beyond BIM regulation, this will open new avenues for investigation of the underlying molecular mechanisms whereby ATAD1 contributes to regulating apoptotic signaling in cancer and other models. With that being said, tempering the writing to better highlight that BIM regulation does not explain the ATAD1 protection observed across cancer cell models (it is the case in some, but not all) would be helpful. While there is value in the new mechanistic insight provided into the potential mechanism of ATAD1-dependent apoptotic regulation, more focus on the specific relationship between ATAD1 deficiency and proteasome inhibitor sensitivity would better suit the current work.

Reviewer #1 (Public Review):

Pathogen effectors promote parasitism either in the apoplast or cytoplasm. Unexpectedly, the work described here suggests that FolSpv1 first interacts with SlPR1 in the apoplast and then translocates SlPR1 into the nucleus of tomato plant cells. The authors suggested that the FolSpv1-mediated translocation of SlPR1 into the nucleus prevented the generation of CAPE1, leading to compromised immunity in tomato plants. The study additionally showed that acetylation of FolSpv1 K167 protects the protein from ubiquitination and proteasome-mediated degradation in both the fungal cell and plant cell. Overexpression of SlPR1 or exogenous application of CAPE1 enhanced resistance to F. oxysporum, indicating that CAPE1 contributes to disease resistance to the pathogen in tomato plants. This is consistent with prior reports that CAPE1 positively regulates plant immunity. Y2H screen followed by BiFC and co-IP supported SlPR1 as a target of FolSpv1. Most importantly, incubation of the SlPR1 recombinant protein with FolSvp1 led to uptake of both FolSvp1 and SlPR1 by tomato root protoplasts and nuclear localization of both proteins. Consistent with their model, NLS sequence is required for FolSpv1 virulence function and re-localization of SlPR1 in the nucleus. Furthermore, disease resistance conferred by SlPR1 overexpression in tomato plants could be reversed by overexpression of FolSpv1 in the fungus. Overall, the work represents a potentially significant advance in effector biology of phytopathogens. However, it is too early to exclude the possibility that the nucleus-dependent virulence function of FolSpv1 is independent of CAPE1. It is a bit strange why nuclear localization of SlPR1 is required for preventing CAPE1 generation. The following concerns need to be addressed.

1. Fig 6E shows that CAPE1 is released only upon Fol infection. This appears to contradict with the notion that FolSpv1 prevents CAPE1 release. However, Fol strain overexpressing FolSpv1 prevented the release of CAPE1. It is necessary to compare WT and the mutant strain in which the FolSvp1 gene is deleted. One would expect that the mutant strain induces significantly more CAPE1 release. Similarly, mutant strain complemented with the nls1 construct needs to be tested to see whether nuclear localization is required for preventing CAPE1 release.<br /> 2. SlPR1 is localized in the apoplast in a manner dependent on the signal peptide (Fig 5-figure supplement 1). Overexpression of SlPR1 with added NLS but lacking the signal peptide failed to enhance disease resistance to Fol infection (Fig 7G). What about overexpression of SlPR1 lacking the signal peptide without the added NLS? Does retention of SlPR1 in the cytoplasm sufficient to abolish its function? It is not even discussed why SlPR1 has to be in the nucleus to prevent CAPE1 release.<br /> 3. FolSvp1 carrying the PR1 signal peptide interacted with SlPR1 in the apoplast (Fig 6D and Fig 6-figure supplement 2). Why weren't these proteins translocated into the nucleus? These seem to contradict the in vitro uptake data. It seems that either no or only a very small proportion of SlPR1 transiently expressed in tobacco cells is located in the nucleus. Fig 7C shows that infection of the WT strain, but not the nls1 mutant strain, allowed detection of SlPR1 in the nucleus of tomato cells. However, it is not clear how much of SlPR1 remain in the apoplast or cytoplasm. Is the FolSpv1 protein secreted by Fol sufficient to translocate a significant portion of SlPR1 into the nucleus? The authors are suggested to examine apoplastic and cytoplasmic protein fractions for the relative amounts of SlPR1 after Fol infection.<br /> 4. Fig 7J and 7K, a better experiment would be to pretreat WT tomato plants with CAPE1 prior to inoculation with WT and FolSpv1 OE strains. The pretreatment should eliminate the virulence function of FolSpv1 OE if the virulence is solely dependent on the prevention of CAPE1 release.

Reviewer #1 (Public Review):

This report describes evidence that the main driving force for stimulation of glycolysis in DGC neurons by electrical activity comes from influx of Na+ including Na+ exchanging into the cell for Ca2+. The findings are presented very clearly and the authors' interpretations seem reasonable. This is important and impactful because it identifies the major energy demand in excited neurons that stimulates glycolysis to supply more ATP.

Strengths are the highly rigorous use of fluorescent probes to directly monitor the concentrations of NADH/NAD, Ca2+ and Na+. The strategies directly test the roles of Na+ and Ca2+.

Reviewer #1 (Public Review):

The 'ForensOMICS' approach is an exciting new area that clearly needs further attention. Despite the current paper being a proof-of-concept, the authors have taken due care and diligence to present the findings of the work in a transparent manner, being careful not to draw hard conclusions based on preliminary experimentation.

Despite being one of the most critical aspects of forensic investigations involving human remains, the estimation of PMI still presents significant challenges. This issue forms the premise of the current work, and this is clearly addressed in both the results and the thorough discussion. The selection of bone tissue as the target matrix is also quite unique and valuable, particularly in scenarios where other more common matrices (like soft tissues) are depleted, as is explained in the work. It is clear that, given further studies and validation, this approach could have a profound impact on the operational world of forensics.

Reviewer #1 (Public Review):

The authors of this manuscript report that human DUX4 and mouse Dux4 interact with STAT1 and inhibit interferon-stimulated gene transcription (ISG). The different functional domains of DUX4 were investigated to evaluate which ones are necessary for ISG. DUX4 transcriptional activity was found not to be necessary for ISG, rather the DUX4 C-terminal domain (CTD) was necessary and sufficient to suppress ISG. Employing liquid chromatography-mass spectroscopy (LC-MS), the DUX4 CTD was found to interact with several polypeptides present in human myoblasts. Two key regulators of innate immune signaling, STAT1, and DDX3X, ranked at the top of the list of candidate DUX4-CTD interactors. Immunoprecipitation confirmed DUX4-CTD interaction with STAT1, DDX3X, and several other polypeptides identified by LC-MS. Two regions of DUX4 were found to mediate interaction with STAT1. Amino acids 271-372 were necessary for co-IP of STAT1, and amino acids 372-424, containing (L)LxxL(L) motifs, could enhance binding to phosphorylated STAT1. IFN-gamma treatment enhanced DUX4-CTD binding to wild-type STAT1 and of the STAT1-S727A mutant. In contrast, IFN-gamma did not enhance the binding of DUX4 to the STAT1-Y701A mutant, indicating that DUX4-CTD and STAT1 interaction is promoted by STAT1 -Y701 phosphorylation. A mechanistic investigation of DUX4-STAT1 interaction was conducted by chromatin immunoprecipitation which revealed reduced IFN-gamma-induced STAT1 binding and Pol-II recruitment at promoters of several ISGs. Treatment with IFN-gamma of myoblasts derived from patients affected by facioscapulohumeral dystrophy (FSHD) showed that myoblasts expressing endogenous DUX4 failed to express the IDO1 gene which was, on the other hand, expressed in FSHD myoblasts not expressing DUX4. The majority of Ewing fusion-negative small blue round cell sarcomas have a genetic rearrangement between the CIC and DUX4 genes creating a fusion protein containing the C-terminal (L)LxxL(L) motif of DUX4. The Kitra-SRS sarcoma cell line expresses CIC-DUX4. IFN-gamma treatment of the Kitra-SRS cells showed very low induction of ISGs. Knock-down of the CIC-DUX4 fusion RNA resulted in a substantially increased IFN-gamma induction of ISGs whereas a corresponding knock-down in human myoblasts, which do not express CIC-DUX4, did not alter ISG induction.

This is an important and compelling study that sheds light on a molecular mechanism by which DUX4 inhibits IFN-mediated immune response with potential translational relevance for the treatment of DUX4-expressing cancers. The experiments are rigorously executed and controlled for, and the conclusions are well supported by the presented data.

Reviewer #1 (Public Review):

The authors sought to identify the relationship between social touch experiences and the endogenous release of oxytocin and cortisol. Female participants who received a touch from their romantic partner before a stranger exhibited a blunted hormonal response compared to when the stranger was the first toucher, suggesting that social touch history and context influence subsequent touch experiences. Concurrent fMRI recordings identified key brain networks whose activity corresponded to hormonal changes and self-report.

The strengths of the manuscript are in the power achieved by collecting multi-faceted metrics: plasma hormones across time, BOLD signal, and self-report. The experiment was cleverly designed and nicely counterbalanced. Data analysis was thorough and statistically sophisticated, making the findings and conclusions convincing.

This work sheds new light on potential mechanisms underlying how humans place social experiences in context, demonstrating how oxytocin and cortisol might interact to modulate higher-level processing and contextualizing of familiar vs. stranger encounters.

Reviewer #1 (Public Review):

Overview:

In this work, the authors set to study the effects of topographic connectivity in a hierarchical model of neural networks. They hypothesize that the topographic connectivity, often observed in cortical networks, is essential for signal propagation and allows faithful transmission of signals.

To study the effects of topographic connectivity on the dynamics, the authors consider a network composed of several layers. Each layer is a recurrent neural network with excitatory and inhibitory subpopulations. The excitatory neurons in each layer enervate a subpopulation of the following layer. The receiving excitatory subpopulation targets a specific group in the next layer and so on. This procedure leads to separate channels that carry the inputs through the network. The authors study how the degree of specificity in each targeted projection, called 'modularity,' affects signal propagation through the network.

The authors find that the network reduces noise above a critical level of network modularity: the deep layers show a clear separation of an active channel and inactive channels, despite the noisy input signal. They study how different dynamical and structural properties affect the signal propagation through the network layers and suggest that the dynamics can implement a winner-takes-all computation.

Strengths and novelty:

- Topographic projections, in which subpopulations of neurons target specific cells in efferent populations, are common in the central nervous system. The dynamic and computation benefits of this organization are not fully understood. With their simple model, the authors were able to quantify the amount of topographic structure and selectivity in the network and study its impact on the network's steady-state. In particular, a bifurcation point suggests a qualitative difference between networks with and without sufficient topographic modularity.<br /> - The theoretical analysis in the paper is rigorous, and the mean-field study shows good agreement with computer simulations of the model.<br /> - The authors describe simulation results of networks with different dynamical properties, including rate-based networks, integrate-and-fire neurons, and more realistic conductance-based spiking neurons. All simulations exhibit similar qualitative behavior, supporting the conclusion that the behavior due to structural modularity will carry to more complex and biologically relevant neural dynamics.<br /> - Overall, the authors convince that the topographic structure of the network can lead to noise reduction, given that the input to the network is provided as distinct channels.

Weaknesses:

The authors support their hypothesis and show a relation between topographic connection and noise reduction in their model. However, I find the study limited and struggle to see the impact it will have on the field. The paper is purely theoretical; it does not provide any physiological evidence that supports the conclusion. On the other hand, and this is the key issue, I do not find real theoretical insights in this work. In the following, I elaborate on why I hold this opinion.

- The hypothesis is that topographic projections in cortical areas allow faithful signal propagation. However, as the authors point out, reliable transmission can be achieved in other ways, such as by direct routing of information (lines 17-19). Furthermore, denoising can be accomplished by a simple feedforward network (e.g., ref 38) without E/I balance and with plasticity rules that do not require topographic connectivity. Thus, I find the computational model not well motivated.<br /> - The task studied here is a simple classification of static inputs: the efferent readout needs to identify the active channel. Again, this could be achieved by a single layer of simple binary neurons [Babadi and Sompolinsky 2014]. The recurrent connectivity and E/I balance suggest that dynamics should play an essential part in the model. However, the task is not well suited for understanding the role of dynamics.<br /> - The authors perform a mean-field study to explain how modularity affects signal propagation. At the heart of their argument is that the E/I network exhibit bistability. However, bistability can be achieved by an excitatory population with a threshold [Renart et al., 2013]. The role of the inhibitory population does not seem crucial for the task and questions the motivations for this analysis.<br /> - Active and inactive channels are decided by the two stable states of the network: the high and the low activity regimes. However, noise fluctuations and their propagation through the network may have a prominent role in the overall dynamics. I find that noise fluctuation analysis is bluntly missing in this work.<br /> - The main finding is a critical level of modularity, m=~0.83, above which the network shows denoising properties of silencing inactive channels and increasing the mean activity of active ones. However, the critical modularity is numerically demonstrated and is not derived theoretically. For a theoretical insight into this transition between denoising and mixing properties of the network, I would have liked to see a more rigorous discussion on the critical value. What does the critical point depend on? The authors show that the single-neuron dynamics do not affect the critical value, but what about other structural elements such as the relative efficacies of the E/I and the feedforward connectivity matrices? Do the authors suggest that m=0.83 is a universal number? I expect a more detailed analysis and discussion of this core issue in a theoretical paper.

To conclude my main criticism, I believe that a theoretical paper should offer a more in-depth analysis and discussion of the core ideas presented and not rely mainly on simulations. For example, to provide theoretical insight, the authors should address central questions such as the origin of the critical modularity, the role of the recurrent balance connectivity, and how the network can facilitate computations other than winner-takes-all among channels. Alternatively, if the authors aim to describe a neural dynamics model without deep theoretical insights, I would expect to see physiological evidence supporting the suggested dynamics.

Conclusions:

The model studied by the authors is novel and provides a valuable way of exploring the effects of modularity and topographic connectivity on signal propagation through hierarchical recurrent neural networks. However, the study lacks theoretical insights into cortical circuit functions in its current version. I believe that for this work to impact the field, it needs to show further analysis and not rely on a numerical study of the model with limited theoretical derivations.

Reviewer #1 (Public Review):

This paper tests the hypothesis that 1/f exponent of LFP power spectrum reflects E-I balance in a rodent model and Parkinson's patients. The authors suggest that their findings fit with this hypothesis, but there are concerns about confirmation bias (elaborated on below) and potential methodological issues, despite the strength of incorporating data from both animal model and neurological patients.

First, the frequency band used to fit the 1/f exponent varies between experiments and analyses, inviting concerns about potentially cherry-picking the data to fit with the prior hypothesis. The frequency band used for fitting the exponent was 30-100 Hz in Experiment 1 (rodent model), 40-90 Hz in Experiment 2 (PD, levodopa), and 10-50 Hz in Experiment 3 (PD, DBS). Ad-hoc reasons were given to justify these choices, such as " to avoid a spectral plateau starting > 50 Hz" in Experiment 3. However, at least in Experiment 3 (Fig. 3), if the frequency range was shifted to 1-10 Hz, the authors would have uncovered the opposite effect, where the exponent is smaller for DBS-on condition.

Second, there are important, fine-grained features in the spectra that are ignored in the analyses, which confounds the interpretation.

One salient example of this is Fig. 2, where based on the plots in B, one would expect that the power of beta-band oscillations to be higher in the Med-On condition, as the oscillatory peaks rise higher above the 1/f floor and reach the same amplitude level as the Med-OFF condition (in other words, similar total power is subtracted by a smaller 1/f power in the Med-ON condition). But this impression is opposite to the model-fitting results in C, where beta power is lower in the Med-ON condition.

Another example is Fig. 1C, where the spectra for high and low STN spiking epochs are identical between 10 and 20 Hz, and the difference in higher frequency range could be well-explained by an overall increase of broadband gamma power (e.g. as observed in Manning et al., J Neurosci 2012, Ray & Maunsell PLoS Biol 2011). This increase of broadband gamma power is trivially expected, as broadband gamma power is tightly coupled with population spiking rate, which was used to define the two conditions.

The above consideration also speaks to a major weakness of the general approach of considering the 1/f spectrum a monolithic spectrum that can be captured by a single exponent. As the authors' Fig. 1C shows, there are distinct frequency regions within the 1/f spectrum that have different slopes. Indeed, this tripartite shape of the 1/f spectrum, including a "knee" feature around 40-70 Hz which is well visible here, was described in multiple previous papers (Miller et al., PLoS Comput Biol 2009; He et al., Neuron 2010), and have been successfully modeled with a neural network model using biologically plausible mechanisms (Chaudhuri et al., Cereb Cortex, 2017). The neglect of these fine-grained features confounds the authors' model fitting, because an overall increase in the broadband gamma power - which can be explained straightforwardly by the change in population firing rates - can result in the exponent, fit over a larger spectral frequency region, to decrease. However, this is not due to the exponent actually changing, but the overall increase of power in a specific sub-frequency-region of the broadband 1/f activity.

Reviewer #1 (Public Review):

The manuscript by Vitet et al. reveals the role of the motor adaptor protein Huntingtin in regulating the pool of synaptic vesicles via its phosphorylation and binding to Kinesin-3 motor protein on one end and synaptic vesicle precursors on the other. The authors use both genetic models of mice harboring mutations in the HTT gene that either mimic constitutive phosphorylation of Huntingtin protein or a phospho-dead version of it. Despite previous reports suggesting no functional outcome for these mutations, using modified motor tests, the authors identified that constitutive phosphorylation of huntingtin impairs the motor skill learning of mice. Next, in a set of elegant and multidisciplinary methods, including electrophysiological recordings in acute slices, TEM imaging, knock-out rescue assay, and biochemical and in-vitro approaches, the authors suggest the mechanism for this dysfunction is through the accumulation of synaptic vesicles in the constitutive phosphorylation mode of huntingtin which increases the release probability and the corticostriatal network. The authors show that this accumulation is mediated by enhanced interaction between vesicular and phosphorylated huntingtin with Kinesin-3 motor proteins which drives the anterograde transport of synaptic vesicle precursors towards the axons and synaptic terminals.

Altogether, this reviewer finds this manuscript well written, well performed, comprehensive and convincing. The new findings in this work are a fundamental addition to the understanding of both basic mechanisms of neuronal function, as well as their dysfunction in neurodegenerative diseases, in this case, Huntington's disease.

Reviewer #1 (Public Review):

In the current manuscript, scRNA data of the early "ventral nerve cord" and optic system of the adult brain are compared. The authors generated scRNAseq data for the embryo and integrated existing data sets from other labs and extracted repo-positive glial sets to present a description of the transcriptional landscape of glial cells. The main message of the paper is that morphological diversity among glial cells in a given class is not a strong predictor of transcriptional identity.

However, the data on embryonic "ventral nerve cord" glia are generated from whole embryos, and even provided that the ventral nerve cord harbors 75% of all glia and thus the majority is ventral nerve cord, the data should not be called vnc-specific. The vnc-specific data set (adult CNS) that is already published (Allen et al., 2020) is strangely not even mentioned in the current manuscript. The idea of having a comprehensive description of glial transcriptional profiles is great - but I was missing the integration of the midline glial cells, which can be considered as ensheathing glial cells that - as the cortex glia - also express wrapper (Stork et al., 2009).

Unfortunately, I found most of what is reported in this work not to be entirely new. The classification of glial diversity in the adult brain was presented by the Meinerzhagen and Gaul labs (Edwards and Meinertzhagen, 2010; Edwards et al., 2012; Kremer et al., 2017). The description of two astrocyte-like cell types is a reduction of data that defined three morphologically distinct astrocyte-like cells (Peco et al., 2016), which is not discussed. Some other aspects were ignored, too. Two other morphological distinct types of ensheathing glia exist, ensheathing glia and ensheathing/wrapping or track-associated glia were described but this is not discussed (Kremer et al., 2017; Peco et al., 2016).

Reviewer #1 (Public Review):

The authors show that metformin reduced the elevated intraocular pressure in mice with steroid-induced ocular hypertension and attenuated damage to the cytoskeleton of the ocular trabecular meshwork. In human trabecular meshwork cells, the authors showed that the protective effects of metformin against oxidative injury were exerted by regulating cytoskeleton remodeling through integrin/ROCK signals.

Strengths of the paper include the rigorous methodology and support of the data for the conclusions. The work has the potential to advance glaucoma research but also the use of metformin for reversing other states of oxidative injury, such as fundamental aging mechanisms, in multiple tissues.

Reviewer #1 (Public Review):

Iyer et al. address the problem of how cells exposed to a graded but noisy morphogen concentration are able to infer their position reliably, in other words how the positional information of a realistic morphogen gradient is decoded through cell-autonomous ligand processing. The authors introduce a model of a ligand processing network involving multiple "branches" (receptor types) and "tiers" (compartments where ligand-bound receptors can be located). Receptor levels are allowed to vary with distance from the source independently of the morphogen concentration. All rates, except for the ligand binding and unbinding rates, are potentially under feedback control. The authors assume that the cells can infer their position from the output of the signalling network in an optimal way. The resulting parameter space is then explored to identify optimal "network architectures" and parameters, i.e. those that maximise the fidelity of the positional inference. The analysis shows how the presence of both specific and non-specific receptors, graded receptor expression and feedback loops can contribute to improving positional inference. These results are compared with known features of the Wnt signalling system in Drosophila wing imaginal disc.

The authors are doing an interesting study of how feedback control of the signalling network reading a morphogen gradient can influence the precision of the read-out. The main strength of this work is the attention to the development of the mathematical framework. While the family of network architectures introduced here is not completely generic, there is enough flexibility to explore various features of realistic signalling systems. It is exciting to find that some network topologies are particularly efficient at reducing the noise in the morphogen gradient. The comparison with the Wnt system in Drosophila is also promising.

Major comments:

- The authors assume that the cell estimates its position through the maximum a posteriori estimate, Eq.(5), which is a well-defined mathematical object; it seems to us however that whether the cell is actually capable of performing this measurement is uncertain (it is an optimal measurement in some sense, but there is no guarantee that the cell is optimal in that respect). Notably, this entails evaluating p(theta), which is a probability distribution over the entire tissue, so this estimate can not be done with purely local measurements. Can the authors comment on this and how the conclusions would change if a different position measurement was performed?

- One of the features of the signalling networks studied in the manuscript is the ability of the system to form a complex (termed a conjugated state, Q) made of two ligands L, one receptor and one non-signalling receptor. While there are clear examples of a single ligand binding to two signalling receptors (e.g. Bmps), are there also known situations where such a complex with two ligands, one receptor, and one non-signalling receptor can form? In the Wnt example (Fig. 10a), it is not clear what this complex would be? In general, it would be great to have a more extended discussion of how the model hypothesis for the signalling networks could relate to real systems.

- The authors consider feedback on reaction rates - it would seem natural to also consider feedback on the total number of receptors; notably, since there are known examples of receptors transcriptionally down-regulated by their ligands (e.g. Dpp/Tkv)? Also it is not clear in insets such as in Fig. 7b, if the concentration plotted corresponds to the concentration of receptors bound to ligands?

- The authors are clear about the fact that they consider the morphogen gradient to be fixed independently of the reaction network; however, that seems like a very strong assumption; in the Dpp morphogen gradient for instance over expression of the Tkv receptor leads to gradient shortening. Can the authors comment on this?

- Fig. 10f is showing an exciting result on the change in endocytic gradient CV in the WT and in DN mutant of Garz. Can the authors check that the Wg morphogen gradient is not changing in these two conditions? And can they also show the original gradient, and not only its CV?

Reviewer #1 (Public Review):

Hyperactivation of WNT/b-catenin signaling has been implicated in cancer. How b-catenin enters the nucleus is not completely understood. Using a heterologous model system of budding yeast, authors find that nuclear translocation of b-catenin is mediated by Kap104, the orthologue of TPO1/2. Authors further showed that a PY like motif in the C-terminus of b-catenin binds TPO1 and serves as a nuclear localization signal (NLS). Mutation of the PY like motif or inhibition of TPO1/2 inhibits b-catenin mediated transcription. Overall, this is an interesting study. The evidence that the PY like motif can serve as a NLS in yeast is convincing. However, how much this motif contributes to nuclear localization of full-length b-catenin in mammalian cells is not clear. Authors have relied on transcription readout of b-catenin, which has many caveats. Direct measurement of the level of b-catenin in the nucleus is important.

Reviewer #1 (Public Review):

This manuscript reports the function of FIO1, a mammalian METTL16 homolog, in Arabidopsis. The authors found FIO1 affects early flower phenotype through regulating splicing via U6 m6A modification. This paper confirmed FIO1-mediated m6A methylation on U6 RNA, consistent with two recently published reports. The manuscript contains quite a thorough splicing analysis on how splicing is affected in the fio1 mutant where U6 m6A is absent, and a detailed explanation of how m6A could affect base pairing and secondary structure involving U6 at different temperatures.

1. FLC mRNA can be m6A methylated. The authors appear to suggest the effect is secondary. More analysis and explanation are required. For instance the authors could measure m6A level on FLC in fio1 mutant, mta mutant, and compare it with that of wt.

2. The authors used nanopore m6A sequencing to map m6A in mRNA from wt and fio1 mutant strains. I would suggest either RIP-seq or mass spectrometry measurement to confirm the loss of fio1 leads to limited mRNA m6A changes.

Reviewer #1 (Public Review):

In the article "Neuroendocrinology of the lung revealed by single cell RNA sequencing", Kuo et. al. described various aspects of pulmonary neuroendocrine cells (PNECs) including the scRNA-seq profile of one human lung carcinoid sample. Overall, although this manuscript does not have any specific storyline, it is informative and would be an asset for researchers exploring various new roles of PNECs.

Major comments:<br /> The major concern about the work is most results are preliminary, and at a descriptive level, conclusions or sub-conclusions are derived from scRNA-seq analysis only, lacking in-depth functional analysis and validation in other methods or systems. There are many open-end results that have been predicted by the authors based on their scRNA-seq data analysis without functional validation. In order to give them a constructive roadmap, it would be better to investigate literature and put them in a potential or probable hypothesis by citing the available literature. This should be done in each section of the result part.<br /> The paper lacks a main theme or specific biology question to address. In addition, the description about the human lung carcinoid by scRNA-seq is somehow disconnected from the main study line. Also, these results are derived from the study on only one single patient, lacking statistical power.

Reviewer #1 (Public Review):

In this manuscript, Dhurandhar, Cecchi and Meyer present a model that aims to predict the discrimination performance of human subjects in an odor mixture discrimination task using low-dimensional features, which include intensity, pleasantness and a set of 19 semantic descriptors. Specifically, the authors aim to find a metric of odor mixture similarity in feature space that accurately captures similarity (or discriminability) as judged by human subjects. The semantic descriptors are obtained from a chemoinformatic model previously developed by the authors. A mixture's feature vector is defined as the average of the features of the individual components. A Mahalanobis distance is defined between two mixtures, whose parameters are fit using experimental data from Bushdid et al, Science, 2016 and applied to three other independent datasets. They show that the RMSE in prediction outperforms a previously published model in two of the datasets.

Strengths:

The idea to relate the embedding vector of individual odor components to the embedding of a mixture so as to predict mixture discrimination performance is novel and interesting.

Weaknesses:

1) The authors claims are not supported by the data presented in the Figures. A trivial model which predicts a constant can potentially achieve better predictive performance:

It is difficult to gauge the performance of the model solely from the RMSE as the data and predictions are not plotted (except in a pooled format in Figure 4b, which is however masked by the density plot). The RMSE should at the minimum be compared to the standard deviation of the dataset and plotted as the fraction of variance unexplained. Without knowledge of the standard deviation of the experimental data, it is not possible to judge the quality of the prediction.

An examination of the inset in Figure 2a and Figures 4 shows that the data spans from ~0.54 to ~0.75. Since this was quite comparable to the RMSE of ~0.17 obtained by the author's prediction, I examined the data from the four datasets provided as a supplement by the authors. It turns out that the standard deviations of the discrimination performance (the output variable) are: Bushdid 0.176, Ravia 0.144, Snitz1 0.124, Snitz2 0.119. As these numbers indicate, simply using the constant mean as a prediction will lead to an RMSE of 0.176 for the Bushdid dataset.

This appears to contradict the Middle inset in Figure 2a, which seemingly looks like a good fit. Closer examination of the two plots shows that the experimental data in the two are not the same (note for example the two datapoints with y < 0.45 in the left plot which are absent in the right). Since the authors have not clarified in the caption whether this is an illustration or if it is actual data, it is unclear how to interpret this plot.

2) The data transformations performed to obtain the mixture embedding vector seem arbitrary. For a mixture of 30 components (or even 10), this involves taking an average of 30 feature vectors, which will very likely average out. The authors should explain the rationale for taking the average and not for instance the most common descriptors that appears in the mixture components.

3) Other comments - i) the authors use linear regression to model a classification task. The justification for this choice is not explained. ii) Although this is not primary data from the authors, the authors should perhaps comment on why the minimal performance is not chance level (33%) but instead around 50 percent, even when the percent overlap between the mixtures is close to 100%. Iii) The authors do not define the Direct model. How is the RMSE of the Direct model on the Ravia dataset (0.45) much larger than the standard deviation of the dataset (0.144)?

Reviewer #1 (Public Review):

The sequencing of a genome is the first step in identifying the functional regions of that genome. The identification of the regions that encode sequences that will become proteins (protein coding genes) is made complicated by the transcription of the DNA into multiple versions of RNA (isoforms) from the same genome locus. Often these RNA isoforms have different start and stop positions in the genome and also have different sequences (exons) that are used for the protein coding process. Taking advantage of considerable improvements in a recently developed computer algorithm that predicts the most stable three-dimensional (3D) folding of protein sequences (AlphaFold2) Sommer, et al describe a strategy to use this information to evaluate among the multiple isoforms generated by each gene. This approach provides additional information along with sequence conservation, synteny and other genes that are co-regulated that can potentially rank order among isoforms to aid in annotating the protein coding human transcriptome. This capability is needed in determining the boundaries, exon sequences, evolutionary relationships of genes to their ancestral homologues, gene function and the structural regions responsible for disease.

A troubling issue of using this approach is pointed out by the authors themselves, namely, the fact that many functional genes express isoforms that make proteins with poor Local Distance Difference Test (pLDDT) scores. Thus, the 3D structures of a proteins arising from two different isoforms cannot be the only criteria used to identify the gene structure encoded in a locus. However, an isoform encoding a protein with a high pLDDT (estimated to be >80/100) is likely to help define at least a conservative set of boundaries and structures for the annotation for a gene. It would have been useful to have some overall estimate as to the false positive and negative rates of using this strategy. Without this information this approach while useful, could be considered an incremental improvement in the annotation process.

Reviewer #1 (Public Review):

The majority of polygenic scores have been developed in individuals of European descent and the analysis of the generalisability and applicability of these PRSes in diverse populations has hitherto been limited. In this study, the authors make an important contribution to addressing this gap by evaluating utility of common PRS, curated in the Polygenic Score (PGS) Catalog, in predicting the risk of the commonly diagnosed cancers with high genetic predisposition (breast, prostate, colorectal, and lung) in a prospective cohort comprising 21,694 participants of East Asian descent in Singapore.

Two major strengths in this paper are that this is one of the largest prospective Asian cohorts with long term follow up data, and the authors have completed the evaluation of a large number of PRSes (although it should be pointed out that not all of which are independent of each other).

However, the authors have only described the results of the best performing PRS and attempted to describe PRSes across 4 major common cancers as a group. In so doing, there is a missed opportunity to describe what lessons we might learn in the applicability of PRSes discovered in one population in another diverse population. In addition, it is not clear what benefits may be gleaned from the analysis of the PRSes as a group, rather than individually.

Reviewer #1 (Public Review):

This paper has significant strengths in taking a rich, quantitative, neurally-grounded approach to the development of human walking. It provides a rich empirical dataset of EMG and kinematic data at this challenging age, as well as sophisticated analyses of these data in terms of motor primitives, which are a concept that has recently been usefully applied to understanding human walking and its development.

STRENGTHS

It builds on emerging literature in this field and adds data at the key age of infancy-toddlerhood.

It takes a longitudinal approach, sampling children at the ages of newborn, 3 months, and newly walking. This is still reasonably rare in developmental research and allows for a powerful, robust interpretation of data: the authors should be commended for taking this approach.

WEAKNESSES

Some aspects of the work could have been more clearly introduced. This includes neural aspects: the location of the CNS control centres at the spinal level, and which higher centres control them (e.g. brainstem); the justification for understanding primitives as modular (no cross-talk or feedback). It also includes developmental aspects: introducing the stepping reflex, and behavioural aspects of infant motor variability (e.g. Adolph, Hoch & Cole, TICS, 2018).

The patterns relate to walking in a stereotypical manner, yet children's walking is full of skips, jumps, and climbs - both in relation to external obstacles and on even ground. Indeed, it is a challenge to get children to 'walk normally' in a lab. Thus, variability is in fact greater than is discussed here and this should be acknowledged.

The analyses are based on a limited sample of the data. (1) I am not clear on what basis the coders selected cycles, and why 5 cycles were selected. (2) It is not clear why certain movement parameters (cycle duration and flexion/extension proportions) and not others (e.g. step length, double support time) were selected. In particular, it is not clear why the authors focus on temporal, rather than spatial, variability. (3) Some data are based on stepping, and some on kicking. Because it's not clear that these are really equivalent, and because there are small samples of each (n<10), it's not clear that there is enough data to allow us to come to strong conclusions. The sample size should be justified - on the basis of power analyses and/or previous work in this area (e.g. Dominici, Science, n=40). From the results, where p values often hover around p=0.06, the paper seems underpowered to detect a decrease in variability with age for stepping kinematics and primitives.

There are some points of interpretation that could have been clearer, for example highlighting how one might distinguish between variability as incidental (motor noise) or purposeful (for exploration); and how studying the time around walking onset can contribute to the broader literature on this topic.

Reviewer #1 (Public Review):

The study focuses on the role of SLC38A5, a neutral amino acid transporter, in retinal angiogenesis. The authors show that Slc38a5 transporter is highly enriched in normal retinal vascular ECs, and upregulated in the ECs in pathogenic neoangiogenesis (the OIR model). Additionally, the authors show that Slc38a5 transcription is regulated by Wnt/β-catenin signaling and deletion of Slc38a5 in mice substantially delays retinal vascular development and suppresses pathological neovascularization in the OIR model by suppressing glutamine uptake and reducing VEGFR2 expression. The authors claim that SLC38A5 is a new metabolic regulator of retinal angiogenesis.

The study is performed carefully and demonstrates clearly an important role for the transporter in retina angiogenesis. However, there are some concerns that need to be addressed as follows:

1) The authors show that Slc38a5 is downregulated in the Lrp5-/- and Ndpy/- retinas (Fig 1A, B); however, there is a discrepancy in Slc38a5 expression levels in the control retinas. The expression of Slc38a5 in the WT retina goes down from P8-P12 and then plateaus through P17 (Fig. 1A). In contrast, in Fig.1B, the expression of Slc38a5 in the Ndpy/+ retina plateaus from P8-P12 and then goes up through P17. The authors need to establish better the temporal expression of the transporter in control (WT) retinas.

2) While it's clear that Slc38a5 mRNA and protein expression is enriched in LCM-isolated retinal vessels, it's unclear whether that expression is exclusively in ECs or also in vessel associated mural cells (Fig.1C, Fig.S1). Although Fig.S1 shows the mining of mouse retinal scRNA-seq database to demonstrate exclusive Slc38a5 expression in ECs, it's necessary to validate that in the tissue using either RNA in situ hybridization or IHC for in combination with an endothelial cell or mural cell marker.

3) Fig.3: The image qualities are poor. The authors need to enhance image qualities to show the vessels clearly in such low magnification.

4) Fig.3F: The images in this panel show more than 50% decrease in the vascular area in the deep plexus between WT and Slc38a5-/- retinas. However, the graph shows a far lower (10-15% at best) decrease in the vascular coverage. The authors need to select representative images to match the graph.

5) The authors show the presence of vessels in the adult Slc38a5-/- retina to claim that vascular abnormalities seen in early development are gone in the adult (Fig. S2). However, the presence of vessels does not mean that there are no vascular abnormalities. The authors should compare established vascular parameters such as branching-density, vascular pruning between adult WT and Slc38a5-/- retinas to justify the claim.

6) While the authors show that there is a decrease in pathological neovascularization in the Slc38a5-/- retina at P17 in the OIR model (Fig4), they do not mention what happens to the Slc38a5-/- retina at P12 immediately after the hyperoxia phase. Is the vaso-obliteration altered in the Slc38a5-/- retina at that time compared to the WT?

7) What happens to the neurovascular unit (pericyte, astrocyte, Müller glia etc) in the Slc38a5-/- retina? How do they respond to altered angiogenesis?

8) Overall, the Discussion needs to emphasize the role of endothelial cell metabolisms in vascular development and maturation and how Slc38a5 may influence these processes.

Reviewer #1 (Public Review):

The core question addressed by this study is whether right IFC damage disrupts stop-signal task performance because it plays a key role in response inhibition per se, or because it is crucial for attending to the need to engage response inhibition. A relatively large sample of patients with damage including right IFC, as well as lesioned and healthy control groups, were assessed on the stop-signal task accompanied by scalp EEG. The behavioral data were analyzed using hierarchical Bayesian modeling. Right IFC damage was associated with more trials where 'stopping' was not initiated, while an EEG hallmark of inhibitory control was present in trials where stopping initiation did occur, arguing that rIFG damage disrupts attention to the stop signal, rather than the inhibition that follows.

This is an interesting study testing a well-defined hypothesis relevant to competing views of the brain basis of inhibitory control. The experimental design is sophisticated and the analysis was preregistered. The acquisition of both behavioral and EEG data in lesion patients provides converging evidence and supports causal inference.

Interpretation of the results hinges on accepting that a hierarchical Bayesian model is appropriate for discriminating trials where stopping was 'triggered' from trials where there was no trigger. Likewise, we need to accept the EEG frontal beta burst pattern is an indicator of response inhibition. Both of these methodological elements have support from existing literature, although I don't think either of these has been applied in chronic focal lesion patients, so there may be technical issues to consider in their interpretation. Finally, as with most human lesion studies, caution should be applied in interpreting the critical lesion location: in this sample, the effects might relate to insula damage, or to white matter disruption within the ventrolateral/lateral frontal lobe or between those regions and subcortical regions. However, these provisos do not detract from the key finding that damage somewhere in these areas affected initiation/attentional processes rather than response control per se.

The results are more consistent with an attentional account of right IFG (or more broadly, right ventral frontal lobe) contributions to stop-signal task performance; this is provocative in light of current views of prefrontal contributions to inhibitory control, although in line with a wider literature implicating right frontoparietal circuitry in selective attention. As the authors suggest, a sharp distinction between attention and inhibition may be somewhat artificial: these processes may be closely interrelated in speeded tasks requiring response interruption. However, the present study cleverly tackles the challenge of disentangling them, applying recent modeling and EEG distinctions with interesting results.

The findings are helpful in further sharpening ideas regarding the neural basis of response control. They also have potential theoretical implications and perhaps direct experimental application in clinical-applied research on disorders of inhibitory control.

Reviewer #1 (Public Review):

The authors report data consistent with a new and unanticipated phenomenon: that Cre or its mRNA may be transmitted between tissues in the mouse. The epididymis appears to be the most common beneficiary of such transport from neural, and some other, tissues. The authors show this in two ways. First, they infect brains with AAV expressing Cre. They see expression of TdTomato in epididymis, from a construct that cannot express unless a loxP-flanked STOP cassette is recombined out. Second, because viral spread is a possible confounding artifact of AAV delivery, the authors also show that transgenes that drive Cre expression in the nervous system or elsewhere can cause TdTomato expression in the epididymis. They rule out that TdTomato is itself transmitted to the epididymis by showing that recombination occurred in the epididymis of the TdTomato-expressing mice.

I believe that the authors saw what they report. The data are beautiful and convincing, the experimental design was excellent in every sense, including the use of multiple alternative Cre lines, viruses, or methods. The expression of TdTomato in the epididymis and sometimes elsewhere was unambiguous, and using PCRs to validate editing in TdTomato expressing cells clinched the case.

What I am less sure of is the interpretation. The creative idea that Cre or its RNA can move between tissues in mouse would be extremely important for future technical exploitation and for demonstrating a previously un-considered complication to interpreting mouse reverse-genetic results. But as the authors note, both experiments to show this have potential caveats: AAV could escape into the circulation and go to other tissues, and promoter-Cre fusions can have leaky expression outside their expected expression zone. The authors argue, appropriately, that these most likely artifacts in the two experiment types differ, so one would have to posit that both types of artifacts occur. But this is not impossible.

I was thus excited for the parabiosis experiment, as it was the perfect way to settle the issue. The choice of strains to link in this way was ideal. Unfortunately, the sample size was small and the results were mixed: 1 of 3 cases showed a result consistent with the authors' hypothesis. Further experiments involving injection of exosomes or serum were similarly suggestive but not conclusive.

The clear and convincing data are a warning to mouse researchers about an unexpected complication of Cre-mediated gene manipulation. The data presented are consistent with the most interesting model, that Cre or its RNA can be transmitted between tissues, but additional data would make this conclusion unassailable.

Reviewer #1 (Public Review):

Jeong and coauthors demonstrate that eukaryotic type II topoisomerases undergo liquid-liquid phase separation (LLPS) under physiological conditions, and that the outcome of type II topoisomerase activity on supercoiled plasmid DNA is altered within condensates. The authors used budding yeast (Saccharomyces cerevisiae) topoisomerase II (scTopoII) to demonstrate LLPS and explore the dependence of LLPS on protein concentration, DNA concentration, and both the presence and phosphorylation sate of the unstructured C-terminal domains (CTDs) of scTopoII. Crucially, the authors verify the fluid-like behavior of the condensates, confirming coalescence of drops directly, and establishing exchange between condensed droplets and the aqueous phase via FRAP experiments. The condensates form under nominally physiological conditions, but the critical concentration decreases significantly when DNA exceeding 100 base pairs is included. As expected, the condensates can be solubilized with increasing salt or DNA concentrations. Based on established phase condensation prediction algorithms, the authors identify the CTDs of the yeast and two human isoforms of topo II as the most likely protein elements driving LLPS. They expand on this prediction by performing a useful alignment of several representative eukaryotic topo II enzymes, which reveals low homology but conservation of disorder and high frequencies of charged amino acids, both of which contribute to LLPS. The authors confirm the importance of the CTDs in LLPS by demonstrating that isolated CTDs can form condensates under a more limited set of conditions than the WT protein, whereas removing the CTDs from scTopoII inhibited LLPS altogether. In contrast, phosphorylation of the CTDs altered the biophysical properties of the condensates (fluidity for example) but not affect the propensity to form condensates. By employing a 2.9 kb negatively supercoiled DNA as the condensation scaffold and adding ATP to the condensates, the authors could measure the effects of LLPS on topo II activity. They demonstrate convincingly that topo II activity is driven towards catenation of circular DNA in condensates with full length topo II and interestingly towards the formation of knotted substrates when comparable concentrations of scTopoII lacking the CTDs was used. The authors round our this elegant work by comparing the results obtained with scTopoII with the two human isoforms hsTopoIIα and hsTopoIIβ. Together these results indicate that eukaryotic topo II enzymes can phase separate with DNA under physiological conditions and that this process can change the outcome of the strand passage reaction catalyzed by the type II enzymes. These findings help explain previous results demonstrating linking and knotting of closed circular DNA by high concentrations of type II topoisomerases in vitro, and may help unravel the roles of these enzymes in both promoting and resolving chromosome entanglements in vivo.

The main thing that others may criticize is the lack of the demonstration of LLPS and its role in vivo, but I think their findings, especially the different activities under LLPS permissive and inhibitory conditions, stand on their own.

The experiments are clear and compelling and the results support the conclusions of the study. The finding of different morphological states with plasmid DNA under some conditions is interesting and should be more fully investigated to understand the nature of this different structure that may be more relevant in vivo than the more conventional condensates observed with short DNA substrates.